Glaucoma is a complex neurodegeneration and a leading cause of blindness worldwide. Current therapeutic strategies, which are all directed towards lowering the intraocular pressure (IOP), do not stop progression of the disease. We have demonstrated that recombinant adeno-associated virus (rAAV) gene delivery of a form of erythropoietin with attenuated erythropoietic activity (EpoR76E) can preserve retinal ganglion cells, their axons, and vision without decreasing IOP. The goal of this study was to determine if modulation of neuroinflammation or oxidative stress played a role in the neuroprotective activity of EPO.R76E. Five-month-old DBA/2J mice were treated with either rAAV.EpoR76E or a control vector and collected at 8 months of age. Neuroprotection was assessed by quantification of axon transport and visual evoked potentials. Microglia number and morphology and cytokine and chemokine levels were quantified. Message levels of oxidative stress-related proteins were assessed. Axon transport and visual evoked potentials were preserved in rAAV.EpoR76E-treated mice. The number of microglia was decreased in retinas from 8-month-old rAAV.EpoR76E-treated mice, but proliferation was unaffected. The blood-retina barrier was also unaffected by treatment. Levels of some pro-inflammatory cytokines were decreased in retinas from rAAV.EpoR76E-treated mice including IL-1, IL-12, IL-13, IL-17, CCL4, and CCL5. TNFα messenger RNA (mRNA) was increased in retinas from 8-month-old mice compared to 3-month-old controls regardless of treatment. Expression of several antioxidant proteins was increased in retinas of rAAV.EpoR76E-treated 8-month-old mice. Treatment with rAAV.EpoR76E preserves vision in the DBA/2J model of glaucoma at least in part by decreasing infiltration of peripheral immune cells, modulating microglial reactivity, and decreasing oxidative stress.
We reported earlier that the levels of Ca 2+ -dependent metalloproteinases are increased in Alzheimer’s disease (AD) specimens, relative to control specimens. Here we show that these enzymes are forms of the matrix metalloproteinase MMP-9 (EC 3.4.24.35 ) and are expressed in the human hippocampus. Affinity-purified antibodies to MMP-9 labeled pyramidal neurons, but not granular neurons or glial cells. MMP-9 mRNA is expressed in pyramidal neurons, as determined with digoxigenin-labeled MMP-9 riboprobes, and the presence of this mRNA is confirmed with reverse transcriptase PCR. The cellular distribution of MMP-9 is altered in AD because 76% of the total 100 kDa enzyme activity is found in the soluble fraction of control specimens, whereas only 51% is detectable in the same fraction from AD specimens. The accumulated 100 kDa enzyme from AD brain is latent and can be converted to an active form with aminophenylmercuric acetate. MMP-9 also is detected in close proximity to extracellular amyloid plaques. Because a major constituent of plaques is the 4 kDa β-amyloid peptide, synthetic Aβ 1–40 was incubated with activated MMP-9. The enzyme cleaves the peptide at several sites, predominantly at Leu 34 -Met 35 within the membrane-spanning domain. These results establish that neurons have the capacity to synthesize MMP-9, which, on activation, may degrade extracellular substrates such as β-amyloid. Because the latent form of MMP-9 accumulates in AD brain, it is hypothesized that the lack of enzyme activation contributes to the accumulation of insoluble β-amyloid peptides in plaques.
Multiple PDZ domain protein 1 (MUPP1), a putative scaffolding protein containing 13 PSD-95, Dlg, ZO-1 (PDZ) domains, was identified by a yeast two-hybrid screen as a serotonin2C receptor (5-HT2C R)-interacting protein (Ullmer, C., Schmuck, K., Figge, A., and Lubbert, H. (1998) FEBS Lett. 424, 63–68). MUPP1 PDZ domain 10 (PDZ 10) associates with Ser458-Ser-Val at the carboxyl-terminal tail of the 5-HT2C R. Both Ser458 and Ser459 are phosphorylated upon serotonin stimulation of the receptor (Backstrom, J. R., Price, R. D., Reasoner, D. T., and Sanders-Bush, E. (2000) J. Biol. Chem. 275, 23620–23626). To investigate whether phosphorylation of these serines in the receptor regulates MUPP1 interaction, we used several approaches. First, we substituted the serines in the receptor carboxyl tail with aspartates to mimic phosphorylation (S458D, S459D, or S458D/S459D). Pull-down assays demonstrated that Asp mutations at Ser458 significantly decreased receptor tail interaction with PDZ 10. Next, serotonin treatment of 5-HT2C R/3T3 cells resulted in a dose-dependent reduction of receptor interaction with PDZ 10. Effects of serotonin on receptor-PDZ 10 binding could be blocked by pretreatment with a receptor antagonist. Alkaline phosphatase treatment reverses the effect of serotonin, indicating that agonist-induced phosphorylation at Ser458 resulted in a loss of MUPP1 association and also revealed a significant amount of basal phosphorylation of the receptor. We conclude that 5-HT2C R interaction with MUPP1 is dynamically regulated by phosphorylation at Ser458. Multiple PDZ domain protein 1 (MUPP1), a putative scaffolding protein containing 13 PSD-95, Dlg, ZO-1 (PDZ) domains, was identified by a yeast two-hybrid screen as a serotonin2C receptor (5-HT2C R)-interacting protein (Ullmer, C., Schmuck, K., Figge, A., and Lubbert, H. (1998) FEBS Lett. 424, 63–68). MUPP1 PDZ domain 10 (PDZ 10) associates with Ser458-Ser-Val at the carboxyl-terminal tail of the 5-HT2C R. Both Ser458 and Ser459 are phosphorylated upon serotonin stimulation of the receptor (Backstrom, J. R., Price, R. D., Reasoner, D. T., and Sanders-Bush, E. (2000) J. Biol. Chem. 275, 23620–23626). To investigate whether phosphorylation of these serines in the receptor regulates MUPP1 interaction, we used several approaches. First, we substituted the serines in the receptor carboxyl tail with aspartates to mimic phosphorylation (S458D, S459D, or S458D/S459D). Pull-down assays demonstrated that Asp mutations at Ser458 significantly decreased receptor tail interaction with PDZ 10. Next, serotonin treatment of 5-HT2C R/3T3 cells resulted in a dose-dependent reduction of receptor interaction with PDZ 10. Effects of serotonin on receptor-PDZ 10 binding could be blocked by pretreatment with a receptor antagonist. Alkaline phosphatase treatment reverses the effect of serotonin, indicating that agonist-induced phosphorylation at Ser458 resulted in a loss of MUPP1 association and also revealed a significant amount of basal phosphorylation of the receptor. We conclude that 5-HT2C R interaction with MUPP1 is dynamically regulated by phosphorylation at Ser458. A growing number of proteins containing PDZ 1The abbreviations used are: PDZ, postsynaptic density-95, Discs large, zonula occludens-1; MUPP1, multiple PDZ domain protein 1; 5-HT2C R, serotonin2C receptor; MAGUK, membrane-associated guanylate kinase; GST, glutathione S-transferase; BOL, 2-bromo-lysergic acid diethylamide; AMPA, α-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate; WT, wild type.1The abbreviations used are: PDZ, postsynaptic density-95, Discs large, zonula occludens-1; MUPP1, multiple PDZ domain protein 1; 5-HT2C R, serotonin2C receptor; MAGUK, membrane-associated guanylate kinase; GST, glutathione S-transferase; BOL, 2-bromo-lysergic acid diethylamide; AMPA, α-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate; WT, wild type. domains have been shown to play important roles in the organization and/or regulation of signaling events in cells. PDZ domains (or GLGF repeats) were named after three proteins identified over a decade ago: postsynaptic density-95, Drosophila Discs large, and zonula occludens-1 (3Cho K.O. Hunt C.A. Kennedy M.B. Neuron. 1992; 9: 929-942Abstract Full Text PDF PubMed Scopus (1001) Google Scholar, 4Woods D.F. Bryant P.J. Cell. 1991; 66: 451-464Abstract Full Text PDF PubMed Scopus (765) Google Scholar, 5Stevenson B.R. Siliciano J.D. Mooseker M.S. Goodenough D.A. J. Cell Biol. 1986; 103: 755-766Crossref PubMed Scopus (1277) Google Scholar). These three proteins belong to the membrane-associated guanylate kinase (MAGUK) family of proteins. Most MAGUK proteins contain three PDZ domains, an Src homology 2 domain, and a guanylate kinase-like domain, each having different cellular roles. PDZ domains range from 80 to 100 amino acids in length and typically bind to the carboxyl-terminal sequence of target proteins including receptors, channels, and various signaling molecules to regulate subcellular localization, trafficking, recycling, and/or signaling (6Sheng M. Sala C. Annu. Rev. Neurosci. 2001; 24: 1-29Crossref PubMed Scopus (1037) Google Scholar, 7Hung A.Y. Sheng M. J. Biol. Chem. 2002; 277: 5699-5702Abstract Full Text Full Text PDF PubMed Scopus (588) Google Scholar, 8Huber A. Eur. J. Neurosci. 2001; 14: 769-776Crossref PubMed Google Scholar, 9Fanning A.S. Anderson J.M. Curr. Top. Microbiol. Immunol. 1998; 228: 209-233PubMed Google Scholar, 10Kornau H.C. Seeburg P.H. Kennedy M.B. Curr. Opin. Neurobiol. 1997; 7: 368-373Crossref PubMed Scopus (312) Google Scholar). MUPP1, a protein containing 13 putative PDZ domains, was isolated in a yeast two-hybrid screening for proteins that bound to the carboxyl-terminal tail of the 5-HT2C R (1Ullmer C. Schmuck K. Figge A. Lubbert H. FEBS Lett. 1998; 424: 63-68Crossref PubMed Scopus (148) Google Scholar). MUPP1 is expressed in many tissues, whereas the 5-HT2C R is a brain-specific protein (1Ullmer C. Schmuck K. Figge A. Lubbert H. FEBS Lett. 1998; 424: 63-68Crossref PubMed Scopus (148) Google Scholar, 11Julius D. MacDermott A.B. Axel R. Jessell T.M. Science. 1988; 241: 558-564Crossref PubMed Scopus (536) Google Scholar). The 5-HT2C R has classically been thought to couple to Gq activation; however, additional G protein families have been implicated, leading to the activation of different downstream signaling pathways including phospholipase A2, C, or D, and various cation channels (12Conn P.J. Sanders-Bush E. J. Neurochem. 1986; 47: 1754-1760Crossref PubMed Scopus (59) Google Scholar, 13Mayer S.E. Sanders-Bush E. Mol. Pharmacol. 1994; 45: 991-996PubMed Google Scholar, 14Kaufman M.J. Hartig P.R. Hoffman B.J. J. Neurochem. 1995; 64: 199-205Crossref PubMed Scopus (58) Google Scholar, 15Chang M. Zhang L. Tam J.P. Sanders-Bush E. J. Biol. Chem. 2000; 275: 7021-7029Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 16Price R.D. Weiner D.M. Chang M.S. Sanders-Bush E. J. Biol. Chem. 2001; 276: 44663-44668Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 17McGrew L. Chang M.S. Sanders-Bush E. Mol. Pharmacol. 2002; 62: 1339-1343Crossref PubMed Scopus (60) Google Scholar). Since PDZ-containing proteins can scaffold many signaling molecules together into a signal transduction complex, the interaction between MUPP1 and the 5-HT2C R was further investigated. The 5-HT2C R contains a PDZ binding motif, Ser458-Ser-Val, at its extreme carboxyl terminus, which is critical for interaction with PDZ 10 of MUPP1 (18Becamel C. Figge A. Poliak S. Dumuis A. Peles E. Bockaert J. Lubbert H. Ullmer C. J. Biol. Chem. 2001; 276: 12974-12982Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). In an alternate approach to the yeast two-hybrid system, we independently show that PDZ 10 of MUPP1 is the primary site of interaction for the 5-HT2C R. Serotonin stimulation has previously been shown to promote phosphorylation of the two serine residues of the 5-HT2C R PDZ binding motif, Ser458 and Ser459 (2Backstrom J.R. Price R.D. Reasoner D.T. Sanders-Bush E. J. Biol. Chem. 2000; 275: 23620-23626Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). We therefore hypothesize that phosphorylation of the carboxyl-terminal serines of the 5-HT2C R regulates receptor interaction with MUPP1. To test this hypothesis, we investigated whether a modification of Ser458 and/or Ser459 of the 5-HT2C R carboxyl-terminal tail would alter PDZ 10 interaction. Ser458 and/or Ser459 of the receptor tail were mutated to aspartate to mimic phosphorylation (i.e. introduction of a negative charge). Next, cells expressing 5-HT2C Rs were treated with agonist or antagonist to assess the interaction of the 5-HT2C R with MUPP1. The results of these experiments support our hypothesis that phosphorylation is a key regulator of 5-HT2C R interaction with MUPP1. Furthermore, the results indicated that a significant amount of basal phosphorylation of the receptor may also play a yet undetermined role in regulating PDZ-protein interactions. Polyclonal anti-peptide antibodies against amino acids 419–435 (amino acids RHTNERVARKANDPEPG) of the rat 5-HT2C R were generated as described previously (19Backstrom J.R. Sanders-Bush E. J. Neurosci. Methods. 1997; 77: 109-117Crossref PubMed Scopus (22) Google Scholar). Anti-glutathione S-transferase (GST) antibody was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Overlapping regions of MUPP1 containing two or three PDZ domains (Fig. 1), or one PDZ domain (PDZ 9, 10, or 11) were generated by reverse transcription-PCR, sequenced, and subcloned into pGEX-4T1 (Amersham Biosciences) for expression of GST fusion proteins. MUPP1 PDZ 9, 10, or 11 were also subcloned into pGEMEX-1 (Promega), a T7 gene 10 fusion protein vector. MUPP1 PDZ 9–11 was also subcloned into pcDNA3 (Invitrogen). The 5-HT2C R carboxyl-terminal tail (last 60 amino acids) with or without the PDZ binding motif (Ser458-Ser-Val) and a truncation mutant at residue 445 were subcloned into pGEMEX-1. The 5-HT2C R carboxyl-terminal tail with the PDZ binding motif was also subcloned into pGEX-4T1. The 5-HT2C R carboxyl tail Ser458-Ser-Val (WT) was modified to S458A, S458D, S459D, or S458D/S459D by PCR site-directed mutagenesis and subcloned into pGEMEX-1. Escherichia coli was transformed with pGEX-4T1 constructs, induced to overexpress fusion proteins with isopropyl β-d-thiogalactoside, and analyzed. Bacterial lysates were obtained by first adding cold lysis buffer (50 mm Tris pH 7.5, 50 mm NaCl, 5 mm MgCl2, 1 mm dithiothreitol, 1 μg/ml leupeptin, 1 μg/ml pepstatin A, 1 μg/ml aprotinin, 1 mm benzamide, 1 mm benzamidine, 1 mm phenylmethylsulfonyl fluoride) to resuspend the pellets. Resuspended pellets were sonicated for 20 s on ice and centrifuged at 15,000 rpm for 30 min at 4 °C. Proteins were resolved on SDS-PAGE to confirm overexpressed GST fusion protein by Coomassie Blue staining and Western blotting using GST antibodies. pGEMEX-1 constructs were used for coupled transcription and translation using the TnT® in vitro translation system (Promega) in the presence of [35S]methionine (PerkinElmer Life Sciences) according to the supplier's protocol to generate 35S-labeled proteins. Ten micrograms of GST fusion proteins or GST were size-fractionated on SDS-PAGE and transferred onto nitrocellulose membrane. Nitrocellulose membranes were blocked with freshly prepared 1% BSA/phosphate-buffered saline for 1 h at room temperature. Solution was then replaced with 35S-labeled fusion proteins in 1% BSA/phosphate-buffered saline buffer and incubated with nitrocellulose membranes for 16 h at 4 °C. Nitrocellulose membranes were rinsed three times for 20 min at room temperature in 1% BSA/phosphate-buffered saline containing 0.2% Triton X-100. Nitrocellulose membranes were air-dried and exposed to x-ray film or a PhosphorImager screen (Amersham Biosciences) to visualize radiolabeled proteins. Western blot analysis using GST antibodies was used to document similar GST protein levels. NIH-3T3 cells stably transfected with the 5-HT2C R (5-HT2C R/3T3) were cultured in Dulbecco's modified Eagle's medium (Invitrogen) until confluent (20Barker E.L. Sanders-Bush E. Mol. Pharmacol. 1993; 44: 725-730PubMed Google Scholar). Cells were washed four times with Hanks' buffered saline solution (with Ca2+/Mg2+) and then serum starved in serum-free Dulbecco's modified Eagle's medium for 16 h. Cells were treated without or with antagonist (1 μm 2-bromolysergic acid diethylamide (BOL)) for 15 min at 37 °C prior to serotonin addition for 30 min at 37 °C. Medium was then removed, 1 ml of Tris buffer (50 mm Tris, pH 7.6, 0.5 mm EDTA, pH 8.0, 5 μm leupeptin, 1 mm phenylmethylsulfonyl fluoride) was added, and cells were scraped from plates and placed in an Eppendorf tube on ice. Membrane extracts were obtained using 300 μl of Tris buffer containing 1% Triton X-100. Membrane protein concentrations were determined by BCA protein assay (Pierce). Equal amounts of protein were added to the affinity columns. Western blot analysis using GST antibodies was used to determine that similar levels of fusion protein were pulled down. Twenty microliters of glutathione-Sepharose beads (Amersham Biosciences) were washed three times with PD buffer (20 mm HEPES, pH 7.6, 100 mm KCl, 10% glycerol, 0.5 mm EDTA, pH 8.0, 1 mm phenylmethylsulfonyl fluoride, 1 mm dithiothreitol, 1% Nonidet P-40). Ten micrograms of GST fusion proteins or GST were incubated with the washed glutathione beads for 1 h at 4 °C. Five microliters of 35S-labeled fusion proteins or 50 μg of membrane extracts were added to the GST-glutathione beads and incubated for 2–3 h or overnight, respectively, at 4 °C. After incubation, GST-glutathione beads were washed six times (for assays with 35S-labeled fusion proteins) or three times (for assays with membrane extracts) with PD buffer. For pull-downs from 5-HT2C R/3T3 cell lysates, precipitated protein, containing the 5-HT2C R, was treated with peptide:N-glycanase F before SDS-PAGE (see below). Loading dye (6% SDS, 1% β-mercaptoethanol, 20 mm Tris, pH 6.8, 10% glycerol plus a little bromphenol blue) was added to elute proteins. Eluates were separated on SDS-PAGE and transferred onto nitrocellulose membranes. Autoradiography or PhosphorImager screen was used to visualize radiolabeled proteins. Western blot analysis using GST and 5-HT2C R antibodies were used to document GST fusion proteins and 5-HT2C R, respectively. After serum starvation, cells were treated with 100 nm serotonin for 30 min at 37 °C. After treatment, medium was aspirated to remove the serotonin, and cells were washed four times with Hanks' buffered saline solution. Serum-free Dulbecco's modified Eagle's medium was then added, and cells were incubated for 10 or 30 min. Cells were then lysed, and a pull-down assay was performed as mentioned above. This assay was performed as previously described by Backstrom et al. (2Backstrom J.R. Price R.D. Reasoner D.T. Sanders-Bush E. J. Biol. Chem. 2000; 275: 23620-23626Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Briefly, cells grown to confluence were serum-starved and treated with increasing amounts of serotonin for 15 min at 37 °C. This incubation time was previously shown to be the minimal amount of time that would result in maximal receptor phosphorylation. Cells were then lysed, and membrane extracts containing receptors were prepared. Western blot analysis using 5-HT2C R antibodies were used to document changes in 40- and 41-kDa bands, representative of unphosphorylated and phosphorylated 5-HT2C R, respectively. Following pull-downs from membrane extracts, beads were pelleted and washed once with PD buffer containing 0.1% SDS. Fifteen microliters of PD buffer containing 1% SDS was added to the beads, and the mixture was incubated for 15 min at 37 °C. Then 58 μl of PD buffer was added, and after mixing, 15 μl of PD buffer containing 10% Triton X-100 was added. Finally, 2 μl of peptide:N-glycanase F (Glyko or New England Biolabs) was added, and samples were incubated for 2 h at 37 °C. After deglycosylation, 15 μl of 4× loading dye were added, and samples were incubated at room temperature for 20 min prior to SDS-PAGE. Membrane extracts (50 μg) of untreated or serotonin-treated cells were incubated in PD buffer plus 50 units of calf intestinal (alkaline) phosphatase (New England Biolabs) for 2 h at room temperature. After incubation, pull-down assays were carried out as described above. Alkaline Phosphatase Detection—Nitrocellulose membranes were blocked in 1% BSA/Tris blot buffer (25 mm Tris, pH 7.5, 150 mm NaCl, 0.05% Tween 20, 0.05% NaN3)for1hat room temperature. Membranes were then incubated with GST (1:1000 dilution) or 5-HT2C R (3–5 μg/ml) antibodies in 1% BSA/Tris blot buffer for 2 h to overnight at 4 °C. Membranes were washed three times with Tris blot buffer alone for 10 min. Alkaline phosphatase-conjugated goat anti-rabbit secondary antibodies (1:1000 dilution; Jackson Immunolaboratories) were incubated with membranes for2hat room temperature. Membranes were washed three times with Tris blot buffer and once with Tris (150 mm pH 9.4) and then developed with 5-bromo-4-chloro-3-indolyl-phosphate and nitro blue tetrazolium. Chemilluminescence Detection—Nitrocellulose membranes were blocked in 8% milk/Tween 20 Tris buffer solution (25 mm Tris, pH 7.4, 137 mm NaCl, 0.27 mm KCl, 0.05% Tween 20) overnight at 4 °C. Membranes were then incubated with GST (1:4000 dilution) or 5-HT2C R (0.5 μg/ml) antibodies in 2% milk/Tween 20 Tris buffer solution overnight at 4 °C. Membranes were washed four times for 5 min with Tween 20 Tris buffer solution. Horseradish peroxidase-conjugated goat anti-rabbit secondary antibodies (1:20,000 dilution; Jackson Immunolaboratories) were incubated with membranes for 45 min at room temperature. Membranes were washed four times for 15 min with Tween 20 Tris buffer solution and developed with the Pierce Supersignal West Dura® kit according to the supplier's protocol. Horseradish peroxidase signal was analyzed by Bio-Rad Flouro-S, and densitometric analysis was performed by QuantityOne (Bio-Rad) software. All bar graph data was analyzed with Graphpad Prism one-way analysis of variance with Tukey's post-test; p < 0.05 is significant, unless otherwise noted in a figure legend. GST alone was the background control for all GST fusion protein experiments, and graph data presented are background-subtracted. Data represent the means ± S.D. from several independent experiments. The 5-HT2CReceptor Selectively Interacts with MUPP1 PDZ 10 —The carboxyl region of MUPP1 containing the last four PDZ domains (PDZ 10 to PDZ 13) was originally identified by yeast two-hybrid screening for 5-HT2C R-interacting proteins (1Ullmer C. Schmuck K. Figge A. Lubbert H. FEBS Lett. 1998; 424: 63-68Crossref PubMed Scopus (148) Google Scholar). However, it was unclear which PDZ domain interacted with the 5-HT2C R. Therefore, we set out to identify which domain(s) of MUPP1 interacts with the 5-HT2C R. Overlapping PDZ domain regions of MUPP1 were generated as GST fusion proteins (Fig. 1). Purified GST-MUPP1 PDZ domains were used to pull-down in vitro translated 35S-labeled 5-HT2C R carboxyl terminus fusion protein, which consists of the last 60 amino acids harboring a PDZ binding motif, Ser458-Ser-Val. Fig. 2A illustrates that significantly more receptor tail interacted with PDZ 9–11 and PDZ 9–13. A weak interaction of the receptor tail over GST alone was observed with PDZ 12 and 13, suggesting that PDZ 9–11 is the primary 5-HT2C R interacting region. In protein overlay assays, GST-MUPP1 PDZ domain fusion proteins were blotted and probed with 35S-labeled 5-HT2C R carboxyl terminus, and the 5-HT2C R tail specifically interacted with PDZ 9–11 and PDZ 9–13; no other PDZ domains displayed a significant interaction with the receptor tail (results not shown). Thus, both protein overlay and pull-down assays consistently indicate that PDZ 9–11 is responsible for interacting with the 5-HT2C R tail. To further determine the specific site of interaction, GST fusion proteins of the individual PDZ domains, 9, 10, and 11 were made (Fig. 1). Unfortunately, GST-PDZ 10 was unstable when overexpressed in bacteria. Therefore, the ability to pull down the 35S-labeled 5-HT2C R tail by GST-PDZ 9 or 11 was compared with GST-PDZ 9–11. GST fusion proteins of PDZ 9 or 11 alone were not able to bind to the receptor tail as compared with GST-PDZ 9–11, which contains PDZ 10 (data not shown). In a complementary pull-down experiment, the individual MUPP1 PDZ domains 9, 10, and 11 were in vitro translated with [35S]methionine and pulled down by the 5-HT2C R carboxyl-terminal tail expressed as a GST fusion protein (Fig. 2B). The carboxyl tail of the receptor specifically interacted with PDZ 10 and not PDZ domain 9 or 11, further supporting PDZ 10 as the interacting region for the receptor tail. Next, we questioned whether regions upstream of the extreme carboxyl terminus of the 5-HT2C R are able to confer binding to PDZ 10. To address this question, we generated GST fusion proteins of the 5-HT2C R carboxyl-terminal tail missing only the last three residues (Δ PDZ) and the 5-HT2C R carboxyl-terminal tail ending at residue 445 (i.e. missing the last 15 amino acids). In an overlay assay, [35S]PDZ 9–11 was incubated with the different GST-5-HT2C R carboxyl-terminal tail fusion proteins. As illustrated in Fig. 3, PDZ 9–11 binds to WT but not the 5-HT2C R carboxyl-terminal truncation mutants. Mutation of Ser458 in the 5-HT2CReceptor Reveals Altered PDZ 10 Interaction—Studies previously demonstrated that Ser458 and Ser459 at the extreme carboxyl tail of the 5-HT2C R, the same region of the receptor necessary for PDZ 10 binding, are phosphorylated upon ligand activation (2Backstrom J.R. Price R.D. Reasoner D.T. Sanders-Bush E. J. Biol. Chem. 2000; 275: 23620-23626Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). A function for Ser459 phosphorylation in receptor resensitization was proposed; however, the role for Ser458 phosphorylation is unknown. Based upon crystal structures of PDZ domains (21Doyle D.A. Lee A. Lewis J. Kim E. Sheng M. MacKinnon R. Cell. 1996; 85: 1067-1076Abstract Full Text Full Text PDF PubMed Scopus (964) Google Scholar, 22Daniels D.L. Cohen A.R. Anderson J.M. Brunger A.T. Nat. Struct. Biol. 1998; 5: 317-325Crossref PubMed Scopus (161) Google Scholar, 23Karthikeyan S. Leung T. Birrane G. Webster G. Ladias J.A. J. Mol. Biol. 2001; 308: 963-973Crossref PubMed Scopus (83) Google Scholar, 24Morais Cabral J.H. Petosa C. Sutcliffe M.J. Raza S. Byron O. Poy F. Marfatia S.M. Chishti A.H. Liddington R.C. Nature. 1996; 382: 649-652Crossref PubMed Scopus (290) Google Scholar, 25Tochio H. Hung F. Li M. Bredt D.S. Zhang M. J. Mol. Biol. 2000; 295: 225-237Crossref PubMed Scopus (92) Google Scholar) and data compiled on PDZ binding motifs (26Songyang Z. Fanning A.S. Fu C. Xu J. Marfatia S.M. Chishti A.H. Crompton A. Chan A.C. Anderson J.M. Cantley L.C. Science. 1997; 275: 73-77Crossref PubMed Scopus (1212) Google Scholar), Ser458 of the 5-HT2C R is predicted to be a critical residue for interacting with PDZ 10. We therefore hypothesized that agonist-mediated phosphorylation of Ser458 disrupts the interaction of MUPP1 PDZ domain 10 and its target, the 5-HT2C R. To test this hypothesis, the serine residues in the receptor tail were replaced with aspartic acid to mimic phosphorylation. The last two serine residues of the 5-HT2C R carboxyl tail (Ser458-Ser459) were modified by PCR site-directed mutagenesis to contain S458A, S458D, S459D, or S458D/S459D substitutions. Wild-type and mutated 5-HT2C R tails were labeled with [35S]methionine and incubated with GST-PDZ 9–11. 5-HT2C R tail mutants containing S458A, S458D, and S458D/S459D substitutions displayed a marked loss of interaction to PDZ 9–11 (Fig. 4A). The S459D mutation, however, retained an ability to interact similar to wild-type interaction (Fig. 4B). These results indicate that Ser458 is an important residue in determining the interaction with PDZ 10. Serotonin Treatment Decreases the Ability of the 5-HT2CReceptor to Interact with PDZ 10 —Results from the 5-HT2C R tail mutants raise the possibility of a dynamic regulation of the interaction between the 5-HT2C R and MUPP1. Thus, we investigated whether agonist stimulation of the 5-HT2C R stably expressed in NIH-3T3 cells would also result in a loss of MUPP1 interaction. To determine whether serotonin stimulation had any effect on MUPP1-receptor interaction, cells were incubated with increasing amounts of serotonin, which have been shown to promote receptor phosphorylation (2Backstrom J.R. Price R.D. Reasoner D.T. Sanders-Bush E. J. Biol. Chem. 2000; 275: 23620-23626Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). The ability of the 5-HT2C R to bind to PDZ 10 was assessed by pull-down assays. Fig. 5A shows that cells treated with serotonin led to a dose-dependent decrease in receptor interaction with MUPP1. A 50% reduction in receptor binding to PDZ 10 was observed with a concentration 100 nm serotonin. Moreover, increasing serotonin concentrations caused a dose-dependent increase in phosphorylated receptor with a concurrent decrease in the amount of unphosphorylated receptor as determined by band shift phosphorylation assays (Fig. 5B). To determine whether the loss of PDZ 10 interaction with the receptor was a consequence of agonist binding with 5-HT2C Rs, cells were preincubated in the absence or presence 1 μm of BOL, a 5-HT2C R antagonist, for 15 min prior to the addition of serotonin. BOL antagonized a subsequent serotonin-mediated decrease in receptor pull-down (Fig. 6), thereby demonstrating that the loss of PDZ 10 interaction is a direct consequence of receptor activation. BOL alone had no effect. Alkaline Phosphatase Treatment of the 5-HT2CReceptor Increases PDZ 10 Interaction and Reveals 5-HT2CReceptor Basal Phosphorylation—The reduction of 5-HT2C R binding to MUPP1 may be the direct result of receptor phosphorylation. We therefore investigated whether treatment of lysate containing receptor with alkaline phosphatase would restore MUPP1 interaction. Cells were treated with agonist, and cell lysates were incubated with alkaline phosphatase prior to pull-down assays. As shown in Fig. 7A, alkaline phosphatase treatment resulted in more receptor pull-down in serotonin-stimulated cells. In the absence of serotonin, alkaline phosphatase treatment doubled the amount of receptor binding to PDZ 10 compared with untreated cells. These findings directly support a role for agonist-induced phosphorylation in disrupting 5-HT2C R binding to MUPP1 as well as uncover a potential function for previously reported basal phosphorylation of the receptor. Phosphorylation of the receptor is reversible; therefore, we investigated the activity of endogenous phosphatases against the receptor by washout experiments. Cells were treated with agonist and then washed thoroughly and incubated in serum-free medium for 10 or 30 min before lysis and pull-down assay. Fig. 7B demonstrates a time-dependent increase in 5-HT2C R binding to MUPP1 (Fig. 7B). These results are consistent with previously published data indicating a time-dependent dephosphorylation of the receptor (2Backstrom J.R. Price R.D. Reasoner D.T. Sanders-Bush E. J. Biol. Chem. 2000; 275: 23620-23626Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). 5-HT2C Rs are implicated in physiological processes such as cerebrospinal fluid production as well as illnesses and disorders including anxiety, migraines, and eating and sleeping disorders (27Roth B.L. Willins D.L. Kristiansen K. Kroeze W.K. Pharmacol. Ther. 1998; 79: 231-257Crossref PubMed Scopus (258) Google Scholar, 28De Vry J. Schreiber R. Neurosci. Biobehav. Rev. 2000; 24: 341-353Crossref PubMed Scopus (153) Google Scholar). Furthermore, the 5-HT2C R is a target for hallucinogenic drugs such as lysergic acid diethylamide (29Burris K.D. Breeding M. Sanders-Bush E. J. Pharmacol. Exp. Ther. 1991; 258: 891-896PubMed Google Scholar, 30Fiorella D. Helsley S. Lorrain D.S. Rabin R.A. Winter J.C. Psychopharmacology. 1995; 121: 364-372Crossref PubMed Scopus (48) Google Scholar). A thorough understanding of intracellular signaling by the 5-HT2C R, including its interaction with PDZ domain containing proteins, may give insight into the cellular mechanisms that underlie these diverse physiological processes. PDZ domain-containing proteins are involved in the localization of potassium channels and glutamate receptors in the synapse as well as localization of numerous other receptors and proteins (31Shieh B.H. Zhu M.Y. Neuron. 1996; 16: 991-998Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar, 32Shieh B.H. Zhu M.Y. Lee J.K. Kelly I.M. Bahiraei F. Proc. Natl. Acad. Sci. U. S. 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We investigated the membrane topology of Bves/Pop1A as a foundation to dissect the molecular basis and function of Bves/Pop1A trafficking during development. Bves contains two asparagine-linked glycosylation sites within the amino terminus and three putative membrane domains. Therefore, glycosylation assays were performed to determine if the amino terminus of Bves is delivered into the endoplasmic reticulum lumen and glycosylated. We establish that Bves from chick heart and transfected cells is glycosylated, implying that the amino terminus of cell surface molecules is extracellular. Three biochemically distinct approaches were utilized to determine the orientation of the carboxyl terminus of Bves. First, glycosylation of Bves at exogenous sites within the carboxyl terminus was only observed in a construct that lacked the third membrane domain, which presumably reversed the orientation of the carboxyl terminus. Second, co-expression of full-length Bves with soluble, carboxyl-terminal Bves constructs that reside in different subcellular compartments revealed that Bves-Bves interactions occur in the cytoplasm. Third, the immunoreactivity of endogenous Bves at the cell surface of epicardial cells was dramatically enhanced with detergent. These results suggest that the membrane topology of cell surface Bves/Pop1A is composed of an extracellular amino terminus, three transmembrane domains, and a cytoplasmic carboxyl terminus. We therefore hypothesize that the carboxyl terminus regulates the cellular distribution of Bves/Pop1A during coronary vessel development. We investigated the membrane topology of Bves/Pop1A as a foundation to dissect the molecular basis and function of Bves/Pop1A trafficking during development. Bves contains two asparagine-linked glycosylation sites within the amino terminus and three putative membrane domains. Therefore, glycosylation assays were performed to determine if the amino terminus of Bves is delivered into the endoplasmic reticulum lumen and glycosylated. We establish that Bves from chick heart and transfected cells is glycosylated, implying that the amino terminus of cell surface molecules is extracellular. Three biochemically distinct approaches were utilized to determine the orientation of the carboxyl terminus of Bves. First, glycosylation of Bves at exogenous sites within the carboxyl terminus was only observed in a construct that lacked the third membrane domain, which presumably reversed the orientation of the carboxyl terminus. Second, co-expression of full-length Bves with soluble, carboxyl-terminal Bves constructs that reside in different subcellular compartments revealed that Bves-Bves interactions occur in the cytoplasm. Third, the immunoreactivity of endogenous Bves at the cell surface of epicardial cells was dramatically enhanced with detergent. These results suggest that the membrane topology of cell surface Bves/Pop1A is composed of an extracellular amino terminus, three transmembrane domains, and a cytoplasmic carboxyl terminus. We therefore hypothesize that the carboxyl terminus regulates the cellular distribution of Bves/Pop1A during coronary vessel development. Bves (blood vessel epicardial substance) was identified from a subtractive cDNA screen to identify clones enriched in heart tissue (1Reese D.E. Zavaljevski M. Streiff N.L. Bader D. Dev. Biol. 1999; 209: 159-171Crossref PubMed Scopus (89) Google Scholar). Chick Bves is a 357-amino acid protein that contains two consensus asparagine-linked glycosylation sites in the amino terminus, three hydrophobic regions that are potential membrane domains, and a large 247-residue carboxyl terminus. Antibodies against chick Bves have labeled the primordial origin of coronary vessels, the proepicardial organ, the epicardium that surrounds the heart, migrating mesenchymal cells derived from the epicardium, and smooth muscle (1Reese D.E. Zavaljevski M. Streiff N.L. Bader D. Dev. Biol. 1999; 209: 159-171Crossref PubMed Scopus (89) Google Scholar). Interestingly, the cellular distribution of Bves changes dynamically from the cell surface in epicardium to a more intracellular location in migrating mesenchyme and later reappears at the cell surface in vascular smooth muscle (2Wada A.M. Reese D.E. Bader D.M. Development. 2001; 128: 2085-2093PubMed Google Scholar). Furthermore, it has been proposed that Bves functions as a cell adhesion molecule based on the results of transfected l-cells (2Wada A.M. Reese D.E. Bader D.M. Development. 2001; 128: 2085-2093PubMed Google Scholar). Thus, we sought to elucidate the membrane topology of Bves to distinguish intracellular regions that may be involved in this dynamic, intracellular trafficking from extracellular regions that may directly participate in cell-cell interactions. The Popeye family of transcripts was independently identified and found to be highly expressed in cardiac and skeletal muscle (3Andrée B. Hillemann T. Kessler-Icekson G. Schmitt-John T. Jockusch H. Arnold H.H. Brand T. Dev. Biol. 2000; 223: 371-382Crossref PubMed Scopus (98) Google Scholar). Two Popeye genes were identified in chick (POP1 and POP3). In addition, four alternatively spliced chick POP1 transcripts were isolated (POP1A through -1D), and POP1A was found to be identical to Bves (3Andrée B. Hillemann T. Kessler-Icekson G. Schmitt-John T. Jockusch H. Arnold H.H. Brand T. Dev. Biol. 2000; 223: 371-382Crossref PubMed Scopus (98) Google Scholar). Although POP1 knockout mice did not exhibit an overt phenotype, treatment of mice that lack POP1 with cardiotoxin displayed delayed skeletal muscle regeneration relative to wild-type mice (4Andrée B. Fleige A. Arnold H.H. Brand T. Mol. Cell. Biol. 2002; 22: 1504-1512Crossref PubMed Scopus (60) Google Scholar). Establishing the membrane topology of Bves/Pop1A would provide a foundation for identifying and characterizing functional domains at the cellular level. The membrane topology of Bves/Pop1A is unknown, but two distinct models have been proposed (2Wada A.M. Reese D.E. Bader D.M. Development. 2001; 128: 2085-2093PubMed Google Scholar, 4Andrée B. Fleige A. Arnold H.H. Brand T. Mol. Cell. Biol. 2002; 22: 1504-1512Crossref PubMed Scopus (60) Google Scholar). Although both groups assumed that all three hydrophobic domains span the membrane, their models present different locations of the termini. Wada et al. (2Wada A.M. Reese D.E. Bader D.M. Development. 2001; 128: 2085-2093PubMed Google Scholar) proposed that the amino terminus is cytoplasmic and the carboxyl terminus is extracellular. In contrast, Andrée et al. (4Andrée B. Fleige A. Arnold H.H. Brand T. Mol. Cell. Biol. 2002; 22: 1504-1512Crossref PubMed Scopus (60) Google Scholar) proposed that the amino terminus is extracellular and the carboxyl terminus is intracellular. Here, we document that the amino terminus of Bves/Pop1A from chick heart and transfected cells is glycosylated at asparagine-linked sites. Furthermore, the orientation of the carboxyl terminus was examined using exogenous glycosylation sites, Bves-Bves interactions, and immunocytochemistry. Our results support a three transmembrane model of Bves/Pop1A in which the amino terminus of cell surface molecules is exposed to the extracellular environment, whereas the carboxyl terminus is exposed to the cytoplasm. Chick Bves cDNA and Mutants—Bves/POP1A was subcloned into the XhoI-SalI sites of pCI-neo (Promega, Madison, WI). PCR was used to add epitope tags and generate mutants. The reagents consisted of the plasmid containing chick Bves/POP1A, recombinant Pfu polymerase (Stratagene, La Jolla, CA), and the appropriate primers (Invitrogen, Carlsbad, CA). The primers used to generate all Bves constructs described in this study are listed in Table I, and a schematic of representative constructs is illustrated in Fig. 1. The FLAG epitope (DYKDDDDK) was added to the extreme carboxyl terminus, yielding Bves-FLAG. The HA 1The abbreviations used are: HA, hemagglutinin; TBS, Tris-buffered saline; WT, wild-type.-epitope tag (YPYDVPDYA) was incorporated into the amino terminus between Leu27 and Lys28 of Bves/Pop1A, yielding HA-Bves. HA-Bves-FLAG was used to compare the immunoreactivity of anti-HA and anti-FLAG antibodies. An insert (GVEVEAVSLLNQTV), based on a glycosylated region of the rat concentrative Na+-nucleoside cotransporter (5Hamilton S.R. Yao S.Y. Ingram J.C. Hadden D.A. Ritzel M.W. Gallagher M.P. Henderson P.J. Cass C.E. Young J.D. Baldwin S.A. J. Biol. Chem. 2001; 276: 27981-27988Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar), was used to replace amino acids 117–137 of Bves in the construct labeled rCNT1. Mutation of the Asn residue within the rCNT1 insert to Ala yielded rCNT1-N/A. The carboxyl-terminal Bves construct Met128-FLAG was generated with primers Xho-Met128 sense, containing a portion of the coding region that starts at Met128, and BstEII antisense. The carboxyl-terminal Bves construct 2C/Met128-FLAG was made with primers that correspond to the end of the amino terminus of the rat serotonin 5-HT2C receptor (2C) and the beginning of Met128-FLAG. The 51-amino acid 2C region encodes a cleavable signal sequence and an endogenous Asn-linked glycosylation site at Asn39. 2J. R. Backstrom, unpublished data. PCR fragments were digested and ligated into Bves plasmid backbones. The sequences of all amplified fragments were determined at the DNA Core in the Stahlman Cardiovascular Research Laboratories.Table ISequences of primers used to generate Bves constructsPrimerDirectionSequence (5′ → 3′)pClneo (1009)SenseCTTTCTCTCC ACAGGTGTCC ACTCCCAGTT CAXho-5′ cBvesSenseAGCCTCGAGA CACGGGGACA GGATTCTTCA AGATGBstEII (640)AntisenseATCATCTGCG ATAATGGTGA CCTGGAA560SenseAATGAAGGTT TCTTATCGAG GGCATEcoR1 (280)SenseCTTGGACATA ATGATCTGGA ATTCTGTGTTcBves-FLAG-Sal-3′AntisenseGGTCGACTCT ATTACTTGTC ATCGTCGTCC TTGTAGTCcBves-Sal-3′AntisenseCTTGTCATCG TCGTCGACGT ATTAAGGCAG CCGCTGCAGC TCAAGN20ASenseGTTATTCCAG ACTTAAAAGC TGCCACCTCT GTGCCTTTCN20AAntisenseGAAAGGCACA GAGGTGGCAG CTTTTAAGTC TGGAATAACN27ASenseGCCACCTCTG TGCCTTTCGC CGAGACTGCA TGTGAAAACN27AAntisenseGTTTTCACAT GCAGTCTCGG CGAAAGGCAC AGAGGTGGCHA-5′f shortAntisenseAGGATATAAG TCAGGAATAA CGCCCAGAGG AGTGAGHA-5′f longAntisenseCGCATAGTCA GGAACGTCGT AAGGATATAA GTCAGGAATA ACGCCHA-3′f shortSenseTATGCGAAAA ATGCCACCTC TGTGCCTTTC AACHA-3′f longSenseTATCCTTACG ACGTTCCTGA CTATGCGAAA AATGCCACCT CTGTGKELS/NETS (119)AntisenseTCTCTTGTAC AGGCTGCTGG TCTCATTCTC TATCTTGATC GGTCTKELS/NETS (119)SenseAGACCGATCA AGATAGAGAA TGAGACCAGC AGCCTGTACA AGAGAMFEP/MNLT (129)AntisenseTGGAGGCACA TGGAGTGTTT CATTCATTCT CTTGTACAGG CTMFEP/MNLT (129)SenseAGCCTGTACA AGAGAATGAA TGAAACACTC CATGTGCCTC CAQRLT/QNLT (141)AntisenseGCAGAATTGC CCAGTTAAGT TTTGAAATAG CTCTGGAGGC ACQRLT/QNLT (141)SenseGTGCCTCCAG AGCTATTTCA AAACTTAACT GGGCAATTCT GCEDKT/KNET (162)AntisenseCCTGTCATCA ACTGATGTTT CATTCTTTGC AGCATAAGCT TGACCEDKT/KNET (162)SenseGGTCAAGCTT ATGCTGCAAA GAATGAAACA TCAGTTGATG ACAGGGEK/NET (209)AntisenseAATGGTGACC TGGAATGTTT CATTACGGTT CATCTGAGTT GATCGrCNT1 5′f shortAntisenseTACTTCCACG CCTTTGATCG GTCTTCTTTT ATACACCAAG TArCNT1 5′f longAntisenseGTTAAGGAGG GACACGGCTT CTACTTCCAC GCCTTTGATC GGTCTTCTrCNT1 3′f shortSenseAACCAAACTG TCCTATTTCA GAGATTAACT GGGCAATTCT GCAACrCNT1 3′f longSenseGTAGAAGCCG TGTCCCTCCT TAACCAAACT GTCCTATTTC AGAGATTAACTD3T 5′fAntisenseGAT CGGTCTT CTGAGTGTAG CCCAAATGAT GAACAATGCD3T/rCNT1 3′fSenseTGGGCTACAC TCAGAAGACC GATCAAAGGC GTGGAAGTArCNT1-N/ASenseGTGGAAGTAG AAGCCGTGTC CCTCCTTGCC CAAACTGTCC TArCNT1-N/AAntisenseTAGGACAGTT TGGGCAAGGA GGGACACGGC TTCTACTTCC ACXho-Met128SenseCCTCGAGAAT GTTTGAACCA CTCCATGTGC2C-Xho 5′fSenseCCTCGAGCAA TCATGGTGAA CCTTGGCAAC GCG2C/Met128-5′fAntisenseTGGCTCAAAC ATATCCGGGA ACTGAAACAA GCGTCCACCA TC2C/Met128-3′fSenseCAGTTCCCGG ATATGTTTGA GCCACTCCAT GTGCCTCCAG AG Open table in a new tab Cells—Monkey kidney COS epithelial cells, mouse NIH/3T3 fibroblasts, and mouse skeletal muscle precursor C2C12 cells were obtained from the ATCC. The rat epicardial cell line EMC (7Eid H. Larson D.M. Springhorn J.P. Attawia M.A. Nayak R.C. Smith T.W. Kelly R.A. Circ. Res. 1992; 71: 40-50Crossref PubMed Scopus (94) Google Scholar, 8Eid H. de Bold M.L. Chen J.H. de Bold A.J. J. Cardiovasc. Pharmacol. 1994; 24: 715-720Crossref PubMed Scopus (46) Google Scholar) was kindly provided by Dr. Hoda Eid at the University of Ottawa, Ottawa, Ontario, Canada. Antibodies—Rabbit polyclonal antibodies against the carboxyl terminus of Bves (B846) have been previously described (2Wada A.M. Reese D.E. Bader D.M. Development. 2001; 128: 2085-2093PubMed Google Scholar). Monoclonal anti-FLAG (M2; 1:1000), polyclonal anti-HA (1:250), and monoclonal anti-β-catenin (1:200) were purchased from Sigma (St. Louis, MO). Rabbit anti-calnexin (SPA-860; 1:200) was purchased from StressGen (San Diego, CA). Goat anti-mouse and goat anti-rabbit conjugated to alkaline phosphatase were from Dako Corp. (Carpenteria, CA). Goat anti-mouse conjugated to Alexa488 was purchased from Molecular Probes, Inc. (Eugene, OR). The remaining antibodies used in this study were purchased from Jackson ImmunoResearch (West Grove, PA). Analysis of Bves from Chick Heart—Chick hearts from day 6 embryos were used to examine glycosylation of Bves. Cell extracts prepared essentially as described (6Backstrom J.R. Price R.D. Reasoner D.T. Sanders-Bush E. J. Biol. Chem. 2000; 275: 23620-23626Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar) were treated with buffer or recombinant N-glycosidase F (Glyko, Novato, CA) and incubated at 37 °C for 3 h. A 4× solution of SDS-sample buffer (250 mm Tris, pH 6.8, containing 8% SDS, 40% glycerol) was added to each tube, and protein concentrations were estimated with the BCA assay (Pierce Chemical Co., Rockford, IL) using bovine serum albumin as the protein standard. Blots of 30 μg of samples (containing 1% 2-mercaptoethanol) were probed with anti-Bves (B846) and immunoreactive bands detected with 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium (Pierce). Transfection of Cells—COS and C2C12 cells were transfected with cDNA prepared in LipofectAMINE (Invitrogen). The DNA solution was replaced with Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and incubated overnight at 37 °C in a humidified chamber containing 5% CO2. NIH/3T3 cells were electroporated with 25 μg of DNA (6Backstrom J.R. Price R.D. Reasoner D.T. Sanders-Bush E. J. Biol. Chem. 2000; 275: 23620-23626Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar) and grown as per the COS and C2C12 cells. For the experiments that involved preventing Asn-linked glycosylation in COS cells, the DNA solution was replaced with Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and 0.2 μg/ml tunicamycin (Calbiochem, La Jolla, CA). Cells were washed once with Hanks' buffered saline solution containing CaCl2 and MgCl2 (Invitrogen) before extracting protein. Extraction of Protein from Cells—Detergent-soluble protein was extracted from cells as described previously (6Backstrom J.R. Price R.D. Reasoner D.T. Sanders-Bush E. J. Biol. Chem. 2000; 275: 23620-23626Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Protein concentrations were estimated with the BCA assay (Pierce). Biotinylation of Cell Surface Protein—COS cells transfected with either Bves-FLAG or rat 5-HT2C receptor cDNA (negative control) were incubated with 0.5 mm NHS-LC-biotin (Pierce) in Hanks' buffered saline solution as per the manufacturer's protocol. Protein was immunoprecipitated with anti-FLAG and subjected to electrophoresis under reducing conditions. Separate lanes were probed with either anti-FLAG to detect total cellular protein or with 0.5 μg/ml streptavidin-phosphatase (Pierce) to detect cell surface protein. Co-immunoprecipitation—COS cells were co-transfected with 4 μg of HA-Bves and 4 μg of either Met128-FLAG or 2C/Met128-FLAG. Anti-FLAG (2 μg) was added to tubes containing 1% Triton-soluble protein and incubated with rocking at 4 °C. After 4 h, 20 μl of anti-mouse Protein G beads (1:2 slurry) was added. The beads were prepared from cross-linking goat anti-mouse IgG to Protein G at a final concentration of 3.2 mg of antibody/ml of beads using Seize-X (Pierce). After an overnight incubation with rocking at 4 °C, supernatants were discarded, and the beads were washed three times with TBS containing 0.1% Triton X-100. Protein was eluted from the beads with 50 μl of SDS-PAGE sample buffer containing 1% 2-mercaptoethanol and subjected to incubation in a 95 °C water bath for 1 min. Two blots were used to analyze 20-μl aliquots of each sample elution (40% of total) and a sample of starting extract (4% of input). Immunocytochemistry—Cells grown in 8-well chamber slides were either fixed and permeabilized with HistoChoice MB (Amresco, Solon, OH) or fixed with 1% paraformaldehyde in phosphate-buffered saline followed by permeabilization with 0.1% Triton X-100 in TBS. The wells were washed once with 2% bovine serum albumin in TBS and blocked with the same solution overnight at 4 °C. Cells were treated with primary antibodies followed by the appropriate secondary antibodies, each for 1 h at room temperature. After three washes with TBS and two washes with water, slides were dried and coverslipped with Aqua Poly-Mount (Polysciences, Inc., Warrington, PA). Fluorescent images were captured using an Olympus BX60 microscope in the Stahlman Cardiovascular Research Laboratories. Glycosylation of Bves from Chick Heart—Chick Bves/Pop1A contains two consensus Asn-linked glycosylation sites within the amino terminus (Asn20 and Asn27; Fig. 1). Because these are the only Asn-linked sites (Asn-X-Ser/Thr), evidence of glycosylation would establish that the amino terminus of Bves reached the lumen of the endoplasmic reticulum, which corresponds to the extracellular environment of cell surface molecules. Thus, extracts prepared from chick heart were incubated in the absence or presence of N-glycosidase F. Protein was subjected to SDS-PAGE, and blots were probed with anti-Bves antibodies (B846). In the absence of N-glycosidase, a diffuse pattern of bands were detected with masses of 56–58 kDa (Fig. 2, lane 1). In contrast, N-glycosidase decreased the size of immunoreactive bands to 43 kDa (lane 2), which is similar to the predicted mass of 41 kDa. An additional minor band was also observed at 50 kDa, which may represent a partially deglycosylated form of Bves. Glycosylation of the Amino Terminus of Bves from Transfected Cells—Glycosylation of Bves was next examined from transfected COS kidney epithelial cells (Fig. 3, top panel), NIH/3T3 fibroblasts (middle panel), and skeletal muscle precursor cells (bottom panel). Wild-type (WT) Bves displayed a predominant immunoreactive band with a mass of 47 kDa (lane 1). Point mutations were made at either one or both consensus Asn-linked sites. Whereas the N20A (lane 2) and N27A (lane 3) mutations produced major bands with masses of 45 kDa, mutation of both sites (N20A/N27A, lane 4) yielded a major band at 43 kDa. These results from transfected cells and chick heart establish that the amino terminus of Bves is glycosylated. Glycosylation at Exogenous Sites within the Carboxyl Terminus of Bves—The glycosylation studies were extended to examine the possibility that the carboxyl terminus of Bves also reaches the lumen of the endoplasmic reticulum. To ensure that this assay would indicate glycosylation at an exogenous site, the endogenous sites within the amino terminus were mutated to alanine (N20A/N27A; Fig. 1). Exogenous glycosylation sites (Asn-Glu-Thr) or (Asn-Ala-Thr) were introduced at five different positions within the carboxyl terminus (Fig. 4A). An additional construct (N20A/N27A/rCNT1) was created that contains a 14-amino acid insert based on a region of the carboxyl terminus of rCNT1 that contains a functional Asn-linked glycosylation site (5Hamilton S.R. Yao S.Y. Ingram J.C. Hadden D.A. Ritzel M.W. Gallagher M.P. Henderson P.J. Cass C.E. Young J.D. Baldwin S.A. J. Biol. Chem. 2001; 276: 27981-27988Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). An increase in size was not observed with any of the constructs containing an exogenous site (Fig. 4B). Thus, either the structure of Bves prevented glycosylation or the carboxyl terminus is exposed to the cytoplasm. We explored the possibility that removing the third membrane domain of Bves would reverse the orientation of the carboxyl terminus and direct it into the endoplasmic reticulum. Our efforts focused on constructs that contain the rCNT1 insert for these sets of experiments. A D3T/rCNT1 construct was created that lacks the predicted third membrane domain (Figs. 1 and 5A) and transfected into COS cells. Deletion of the third transmembrane domain yielded bands on immunoblots with masses of 41 and 43 kDa (Fig. 5B , lane 3). The 2-kDa difference in mass was consistent with the results obtained for each site in the amino terminus of wild-type Bves (lane 1). Although the D3T/rCNT1 construct lacks endogenous glycosylation sites within the amino terminus, two strategies were employed to confirm that formation of the minor, 43-kDa band of D3T/rCNT1 was due to glycosylation of the 41-kDa protein. First, cells were grown in the presence of the Asn-linked glycosylation inhibitor tunicamycin to prevent glycosylation. Tunicamycin prevented the appearance of the larger immunoreactive bands in D3T/rCNT1 (lane 4) as well as in wild-type Bves (lane 2). Second, the Asn residue within the rCNT1 insert was mutated to Ala (D3T/rCNT1-N/A). Whereas two bands were detected from D3T/rCNT1 (Fig. 5C , lane 2), only a single immunoreactive band was observed from D3T/rCNT1-N/A (lane 3), confirming that the Asn residue within the rCNT1 insert provided a functional glycosylation site. These results provide evidence that the carboxyl terminus of wild-type Bves is located in the cytoplasm, and removal of the third membrane domain reverses its orientation into the endoplasmic reticulum lumen. Surface Biotinylation of Bves—Biotinylation experiments were performed to determine whether Bves reaches the cell surface of COS cells. Cells expressing either Bves-FLAG or a negative control construct were incubated with NHS-LC-biotin to label cell surface protein. Cell extracts were incubated with anti-FLAG antibodies and anti-mouse beads. Precipitated proteins were electrophoresed, and the resulting blots were probed with either anti-FLAG to detect total cellular Bves (Fig. 6, lane 1) or streptavidin-phosphatase to detect cell surface Bves (lane 2). These results document that Bves is localized primarily within intracellular compartments of COS cells, whereas only low levels are present at the cell surface. Therefore, approaches to determine the membrane topology of Bves in COS cells examine properties of Bves within intracellular compartments. Oligomerization of Bves—During the course of characterizing immunoreactive Bves on immunoblots, we found that the banding pattern of Bves derived from cell extracts electrophoresed under reducing versus non-reducing conditions was different. Under reducing conditions (1% 2-mercaptoethanol), a predominant, monomeric form of Bves-FLAG was observed (Fig. 7, lane 4). Under non-reducing conditions, several higher molecular mass forms of Bves-FLAG were observed in addition to the monomeric form (lane 1). The amount of monomer from both electrophoretic conditions was compared with indirectly estimate the relative level of oligomers. The monomer from 4 μg of protein run under non-reducing conditions displayed equivalent immunoreactivity to that from 1 μg of protein run under reducing conditions (data not illustrated). These results imply that ∼80% of the Bves protein from COS cells exists in an oligomeric form. Similar results were obtained from C2C12 skeletal muscle precursor cells transfected with Bves-FLAG (data not illustrated). Characterization of Bves Carboxyl-terminal Constructs— Next, we examined whether oligomerization of Bves occurs within the carboxyl terminus. If so, we could exploit this interaction to determine the orientation of Bves. Similar to the results with full-length Bves-FLAG (Fig. 7, lane 1), a carboxyl-terminal Bves construct that lacks the membrane domains and starts at Met128 (Met128-FLAG; Fig. 1) also formed oligomers under non-reducing conditions (lane 2) and monomers under reducing conditions (lane 5). We postulated that wild-type Bves would interact with a carboxyl-terminal Bves construct if the construct was delivered to the subcellular compartment where the carboxyl-terminal tail of wild-type Bves resides. For example, if the carboxyl terminus of Bves is located in the cytoplasm, it could potentially interact with Met128-FLAG, but not if Met128-FLAG was delivered to the lumen of the endoplasmic reticulum. Thus, a chimeric protein was created that contains the amino terminus of the serotonin 5-HT2C receptor fused to Met128-FLAG (2C/Met128-FLAG; Fig. 1). The 2C region contains an endogenous Asn-linked glycosylation site; therefore, glycosylation of 2C/Met128-FLAG would confirm delivery into the endoplasmic reticulum lumen. Importantly, 2C/Met128-FLAG formed oligomers under non-reducing conditions (Fig. 7, lane 3) and monomers under reducing conditions (lane 6). Immunocytochemistry was utilized to determine whether the gross cellular distribution of the carboxyl-terminal constructs Met128-FLAG and 2C/Met128-FLAG are clearly different. COS cells were transiently transfected with the Bves constructs and labeled with mouse anti-FLAG followed by Cy3-labeled anti-mouse (Fig. 8). Full-length Bves-FLAG (A) displayed a similar fibrous labeling pattern as the chimeric carboxyl-terminal construct 2C/Met128-FLAG (B). Furthermore, the results from double-labeling experiments revealed that the distribution of Bves-FLAG and 2C/Met128-FLAG were strikingly similar to the endoplasmic reticulum protein calnexin (data not illustrated). In contrast, the carboxyl-terminal construct Met128-FLAG (C) displayed a diffuse labeling pattern consistent with a cytoplasmic location. A glycosylation assay was used to confirm that 2C/Met128-FLAG reaches the lumen of the endoplasmic reticulum. COS cells transfected with either Bves-FLAG or 2C/Met128-FLAG were incubated in the absence or presence of tunicamycin. In the absence of tunicamycin, the fully glycosylated form of Bves-FLAG had the expected mass of 47 kDa (Fig. 9, lane 2), whereas that of 2C/Met128-FLAG was 41 kDa (lane 4). In the presence of tunicamycin, Bves-FLAG and 2C/Met128-FLAG had smaller masses of 43 kDa (lane 1) and 39 kDa (lane 3), respectively. In summary, these results in COS cells establish that Met128-FLAG localizes to the cytoplasm, whereas 2C/Met128-FLAG reaches the lumen of the endoplasmic reticulum and both carboxyl-terminal constructs form oligomers. Co-immunoprecipitation of Bves—Because Met128-FLAG is localized in the cytoplasm, whereas 2C/Met128-FLAG reaches the endoplasmic reticulum lumen (illustrated in Fig. 10A), selective co-immunoprecipitation of HA-Bves would delineate the cellular location of its carboxyl terminus. Initial characterization studies of the antibodies directed against the HA and FLAG epitopes were performed to determine which antibody should be used for immunoprecipitation and which antibody should be used for detection on blots. HA-Bves-FLAG, containing both epitope tags (Fig. 1), was expressed in COS cells and the cell extracts used to test the antibodies. The mouse anti-FLAG antibody immunoprecipitated ∼6-fold more immunoreactive HA-Bves-FLAG protein relative to the rabbit anti-HA antibody (data not illustrated). In contrast, the anti-HA antibody was only ∼2-fold less reactive on blots against HA-Bves-FLAG relative to anti-FLAG (data not illustrated). Additionally, anti-HA detected HA-Bves but not Bves-FLAG on blots (data not illustrated). Therefore, anti-FLAG was used to immunoprecipitate the carboxyl-terminal FLAG-tagged constructs from cell extracts and anti-HA was used to detect potential associated HA-Bves. HA-Bves was co-expressed with either Met128-FLAG or 2C/Met128-FLAG in COS cells. Mouse anti-FLAG was added to each cell extract and immunoprecipitated with goat anti-mouse cross-linked to Protein G beads. After washing the beads, protein was eluted with sample buffer containing 1% 2-mercaptoethanol. Separate blots were probed with anti-FLAG and anti-HA (Fig. 10B). The cell extracts contained considerably less Met128-FLAG relative to 2C/Met128-FLAG (top panel, lanes 1 and 2), whereas HA-Bves was expressed at similar levels from both extracts (bottom panel, lanes 1 and 2). In agreement with their expression levels, the anti-FLAG immunoprecipitates yielded lower levels of Met128-FLAG (top panel, lane 3) relative to 2C/Met128-FLAG (lane 4). However, co-immunoprecipitated HA-Bves was detected in association with Met128-FLAG (bottom panel, lane 3) but was not detected in association with 2C/Met128-FLAG (lane 4). Similar results were obtained when anti-HA was used with goat anti-rabbit beads to immunoprecipitate HA-Bves and anti-FLAG detected an association with only Met128-FLAG (data not illustrated). However, the level of co-immunoprecipitated Met128-FLAG was barely detected above background, which was consistent with the initial antibody characterization studies. Immunocytochemistry of Endogenous Bves in Rat Epicardial Cells—The results thus far, based on properties of Bves within intracellular compartments, predict that the carboxyl terminus of cell surface Bves would be exposed to the cytoplasm rather than to the extracellular environment. To directly address this issue, we utilized an epithelial cell line derived from rat heart epicardium (epicardial cells) that expresses endogenous Bves. It was previously demonstrated that confluent cultures of epicardial cells express the majority of Bves immunoreactivity at the cell surface (2Wada A.M. Reese D.E. Bader D.M. Development. 2001; 128: 2085-2093PubMed Google Scholar). Thus, we could determine the effect of non-ionic detergent on the availability of a carboxyl-terminal epitope. The optimal concentration of fixative was found to be 1% paraformaldehyde using antibodies against β-catenin as a control to label an intracellular protein localized at the cell surface (data not illustrated). Thus, epicardial cells were fixed with 1% paraformaldehyde and then treated in the absence or presence of 0.1% Triton X-100. Bves was labeled with rabbit anti-Bves (B846), directed against amino acids 263–278 of the carboxyl terminus, and mouse anti-β-catenin. Immunoreactive protein was visualized with Cy3-labeled anti-rabbit and Alexa488-labeled anti-mouse antibodies. In the first set of experiments, antibodies were used to label separate wells of epicardial cells (Fig. 11). Faint Bves and β-catenin labeling was detected in untreated cells (panels A and B, respectively), whereas robust labeling of both antibodies was detected in detergent-treated cells (panels C and D, respectively). In the second set of experiments, cells were double-labeled with both antibodies (data not illustrated). Enhanced immunoreactivity of anti-Bves and anti-β-catenin was again observed in detergent-treated cells relative to untreated cells. These results support the prediction that the carboxyl terminus of cell surface Bves is exposed to the cytoplasm. Bves/POP1A is a member of the recently discovered Popeye (POP) family of transcripts that are expressed in several tissue types (1Reese D.E. Zavaljevski M. Streiff N.L. Bader D. Dev. Biol. 1999; 209: 159-171Crossref PubMed Scopus (89) Google Scholar, 3Andrée B. Hillemann T. Kessler-Icekson G. Schmitt-John T. Jockusch H. Arnold H.H. Brand T. Dev. Biol. 2000; 223: 371-382Crossref PubMed Scopus (98) Google Scholar). Whereas POP1, POP2, and POP3 transcripts have been discovered from mouse, only POP1 and POP3 have been isolated thus far from chicken (3Andrée B. Hillemann T. Kessler-Icekson G. Schmitt-John T. Jockusch H. Arnold H.H. Brand T. Dev. Biol. 2000; 223: 371-382Crossref PubMed Scopus (98) Google Scholar). In addition, splice variants of chick POP1 have been identified, including POP1A (identical to Bves), 1B, 1C, and 1D (3Andrée B. Hillemann T. Kessler-Icekson G. Schmitt-John T. Jockusch H. Arnold H.H. Brand T. Dev. Biol. 2000; 223: 371-382Crossref PubMed Scopus (98) Google Scholar). Interestingly, POP1C is predicted to encode a soluble protein homologous to a region of the carboxyl terminus, whereas the other isoforms contain hydrophobic membrane domains. An overt phenotypic difference was not observed in POP1 knockout mice relative to wild-type littermates, but regeneration of cardiotoxin-treated skeletal muscle was delayed in mice that lack POP1 (4Andrée B. Fleige A. Arnold H.H. Brand T. Mol. Cell. Biol. 2002; 22: 1504-1512Crossref PubMed Scopus (60) Google Scholar). Thus, it is possible that other Pop proteins compensate for a loss of Pop1 function in tissues that co-express more than one Pop member. It is important to resolve whether other Pop members compensate for the loss of Pop1 function during development as well as determine how Bves/Pop1A functions at the cellular level. In addition to muscle tissue, Bves/Pop1A is also found in cells that develop coronary vessels. Coronary blood vessels develop from transitory epithelium of the proepicardial organ. This epithelium provides the epicardial covering of the heart and precursors that form various differentiated cell types of coronary vessels. A subpopulation of epicardial cells delaminate from the epithelial cell layer, migrate into the subepicardial connective tissue and myocardium (9Manasek F.J. J. Morphol. 1968; 125: 329-366Crossref PubMed Scopus (278) Google Scholar, 10Viragh S. Challice C.E. Anat. Rec. 1981; 201: 157-168Crossref PubMed Scopus (262) Google Scholar, 11Hiruma T. Hirakow R. Am. J. Anat. 1989; 184: 129-138Crossref PubMed Scopus (131) Google Scholar, 12Manner J. Anat. Embryol. 1993; 187: 281-289Crossref PubMed Scopus (113) Google Scholar), and differentiate into vascular endothelium, vascular smooth muscle, or fibroblasts of coronary vessels (13Mikawa T. Gourdie R.G. Dev. Biol. 1996; 174: 221-232Crossref PubMed Scopus (528) Google Scholar, 14Dettman R.W. Denetclaw W. Ordahl C.P. Bristow J. Dev. Biol. 1998; 193: 169-181Crossref PubMed Scopus (461) Google Scholar). Bves is found in the epicardium, migrating mesenchyme, and vascular smooth muscle (1Reese D.E. Zavaljevski M. Streiff N.L. Bader D. Dev. Biol. 1999; 209: 159-171Crossref PubMed Scopus (89) Google Scholar). Furthermore, Bves at the cell surface of epicardial cells later accumulates in an intracellular compartment of migrating mesenchymal cells and then reappears at the cell surface of smooth muscle cells (2Wada A.M. Reese D.E. Bader D.M. Development. 2001; 128: 2085-2093PubMed Google Scholar). Thus, Bves may be functional at only one cellular location or have a unique function at each location. Interestingly, Bves enhances the adhesive property of non-adherent l-cells, which suggests that Bves has a function at the cell surface (2Wada A.M. Reese D.E. Bader D.M. Development. 2001; 128: 2085-2093PubMed Google Scholar). Clearly, it is critical to elucidate the membrane topology of Bves to identify regions that likely contain functional domains. The findings that Bves promotes cell adhesion and its distribution is dynamically regulated during development suggest that several functional domains are involved. Thus, the goal of this study was to elucidate the membrane topology of Bves to discriminate extracellular regions that may directly participate in cell-cell interactions from intracellular regions that may be involved in trafficking. This functional distinction does not exclude the possibility that intracellular domains participate in regulating adhesive properties, which has been documented for the prototypic cell adhesion molecule E-cadherin (reviewed in Ref. 15Gumbiner B.M. J. Cell Biol. 2000; 148: 399-403Crossref PubMed Scopus (688) Google Scholar). Nonetheless, defining the membrane topology of Bves/Pop1A would provide a basis for subsequent functional studies. Two different models have been proposed for the membrane topology of Bves/Pop1A. Andrée et al. (4Andrée B. Fleige A. Arnold H.H. Brand T. Mol. Cell. Biol. 2002; 22: 1504-1512Crossref PubMed Scopus (60) Google Scholar) proposed that the amino terminus is extracellular. The data from in vitro translation of Bves/Pop1A in the presence of microsomes (2Wada A.M. Reese D.E. Bader D.M. Development. 2001; 128: 2085-2093PubMed Google Scholar, 3Andrée B. Hillemann T. Kessler-Icekson G. Schmitt-John T. Jockusch H. Arnold H.H. Brand T. Dev. Biol. 2000; 223: 371-382Crossref PubMed Scopus (98) Google Scholar) clearly demonstrated an increase in mass that could be due to Asn-linked glycosylation. Because the amino terminus of Bves is the only region that contains consensus Asn-linked sites, glycosylation would establish that the amino terminus of cell surface Bves is exposed to the extracellular environment. Here, we thoroughly examined glycosylation of Bves from chick heart and transfected cells. Whereas Andrée et al. (4Andrée B. Fleige A. Arnold H.H. Brand T. Mol. Cell. Biol. 2002; 22: 1504-1512Crossref PubMed Scopus (60) Google Scholar) proposed that the carboxyl terminus of Bves/Pop1A is intracellular, Wada et al. (2Wada A.M. Reese D.E. Bader D.M. Development. 2001; 128: 2085-2093PubMed Google Scholar) proposed that it is extracellular, based on the results from antibody blocking experiments. Antibodies against the carboxyl terminus of Bves (B846) blocked cell migration in primary cultures of chick proepicardia, whereas antibodies against another carboxyl-terminal epitope (D033) were without effect (2Wada A.M. Reese D.E. Bader D.M. Development. 2001; 128: 2085-2093PubMed Google Scholar). Here, we utilized three different approaches to determine the orientation of the carboxyl terminus of Bves. Glycosylation of Bves was examined to determine whether the amino terminus reaches the lumen of the endoplasmic reticulum and is glycosylated. Bves from chick heart was glycosylated, and mutating the two Asn-linked glycosylation sites prevented glycosylation of Bves. These results establish that the amino terminus of Bves/Pop1A, the only region that contains Asn-linked sites, reaches the lumen of the endoplasmic reticulum, implying that the amino terminus of cell surface molecules would be exposed to the extracellular environment. Three biochemically distinct approaches were utilized to determine the orientation of the carboxyl terminus of Bves/Pop1A. The first two approaches examined Bves within intracellular compartments of transfected cells, whereas the third approach examined cell surface Bves in a native cell background. First, glycosylation assays were utilized to examine Asn-linked glycosylation at exogenous sites within the carboxyl terminus of Bves. Second, oligomerization of Bves within the carboxyl terminus was exploited to investigate interactions between wild-type Bves and a soluble, carboxyl-terminal construct that either resides in the cytoplasm or is delivered to the endoplasmic reticulum lumen. Third, immunocytochemistry was performed with antibodies against the carboxyl terminus of Bves to examine endogenous protein at the cell surface of epicardial cells. Although each approach has inherent caveats, together, the results provide compelling evidence that the carboxyl terminus of Bves is exposed to the cytoplasm. The results from this study support a topological model in which the amino terminus of cell surface Bves/Pop1A is exposed to the extracellular environment, whereas the carboxyl terminus is exposed to the cytoplasm, a membrane topology consistent with three membrane domains. The data presented here agree with the model proposed by Andrée et al. (4Andrée B. Fleige A. Arnold H.H. Brand T. Mol. Cell. Biol. 2002; 22: 1504-1512Crossref PubMed Scopus (60) Google Scholar) that was based on data that did not establish the orientation of the carboxyl terminus. In contrast, Wada et al. (2Wada A.M. Reese D.E. Bader D.M. Development. 2001; 128: 2085-2093PubMed Google Scholar) proposed that the carboxyl terminus of Bves was extracellular based on their results from antibody blocking experiments. The same polyclonal anti-Bves carboxyl-terminal antibodies (B846) that blocked cell migration of chick proepicardial explants (2Wada A.M. Reese D.E. Bader D.M. Development. 2001; 128: 2085-2093PubMed Google Scholar) were utilized here in the immunocytochemistry experiments with rat epicardial cells. A potential explanation for these apparently conflicting results is that the Bves antibodies neutralized a secreted Pop member, possibly Pop1C. We are currently investigating several avenues to reconcile this important issue. Significantly, the current results from three biochemically distinct approaches indicate that the carboxyl terminus of Bves/Pop1A is exposed to the cytoplasm. The membrane topology of Bves/Pop1A provides regional discrimination of functional domains. First, we predict that direct cell-cell interactions occur between the amino termini of opposing Bves molecules. Second, we predict that the dynamic redistribution of Bves during coronary vessel development is regulated within the carboxyl terminus. Our goal is to identify the functional domain(s) within each of these regions. Establishing the membrane topology of Bves/Pop1A has provided a foundation to dissect the cellular functions of Bves/Pop1A. We thank Kristin Price, Brian Robertson, Nada Jaffal, and Tianli Zhu for expert technical assistance and Dr. Maureen Gannon for critically reading the manuscript.
Phosphorylation-deficient serotonin 5-HT2C receptors were generated to determine whether phosphorylation promotes desensitization of receptor responses. Phosphorylation of mutant 5-HT2C receptors that lack the carboxyl-terminal PDZ recognition motif (Ser458-Ser-Val-COOH; ΔPDZ) was not detectable based on a band-shift phosphorylation assay and incorporation of 32P. Treatment of cells stably expressing ΔPDZ or wild-type 5-HT2C receptors with serotonin produced identical maximal responses and EC50 values for eliciting [3H]inositol phosphate formation. In calcium imaging studies, treatment of cells expressing ΔPDZ or wild-type 5-HT2C receptors with 100 nm serotonin elicited initial maximal responses and decay rates that were indistinguishable. However, a second application of serotonin 2.5 min after washout caused maximal responses that were ∼5-fold lower with ΔPDZ receptors relative to wild-type 5-HT2C receptors. After 10 min, responses of ΔPDZ receptors recovered to wild-type 5-HT2Creceptor levels. Receptors with single mutations at Ser458(S458A) or Ser459 (S459A) decreased serotonin-mediated phosphorylation to 50% of wild-type receptor levels. Furthermore, subsequent calcium responses of S459A receptors were diminished relative to S458A and wild-type receptors. These results establish that desensitization occurs in the absence of 5-HT2C receptor phosphorylation and suggest that receptor phosphorylation at Ser459 enhances resensitization of 5-HT2Creceptor responses. Phosphorylation-deficient serotonin 5-HT2C receptors were generated to determine whether phosphorylation promotes desensitization of receptor responses. Phosphorylation of mutant 5-HT2C receptors that lack the carboxyl-terminal PDZ recognition motif (Ser458-Ser-Val-COOH; ΔPDZ) was not detectable based on a band-shift phosphorylation assay and incorporation of 32P. Treatment of cells stably expressing ΔPDZ or wild-type 5-HT2C receptors with serotonin produced identical maximal responses and EC50 values for eliciting [3H]inositol phosphate formation. In calcium imaging studies, treatment of cells expressing ΔPDZ or wild-type 5-HT2C receptors with 100 nm serotonin elicited initial maximal responses and decay rates that were indistinguishable. However, a second application of serotonin 2.5 min after washout caused maximal responses that were ∼5-fold lower with ΔPDZ receptors relative to wild-type 5-HT2C receptors. After 10 min, responses of ΔPDZ receptors recovered to wild-type 5-HT2Creceptor levels. Receptors with single mutations at Ser458(S458A) or Ser459 (S459A) decreased serotonin-mediated phosphorylation to 50% of wild-type receptor levels. Furthermore, subsequent calcium responses of S459A receptors were diminished relative to S458A and wild-type receptors. These results establish that desensitization occurs in the absence of 5-HT2C receptor phosphorylation and suggest that receptor phosphorylation at Ser459 enhances resensitization of 5-HT2Creceptor responses. Dulbecco's modified Eagle's medium Tris-buffered saline bovine serum albumin 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid Serotonin 5-HT2C (formerly 5-HT1C) receptors exists as several isoforms throughout the brain, due to RNA editing and alternative splicing, and function to stimulate phospholipase C through activation of the G protein Gq (1Chang M. Zhang L. Tam J.P. Sanders-Bush E. J. Biol. Chem. 2000; 275: 7021-7029Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Whereas RNA editing generates isoforms in the second intracellular loop that modify receptor signaling (2Burns C.M. Chu H. Rueter S.M. Hutchinson L.K. Canton H. Sanders-Bush E. Emeson R.B. Nature. 1997; 387: 303-308Crossref PubMed Scopus (860) Google Scholar, 3Herrick-Davis K. 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Phosphorylation of G protein-coupled receptors regulates signaling through multiple mechanisms including receptor desensitization, which attenuates second messenger responses. In the case of phospholipase C-linked receptors, phosphorylation has been demonstrated to promote desensitization based on observations that receptors lacking the corresponding phosphorylation sites display sustained phosphoinositide hydrolysis responses relative to wild-type receptors (8Alblas J. van Etten I. Khanum A. Moolenaar W.H. J. Biol. Chem. 1995; 270: 8944-8951Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 9Diviani D. Lattion A.L. Cotecchia S. J. Biol. Chem. 1997; 272: 28712-28719Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 10Lattion A.L. Diviani D. Cotecchia S. J. Biol. Chem. 1994; 269: 22887-22893Abstract Full Text PDF PubMed Google Scholar, 11Rao R.V. Roettger B.F. Hadac E.M. Miller L.J. Mol. 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Chem. 1997; 272: 15213-15219Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar) by phosphorylation-deficient mutants relative to wild-type receptors, illustrating that mutation of phosphorylation sites involved in desensitization enhances both initial and secondary responses. Although desensitization of 5-HT2C receptor-mediated responses has been observed in assays that examine phosphoinositide hydrolysis (7Westphal R.S. Backstrom J.R. Sanders-Bush E. Mol. Pharmacol. 1995; 48: 200-205PubMed Google Scholar, 17Briddon S.J. Leslie R.A. Elliott J.M. Br. J. Pharmacol. 1998; 125: 727-734Crossref PubMed Scopus (26) Google Scholar), release of intracellular calcium (18Akiyoshi J. Nishizono A. Yamada K. Nagayama H. Mifune K. Fujii I. J. Neurochem. 1995; 64: 2473-2479Crossref PubMed Scopus (34) Google Scholar, 19Watson J.A. Elliott A.C. Brown P.D. Cell Calcium. 1995; 17: 120-128Crossref PubMed Scopus (22) Google Scholar), and Ca2+-activated currents (20Boddeke H.W. Hoffman B.J. Palacios J.M. Hoyer D. Naunyn-Schmiedebergs Arch. Pharmacol. 1993; 348: 221-224Crossref PubMed Scopus (17) Google Scholar), it is unknown whether phosphorylation of the 5-HT2C receptor is involved in desensitization. To address this issue, we identified a 5-HT2C receptor domain that is required for receptor phosphorylation and performed functional assays with phosphorylation-deficient receptor mutants. First, we determined that phosphorylation of the 5-HT2C receptor requires the carboxyl-terminal PDZ (PSD-95 discs-largeZO-1) recognition motif, a domain present in nonedited and edited isoforms of the 5-HT2C receptor. Next, we found that phosphorylation-deficient receptors display identical initial responses as the wild-type 5-HT2C receptor in phosphoinositide hydrolysis and calcium release assays. However, phosphorylation-deficient receptors exhibit diminished secondary responses and a delayed recovery relative to wild-type 5-HT2C receptors. Cells stably expressing 5-HT2C receptors with a single serine-to-alanine mutation also display decreased receptor phosphorylation and diminished secondary calcium responses. We therefore propose that 5-HT2C receptor phosphorylation promotes resensitization of 5-HT2C receptor-mediated responses. The production, purification, and characterization of anti-peptide antibodies against rat 5-HT2C receptors have been described (21Backstrom J.R. Westphal R.S. Canton H. Sanders-Bush E. Mol. Brain Res. 1995; 33: 311-318Crossref PubMed Scopus (34) Google Scholar, 22Backstrom J.R. Sanders-Bush E. J. Neurosci. Methods. 1997; 77: 109-117Crossref PubMed Scopus (22) Google Scholar). Antibodies against a region of the third intracellular loop (referred to below as anti-2C-IC antibodies) were generated against amino acids 270–288 (NH2-CKKNGGEEENAPNPNPDQK-COOH) of the rat sequence and purified against a shorter peptide (NH2-CKKNGGEEENAPN-COOH). Antibodies against a region of the carboxyl terminus (referred to below as anti-2C-CT antibodies) were generated against amino acids 419–435 of the rat sequence (NH2-RHTNERVARKANDPEPGC-COOH, with cysteine added to the carboxyl terminus of peptide) and purified against the same peptide. Mutant 5-HT2C receptors were generated from wild-type rat receptor cDNA (INI isoform) (2Burns C.M. Chu H. Rueter S.M. Hutchinson L.K. Canton H. Sanders-Bush E. Emeson R.B. Nature. 1997; 387: 303-308Crossref PubMed Scopus (860) Google Scholar) using recombinant Pfupolymerase (Promega, Madison, WI), a forward primer (primer 16.3, 5′-TTGGCATTGTATTCTTTGTGTTTCTGA-3′), and a reverse primer containing anXbaI site. For mutants lacking the PDZ recognition motif (ΔPDZ), polymerase chain reaction was performed with primer 16.3 and the reverse primer 5′-TGTCTAGATTTAAATCCTCTCGCTGACCACATTAG-3′. For the S458A and S459A mutants, two rounds of polymerase chain reaction were performed with primer 16.3 and overlapping reverse primers. The first round was performed with primer 16.3 and 5′-TTACACACTGGCAATCCTCTCGCTGACCACATTAGA-3′ (S458A) or 5′-TTACACGGCACTAATCCTCTCGCTGACCACATT-3′ (S459A), and the second round with primer 16.3 and 5′-TGTCTAGATATTACACACTGGCAATCCTCTCGCT-3′ (S458AXba) or 5′-TGTCTAGAATTTACACGGCACTAATCCTCTCGCT-3′ (S459AXba). For mutants lacking the carboxyl terminus except for the immunodominant region of the 2C-CT epitope and the conserved cysteine (Δ375/CT), two rounds of polymerase chain reaction were performed as described in detail elsewhere. 1J. R. Backstrom, manuscript in preparation. Amplified DNA was digested with StuI and XbaI and ligated into the StuI/XbaI sites of the wild-type 5-HT2C receptor cDNA in a Bluescript plasmid (Stratagene, La Jolla, CA). Sequences were confirmed by automated sequencing at the Core Facility of the Center for Molecular Neuroscience at Vanderbilt University. Receptor cDNA was isolated from the Bluescript plasmid with KpnI and XbaI and ligated into the mammalian expression plasmid pCMV2 (a gift of Dr. David Russell). The carboxyl-terminal sequences of wild-type, ΔPDZ, Δ375/CT, S458A, and S459A 5-HT2Creceptors are illustrated in Fig. 1. NIH 3T3 fibroblasts were transiently transfected with wild-type or mutant 5-HT2C receptor cDNA to determine whether the cellular distribution of mutant receptors differed from that of wild-type 5-HT2C receptors. Fibroblasts electroporated with 25 μg of receptor cDNA were seeded in eight-well Lab-Tek II chamber slides (Nalge Nunc International, Naperville, IL) containing Dulbecco's modified Eagle's medium (DMEM2; Life Technologies, Inc.) with 9% bovine serum (Hyclone Laboratories, Logan, UT), 5 units/ml penicillin, and 5 μg/ml streptomycin (Life Technologies, Inc.). Cells were incubated at 37 °C in a humid chamber containing 5% CO2 for 2 days. The medium was discarded and replaced with phosphate-buffered saline containing freshly prepared 4% (w/v) paraformaldehyde and 4% (w/v) sucrose. After 15 min, fixative was discarded and replaced with phosphate-buffered saline containing 0.1% (v/v) Triton X-100 (Sigma). After an additional 15 min, the medium was discarded, and the wells were washed once with Tris-buffered saline (TBS; 50 mmTris, pH 7.6, and 150 mm NaCl) containing 2% bovine serum albumin (BSA; Sigma) before adding a second aliquot of TBS/BSA. Slides were incubated at 4 °C for 1–2 days to block nonspecific sites. Primary antibodies against the 5-HT2C receptor (anti-2C-IC or anti-2C-CT) at a final concentration of 0.5 μg/ml in TBS containing 1% BSA were added to wells and incubated for 2 h at room temperature. After three washes with TBS, Cy3-labeled donkey anti-rabbit antibody (Jackson ImmunoResearch, West Grove, PA) at a dilution of 1:1000 in TBS containing 1% BSA was added to wells and incubated for 1 h at room temperature. After three washes with TBS and two washes with water, slides were dried and coverslipped with Aqua PolyMount (Polysciences, Inc., Warrington, PA). Confocal images were captured with a Zeiss LSM410 confocal microscope in the Cell Imaging Core at Vanderbilt University. Stable clones were derived from transfection of NIH 3T3 fibroblasts with rat wild-type (INI isoform) (2Burns C.M. Chu H. Rueter S.M. Hutchinson L.K. Canton H. Sanders-Bush E. Emeson R.B. Nature. 1997; 387: 303-308Crossref PubMed Scopus (860) Google Scholar) or mutant 5-HT2C receptor cDNA. Mutant receptor cDNA (25 μg) was co-electroporated with empty pcDNA3 plasmid (0.3 μg; Invitrogen, Carlsbad, CA), and cells were selected with G418 (Geneticin, Life Technologies, Inc.). Stable cell lines were grown in DMEM containing 9% bovine serum, 0.5 mg/ml G418, 5 units/ml penicillin, and 5 μg/ml streptomycin. Positive clones were identified by immunocytochemistry. Mutant clones with receptor levels similar to the wild-type 5-HT2C receptor cell line were utilized in subsequent functional assays. Mutant ΔPDZ receptors demonstrated identical affinities for the agonists serotonin (5-HT), 2,5-dimethoxy-4-iodoamphetamine (DOI), and lysergic acid diethylamide (LSD) as wild-type 5-HT2C receptors in competition binding experiments with [3H]mesulergine (data not illustrated). Cells grown in the presence of serum were washed four times with serum-free DMEM and replaced with 5 ml of DMEM. Tunicamycin (Roche Molecular Biochemicals) was added to a final concentration of 2 μg/ml of medium (7Westphal R.S. Backstrom J.R. Sanders-Bush E. Mol. Pharmacol. 1995; 48: 200-205PubMed Google Scholar). After 6 h, agonist was added, and incubation was continued for an additional 15 min at 37 °C. Cell supernatants were discarded and replaced with 1 ml of phosphate/EDTA (PE) extraction buffer (50 mmNaH2PO4/Na3PO4, pH 7.2, containing 5 mm EDTA, 1 mm EGTA, 1 mm phenylmethylsulfonyl fluoride, 5 μmleupeptin, 5 mmNa4P2O7, and 1 mmNa3VO4). The cells were scraped, and cell suspensions were placed in a 1.7-ml tube, sonicated, and then centrifuged at 14,000 × g for 10 min at 4 °C. The supernatants were discarded, and the pellets were treated with PE extraction buffer containing 10 mm CHAPS and sonicated. Detergent-soluble proteins were collected after centrifugation as described above. Protein concentrations were determined with the BCA protein assay (Pierce) using BSA as the protein standard. Protein samples (25 μg) were electrophoresed, blotted, and probed with anti-2C-IC or anti-2C-CT antibodies as described below. Detergent-soluble protein solutions were diluted with 4× sample buffer (250 mm Tris, pH 6.8, containing 8% SDS, 40% glycerol, 4% 2-mercaptoethanol, and bromphenol blue) and separated on 10% SDS-polyacrylamide gels. Proteins were transferred from gels to nitrocellulose membranes Amersham Pharmacia Biotech) in a modified Towbin transfer buffer (25 mm Tris and 192 mm glycine, pH ≈8.6). Membranes were probed with anti-2C-IC or anti-2C-CT antibodies, and the immunoreactive bands were detected using alkaline phosphatase-conjugated secondary antibodies (Dako Corp., Carpinteria, CA) as described (21Backstrom J.R. Westphal R.S. Canton H. Sanders-Bush E. Mol. Brain Res. 1995; 33: 311-318Crossref PubMed Scopus (34) Google Scholar). Band intensities were quantitated on a Macintosh computer using the public domain NIH Image program developed at the United States National Institutes of Health. Cells stably expressing wild-type or mutant ΔPDZ 5-HT2C receptors were labeled with [32P]orthophosphate as described (7Westphal R.S. Backstrom J.R. Sanders-Bush E. Mol. Pharmacol. 1995; 48: 200-205PubMed Google Scholar), except cells were incubated with 0.2 mCi of 32P/ml for 2 h. Cells were untreated or treated with 1 μm 5-hydroxytryptamine for 15 min. Membrane fractions of cells were prepared in PE extraction buffer, and detergent-soluble proteins were extracted with 0.3 ml of PE extraction buffer containing 150 mm NaCl, 1% Triton X-100, and 0.1% SDS. For immunoprecipitation of receptors, 1 μg of antibody was added to detergent-soluble protein and incubated overnight at 4 °C with rocking. Ten μl of goat anti-rabbit antibody beads (generated by conjugating Fc fragment-specific antibodies (Jackson ImmunoResearch Laboratories, Inc.) to protein G-agarose (Pierce) in 20 mm dimethyl pimelimidate to a final concentration of 4 mg of antibodies/ml of gel) was added, and incubation was continued for 4 h at 4 °C with rocking. The beads were pelleted and washed sequentially with PE extraction buffer containing the following: 0.5% Triton X-100 and 0.5 m NaCl; 0.25% Triton X-100 and 0.1m NaCl; 0.1% Triton X-100; and 0.1% SDS (one wash each). Fifteen μl of PE extraction buffer containing 1% SDS was added to the beads, and the mixture was incubated for 5 min at room temperature. Then, 15 μl of PE extraction buffer containing 5% Nonidet P-40 was added to the beads; and after mixing, 90 μl of PE extraction buffer and 2 μl (0.4 units) of recombinant N-glycosidase F (Roche Molecular Biochemicals) were added. After incubation for 2 h at 37 °C, 40 μl of 4× sample buffer was added to the suspension, and 70 μl of each fraction was electrophoresed on two 10% gels. One gel was transferred to nitrocellulose, and membranes were probed with anti-2C-IC antibodies; and the other gel was dried and exposed to a PhosphorImager cassette (Molecular Dynamics, Inc.). Cells stably expressing ΔPDZ (clone ΔPDZ-2) or wild-type 5-HT2C receptors were grown in serum-free medium in the presence of myo-[3H]inositol as described (23Barker E.L. Westphal R.S. Schmidt D. Sanders-Bush E. J. Biol. Chem. 1994; 269: 11687-11690Abstract Full Text PDF PubMed Google Scholar). Cells were stimulated with increasing concentrations of serotonin in the presence of 10 mm lithium chloride and 10 μm pargyline for 30 min. [3H]Inositol monophosphates were extracted, isolated by anion-exchange chromatography, and quantitated by liquid scintillation counting. Concentration-response curves and EC50 values were calculated with GraphPAD Prism software. Calcium imaging was performed with cells in 35-mm plastic culture dishes. Cells in DMEM containing 9% fetal bovine serum were grown to 50–70% confluence. The medium was replaced with serum-free DMEM and incubated overnight. Cells were loaded with the calcium indicator fura-2/AM (2 μg/ml; Molecular Probes, Inc., Eugene OR) for 1 h at room temperature in HEPES/calcium buffer. Loaded cells were continuously superfused with HEPES/calcium buffer and visualized using a Nikon inverted microscope attached to a Compix calcium imaging system. The imaging system consists of a CCD-72 camera (Dage-MTI, Inc., Michigan City, IN) attached to an IBM compatible computer executing SIMCA C-imaging software (Compix, Inc.). Intracellular calcium was visualized by fluorescence ratio measurements at wavelengths of 340 and 380 nm. The response to the initial challenge with 100 nm serotonin was monitored for 10 min. Cells were then washed with HEPES/calcium buffer for 2.5, 5, or 10 min before a second application of 100 nm serotonin. The half-lives for decay of the initial responses were calculated as the time between maximal and half-maximal responses. The second peak response of a cell was divided by the first peak response to determine the percent initial response for each cell. Responses were obtained from 20–80 cells/experiment. Results from three independent experiments (means ± S.D.) were plotted versus the time interval between agonist challenges. Fibroblasts stably expressing the wild-type 5-HT2C receptor were grown in serum-free medium containing tunicamycin to generate newly synthesized, unglycosylated 5-HT2C receptors. Cells were either untreated or treated with serotonin or with serotonin in the presence of the antagonist mianserin. Membrane proteins were solubilized with CHAPS detergent and electrophoresed on SDS-polyacrylamide gels. Immunoblots were probed with 5-HT2C receptor antibodies against a region of the third intracellular loop (anti-2C-IC) or carboxyl terminus (anti-2C-CT). Untreated cells contained an immunoreactive protein with a mass of 40 kDa (Fig. 2 A,first lane). Treatment of cells with serotonin caused formation of an additional 41-kDa immunoreactive protein (Fig.2 A, second lane). The serotonin-mediated shift in mass from 40 to 41 kDa was blocked by preincubation with the antagonist mianserin (Fig. 2 A, third lane). The amount of 41-kDa protein was dependent on serotonin concentration (Fig.2 B) and was maximal after treatment with 100 nmserotonin. Experiments were also performed on glycosylated receptors that were treated with N-glycosidase F to removeN-linked sugars before electrophoresis. Although this approach also demonstrated an agonist-mediated increase in receptor mass, treatment of cells with tunicamycin produced better resolution between the 40- and 41-kDa bands than did treatment of cell extracts with N-glycosidase F. The possibility that the 41-kDa immunoreactive protein was a phosphorylated form of 40-kDa receptors was examined. Alkaline phosphatase or buffer was added to CHAPS-soluble extracts prepared from cells pretreated with serotonin (Fig.2 C). Increasing concentrations of alkaline phosphatase progressively decreased the mass of the 41-kDa protein to 40 kDa. Additional experiments were performed to determine the time course of serotonin-mediated receptor phosphorylation and dephosphorylation. To examine phosphorylation, cells were treated with serotonin for increasing times before extracting CHAPS-soluble protein. Formation of the phosphorylated 41-kDa protein was complete by 10 min of agonist treatment, the earliest time point examined (Fig.3 A, second lane). Treatment with agonist for 1 h (Fig. 3 A, third lane) did not change the amount of 41-kDa protein. To examine dephosphorylation, cells were treated with serotonin for 15 min and washed four times. After the washes, cells were incubated for increasing times in the absence of serotonin before extraction of CHAPS-soluble protein. The amount of 41-kDa protein decreased with a half-life of 10 ± 1 min (n = 3), whereas the amount of 40-kDa protein increased (Fig. 3 B). These results are consistent with the reversible actions of protein phosphorylation and dephosphorylation and establish that the 41-kDa protein is a phosphorylated form of unglycosylated 5-HT2C receptors.Figure 3Time course of 5-HT2C receptor phosphorylation and dephosphorylation. A, the level of 5-HT2C receptor phosphorylation after treatment of cells with 100 nm serotonin for 10 min (second lane) was similar to that after 1 h (third lane). Data are representative of three independent experiments. B, cells treated with 100 nm serotonin for 15 min were washed four times with serum-free medium and incubated for the indicated times. After agonist washout, the half-life of 5-HT2C receptor phosphorylation was 10 ± 1 min (n = 3). The masses of immunoreactive proteins are indicated.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To determine which region of the 5-HT2C receptor was phosphorylated, tryptic digests from intact cells were evaluated in the band-shift phosphorylation assay using anti-peptide antibodies against the third intracellular loop (anti-2C-IC) and the carboxyl terminus (anti-2C-CT). The results were consistent with phosphorylation within the carboxyl terminus (data not shown). Thus, two mutant 5-HT2C receptor constructs were created that lack either two (ΔPDZ) or all (Δ375/CT) potential phosphorylation sites in the carboxyl terminus (Fig. 1). The Δ375/CT receptor was created by deletion of all 86 amino acids and replacement with a region of the 2C-CT epitope and the conserved cysteine 13 residues from the seventh transmembrane domain. To determine whether the cellular distribution of truncation mutants differs from that of wild-type 5-HT2Creceptors, fibroblasts were transiently transfected with receptor cDNA, and immunoreactive receptors were detected with anti-2C-IC antibodies. Receptors that lack the terminal PDZ recognition motif (Ser458-Ser-Val-COOH; ΔPDZ) were distributed throughout transiently transfected cells (Fig.4 B) in a pattern similar to that of wild-type receptors (Fig. 4 A). In contrast, receptors that lack 72 residues of the 86-amino acid tail (Δ375/CT) were localized primarily in punctate structures surrounding the nucleus (Fig. 4 C). Identical results were observed when receptors were labeled with anti-2C-CT antibodies (data not shown). Since the intracellular distribution of Δ375/CT receptors would make it difficult to interpret results from phosphorylation assays, only ΔPDZ receptor cell lines were evaluated further. Fibroblasts stably expressing ΔPDZ or wild-type 5-HT2C receptors were untreated or treated with 1 μm serotonin and examined in two phosphorylation assays. Phosphorylation was not detected in the band-shift assay using two independent ΔPDZ clones (ΔPDZ-2 and ΔPDZ-19) (Fig.5 A, lanes 1 and 2), whereas wild-type receptors were phosphorylated as determined by the increase in mass to 41 kDa (lane 3). To confirm the lack of phosphorylation of the ΔPDZ receptor, 32P incorporation studies were performed with glycosylated receptors in the absence of tunicamycin. Cells labeled with 32P were treated with 1 μmserotonin or vehicle (water), and receptors were immunoprecipitated with anti-2C-IC antibodies. The immunoprecipitates were treated with N-glycosidase F, electrophoresed, and either exposed to a PhosphorImager cassette to determine incorporation of 32P (Fig. 5 B) or blotted onto nitrocellulose and probed with anti-2C-IC antibodies to determine the total amount of receptors (data not shown). Consistent with the results from the band-shift assay, incorporation of 32P was not detected in ΔPDZ receptors from either cell line (Fig. 5 B, lanes 1 and 2), whereas the wild-type receptor was phosphorylated (lane 3). To test the hypothesis that phosphorylation of the 5-HT2C receptor promotes desensitization, a phosphoinositide hydrolysis assay was performed with cell lines expressing either ΔPDZ receptors (ΔPDZ-2) or wild-type 5-HT2C receptors (Fig. 6). Both receptors demonstrated an ∼4-fold increase in basal inositol monophosphate accumulation with identical EC50 values (ΔPDZ, 4.7 ± 1.1 nm; and wild-type, 4.7 ± 1.2 nm; n = six experiments). Thus, deletion of the PDZ recognition motif did not alter the EC50 or maximal response during a single 30-min application of serotonin relative to the wild-type 5-HT2C receptor. To determine whether treatment of cells with serotonin promotes desensitization of a subsequent response, we utilized a protocol that was previously demonstrated to promote desensitization of wild-type 5-HT2Creceptor responses (7Westphal R.S. Backstrom J.R. Sanders-Bush E. Mol. Pharmacol. 1995; 48: 200-205PubMed Google Scholar). Cells were either untreated or treated with 100 nm serotonin, a concentration that causes maximal receptor phosphorylation (Fig. 2), for 16 h. After washing four times with serum-free DMEM, phosphoinositide hydrolysis was determined with increasing concentrations of serotonin (10−10to 10−5m) to generate concentration-response curves. Table Iillustrates that pretreatment of cells with serotonin caused a 3-fold increase in the EC50 of wild-type receptors and an 8-fold increase in the EC50 of ΔPDZ receptors. In these experiments, cells were treated with serotonin for 16 h; however, maximal 5-HT2C receptor phosphorylation occurs within 10 min of serotonin treatment (Fig. 3). Therefore, calcium imaging was utilized to examine dynamic 5-HT2C receptor responses in a more relevant time frame.Table IAgonist pretreatment increases the EC50 of serotonin for ΔPDZ and wild-type 5-HT2C receptors5-HT2CreceptorEC50pvalue1-aComparison between values obtained without and with serotonin.pvalue1-bComparison between wild-type and ΔPDZ, values obtained with serotonin.−Serotonin+SerotoninnmWild-type4.9 ± 1.414 ± 4.60.0091ΔPDZ4.8 ± 1.541 ± 130.00150.0078Cells stably expressing ΔPDZ (ΔPDZ-2) or wild-type 5-HT2Creceptors were labeled overnight with myo-[3H]inositol in the absence (−) or presence (+) of 100 nm serotonin for 16 h. Wells were washed four times with serum-free medium and stimulated with 10−10 to 10−5m serotonin in triplicate wells for 30 min. Radiolabeled inositol monophosphates were isolated by anion-exchange chromatography and subjected to scintillation counting. The data (mean ± S.D.) from four independent experiments were statistically analyzed by two-way analysis of variance, followed by unpaired two-tailed Student's t test.1-a Comparison between values obtained without and with serotonin.1-b Comparison between wild-type and ΔPDZ, values obtained with serotonin. Open table in a new tab Cells stably expressing ΔPDZ (ΔPDZ-2) or wild-type 5-HT2Creceptors were labeled overnight with myo-[3H]inositol in the absence (−) or presence (+) of 100 nm serotonin for 16 h. Wells were washed four times with serum-free medium and stimulated with 10−10 to 10−5m serotonin in triplicate wells for 30 min. Radiolabeled inositol monophosphates were isolated by anion-exchange chromatography and subjected to scintillation counting. The data (mean ± S.D.) from four independent experiments were statistically analyzed by two-way analysis of variance, followed by unpaired two-tailed Student's t test. Calcium imaging was performed with cells expressing ΔPDZ (ΔPDZ-2) or wild-type 5-HT2Creceptors to examine the dynamics of receptor desensitization. Cells were treated with 100 nm serotonin for 10 min, washed for 2.5 min, and restimulated with 100 nm serotonin. Wild-type 5-HT2C receptors (Fig.7 A) and ΔPDZ receptors (Fig.7 B) demonstrated a robust, transient release of intracellular calcium. Furthermore, wild-type and ΔPDZ receptors displayed similar maximal responses (Fig. 7) and indistinguishable half-lives for decay of the initial responses (wild-type, 62.4 ± 7.9 s; and ΔPDZ, 75.2 ± 9.5 s; n = 10; p = 0.31 by unpaired Student's t test). After washing for 2.5 min, a second application of serotonin to cells expressing wild-type receptors promoted calcium release at 37 ± 9% of the initial response (Figs. 7 A and 8). In contrast, a second application of serotonin to cells expressing ΔPDZ receptors produced responses that were only 13 ± 9% of the initial signal (Figs. 7 B and 8). To further investigate this observation, longer washout periods were examined. Fig.8 illustrates that after washing for 5 min, secondary responses of wild-type and ΔPDZ receptors were 50 ± 21 and 7 ± 10% of the initial response, respectively. After washing for 10 min, secondary responses of wild-type and ΔPDZ receptors were indistinguishable.Figure 8ΔPDZ receptors display delayed recovery of calcium responses relative to wild-type 5-HT2Creceptors. Cells stably expressing either wild-type or ΔPDZ 5-HT2C receptors were incubated in serum-free medium overnight and then loaded with fura-2 for 1 h. Cells were challenged twice with 100 nm serotonin (see Fig. 7) at the interval indicated on the x axis. A ratio (shown on the y axis) was generated by dividing the peak height of the second calcium response (peak 2) by the first response (peak 1). Cells expressing ΔPDZ receptors demonstrated slower functional resensitization of calcium responses at 2.5 and 5 min. **, p < 0.005; *, p < 0.05 (unpaired two-tailed Student's t test). Error bars represent S.D. Data for each time point are representative of at least three independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Since ΔPDZ receptors lack two potential phosphorylation sites (Ser458-Ser-Val-COOH), serine-to-alanine point mutations were created, and cell lines expressing S458A or S459A receptors were examined in phosphorylation and calcium release assays. For the phosphorylation assay, cells were treated with 1 μmserotonin, and the levels of phosphorylated receptors were determined in the band-shift assay (Fig.9 A). Both S458A and S459A receptors decreased serotonin-mediated phosphorylation to 50% of wild-type receptor levels (Fig. 9 B, white bars). Identical results were obtained with 100 nm serotonin (data not shown). For the calcium release assay, cells were treated with 100 nm serotonin for 10 min, washed for 5 min, and restimulated with 100 nm serotonin. Whereas the peak secondary responses of S458A receptors were similar to those of wild-type receptors, those of S459A receptors were diminished (Fig. 9 B, gray bars). Thus, mutation of Ser459 decreased receptor phosphorylation and diminished subsequent calcium responses relative to wild-type 5-HT2C receptors. Treatment of cells expressing serotonin 5-HT2Creceptors with serotonin promotes phosphoinositide hydrolysis (24Conn P.J. Sanders-Bush E. Hoffman B.J. Hartig P.R. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 4086-4088Crossref PubMed Scopus (247) Google Scholar), release of intracellular calcium (18Akiyoshi J. Nishizono A. Yamada K. Nagayama H. Mifune K. Fujii I. J. Neurochem. 1995; 64: 2473-2479Crossref PubMed Scopus (34) Google Scholar, 19Watson J.A. Elliott A.C. Brown P.D. Cell Calcium. 1995; 17: 120-128Crossref PubMed Scopus (22) Google Scholar, 25Porter R.H.P. Benwell K.R. Lamb H. Malcolm C.S. Allen N.H. Revell D.F. Adams D.R. Sheardown M.J. Br. J. Pharmacol. 1999; 128: 13-20Crossref PubMed Scopus (324) Google Scholar), and receptor phosphorylation (7Westphal R.S. Backstrom J.R. Sanders-Bush E. Mol. Pharmacol. 1995; 48: 200-205PubMed Google Scholar). Although subsequent responses to agonist application are attenuated (desensitized) relative to the first response (7Westphal R.S. Backstrom J.R. Sanders-Bush E. Mol. Pharmacol. 1995; 48: 200-205PubMed Google Scholar, 17Briddon S.J. Leslie R.A. Elliott J.M. Br. J. Pharmacol. 1998; 125: 727-734Crossref PubMed Scopus (26) Google Scholar, 18Akiyoshi J. Nishizono A. Yamada K. Nagayama H. Mifune K. Fujii I. J. Neurochem. 1995; 64: 2473-2479Crossref PubMed Scopus (34) Google Scholar, 19Watson J.A. Elliott A.C. Brown P.D. Cell Calcium. 1995; 17: 120-128Crossref PubMed Scopus (22) Google Scholar), it is not known whether phosphorylation of the 5-HT2C receptor plays a role in desensitization. Therefore, we created 5-HT2C receptor phosphorylation-deficient mutants and examined serotonin responses in phosphoinositide hydrolysis and calcium release experiments. Here, we demonstrate that deletion of the terminal three amino acids, which include a PDZ recognition motif (Ser458-Ser-Val-COOH; ΔPDZ), abrogates serotonin-mediated phosphorylation and, unexpectedly, delays the recovery (resensitization) of desensitized 5-HT2C receptor responses. Immunoreactive 5-HT2C receptors have masses of 51–68 kDa; and after treatment of cells with tunicamycin to preventN-linked glycosylation, receptors are detected with masses of 40 and 41 kDa (21Backstrom J.R. Westphal R.S. Canton H. Sanders-Bush E. Mol. Brain Res. 1995; 33: 311-318Crossref PubMed Scopus (34) Google Scholar). Since two bands are detected from cells maintained in medium containing serum (21Backstrom J.R. Westphal R.S. Canton H. Sanders-Bush E. Mol. Brain Res. 1995; 33: 311-318Crossref PubMed Scopus (34) Google Scholar), we examined the possibility that serotonin, present in serum, alters the migration of receptors. Here, we demonstrate that the 41-kDa protein is a phosphorylated form of 40-kDa 5-HT2C receptors. Cells in serum-free medium contain 5-HT2C receptors with a mass of 40 kDa, whereas treatment of cells with the agonist serotonin causes the appearance of the 41-kDa protein. This agonist effect is blocked by co-incubation with mianserin, a 5-HT2A/2C receptor antagonist, and reversed by either agonist washout or treatment of cell extracts with alkaline phosphatase. These results establish that the 41-kDa protein reflects phosphorylated 5-HT2C receptors. We exploited the band-shift phosphorylation assay to identify a domain of 5-HT2C receptor that is required for receptor phosphorylation. Two truncated 5-HT2C receptors were generated that lack either all potential phosphorylation sites in the carboxyl terminus (Δ375/CT) or the terminal two serine residues (ΔPDZ). The mutant Δ375/CT receptor was created by truncation after the seventh transmembrane domain to remove all 86 amino acids of the carboxyl terminus and replacement with a region of the 2C-CT epitope that lacks serine, threonine, and tyrosine residues. The cysteine located 13 residues from the predicted membrane domain was conserved in this construct because this residue has been shown to be important for the function of 5-HT2A receptors (26Buck F. Meyerhof W. Werr H. Richter D. Biochem. Biophys. Res. Commun. 1991; 178: 1421-1428Crossref PubMed Scopus (20) Google Scholar). The mutant ΔPDZ receptor lacks only three amino acids (Ser458-Ser-Val-COOH). Mutant 5-HT2C receptor cDNA was transiently expressed in fibroblasts, and the distribution of immunoreactive receptors was compared with that of wild-type 5-HT2C receptors. Two different phenotypes were observed: Δ375/CT receptors were predominantly intracellular, whereas ΔPDZ and wild-type 5-HT2C receptors were distributed in a similar pattern throughout cells. Cell lines expressing ΔPDZ receptors were therefore a valid system for evaluating the contribution of the PDZ recognition motif in phosphorylation assays. The band-shift phosphorylation assay and incorporation of32P were used to examine phosphorylation of two independent cell lines expressing ΔPDZ 5-HT2C receptors. In the absence of serotonin, wild-type and ΔPDZ receptors had an apparent mass of 40 kDa. Treatment of cells with serotonin caused the appearance of the phosphorylated 41-kDa form in wild-type receptors, whereas no increase in mass was detected in ΔPDZ receptors. In support of the results obtained with the band-shift assay, serotonin-mediated incorporation of 32P was not detected in ΔPDZ receptors, whereas wild-type receptors demonstrated robust phosphorylation. These results further confirm that the band-shift phosphorylation assay reflects 5-HT2C receptor phosphorylation and establish that the PDZ recognition motif is required for receptor phosphorylation. Previously, we demonstrated that overnight pretreatment of cells stably expressing wild-type 5-HT2C receptors with serotonin increases the EC50 value for stimulating phosphoinositide hydrolysis without changing the maximal response to serotonin (7Westphal R.S. Backstrom J.R. Sanders-Bush E. Mol. Pharmacol. 1995; 48: 200-205PubMed Google Scholar). To determine whether receptor phosphorylation is involved in the observed desensitization, cells stably expressing ΔPDZ or wild-type 5-HT2C receptors were either untreated or treated with 100 nm serotonin overnight, washed, and then stimulated with increasing concentrations of serotonin. Interestingly, pretreatment with serotonin promoted a significantly greater increase in the EC50 value of serotonin for ΔPDZ receptors (8-fold) than for wild-type 5-HT2C receptors (3-fold). This result is not consistent with the hypothesis that 5-HT2C receptor phosphorylation promotes desensitization and raises the intriguing possibility that phosphorylation may actually attenuate desensitization or promote resensitization of 5-HT2C receptor responses. The time of pretreatment with serotonin was much longer in the functional assays (hours) than in the phosphorylation assays (minutes), and it is possible that additional 5-HT2C receptor sites were phosphorylated during prolonged serotonin treatment. Therefore, calcium imaging was used to examine the dynamics of 5-HT2Creceptor desensitization and resensitization within a time frame that corresponds to receptor phosphorylation. Both ΔPDZ and wild-type 5-HT2C receptors displayed robust, transient increases in intracellular calcium with similar maximal responses. In the continued presence of serotonin, responses of wild-type and ΔPDZ receptors decayed with indistinguishable half-lives of ∼1 min. These results lend additional support, within a relevant time frame, that desensitization of 5-HT2C receptor responses occurs in the absence of receptor phosphorylation. After washing cells with agonist-free medium, dramatically different recovery responses were observed. After 5 min, wild-type 5-HT2C receptor responses recovered to 50% of the initial response, whereas ΔPDZ receptor responses recovered to only 7% of the initial response. After 10 min, which corresponds to the half-life for reversal of wild-type 5-HT2C receptor phosphorylation, significant differences between ΔPDZ and wild-type 5-HT2C receptors were not observed. Thus, the results from phosphoinositide hydrolysis and calcium release experiments suggest that phosphorylation of the 5-HT2C receptor enhances resensitization of 5-HT2C receptor responses rather than altering desensitization kinetics. Since ΔPDZ 5-HT2C receptors lack two potential phosphorylation sites, single point mutants were created at Ser458, the PDZ recognition motif serine, or Ser459 to determine if mutation of either residue alters receptor phosphorylation and/or calcium responses. Cell lines expressing identical densities of S458A or S459A 5-HT2Creceptors were first examined in the band-shift phosphorylation assay. Both S458A and S459A receptors decreased serotonin-mediated phosphorylation to 50% of wild-type 5-HT2C receptor levels, suggesting that both Ser458 and Ser459are phosphorylated. Next, calcium release responses were examined. Interestingly, responses of S459A receptors to a second application of serotonin were diminished relative to wild-type 5-HT2Creceptors, whereas responses of S458A receptors reproduced the wild-type phenotype. These results are consistent with a major role of Ser459 rather than the PDZ recognition motif per se in resensitization of 5-HT2C receptor responses. In contrast, the PDZ recognition motif of β2-adrenergic receptors (Ser-Leu-Leu-COOH) is involved in efficient recycling of internalized receptors to the cell surface, and mutations within this motif enhance agonist-mediated receptor degradation (27Cao T.T. Deacon H.W. Reczek D. Bretscher A. von Zastrow M. Nature. 1999; 401: 286-290Crossref PubMed Scopus (571) Google Scholar). Our observations are consistent with a role of Ser459 in resensitization of 5-HT2C receptor responses and suggest that phosphorylation at this site regulates resensitization. We thank Ann Westphal and Antoinette Poindexter for expert technical assistance and Dr. Colleen Niswender for providing technical guidance and wisdom.
