Secretory Carrier Membrane Protein 2 Regulates Exocytic Insertion of NKCC2 into the Cell Membrane
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The renal-specific Na-K-2Cl co-transporter, NKCC2, plays a pivotal role in regulating body salt levels and blood pressure. NKCC2 mutations lead to type I Bartter syndrome, a life-threatening kidney disease. Regulation of NKCC2 trafficking behavior serves as a major mechanism in controlling NKCC2 activity across the plasma membrane. However, the identities of the protein partners involved in cell surface targeting of NKCC2 are largely unknown. To gain insight into these processes, we used a yeast two-hybrid system to screen a kidney cDNA library for proteins that interact with the NKCC2 C terminus. One binding partner we identified was SCAMP2 (secretory carrier membrane protein 2). Microscopic confocal imaging and co-immunoprecipitation assays confirmed NKCC2-SCAMP2 interaction in renal cells. SCAMP2 associated also with the structurally related co-transporter NCC, suggesting that the interaction with SCAMP2 is a common feature of sodium-dependent chloride co-transporters. Heterologous expression of SCAMP2 specifically decreased cell surface abundance as well as transport activity of NKCC2 across the plasma membrane. Co-immunolocalization experiments revealed that intracellularly retained NKCC2 co-localizes with SCAMP2 in recycling endosomes. The rate of NKCC2 endocytic retrieval, assessed by the sodium 2-mercaptoethane sulfonate cleavage assay, was not affected by SCAMP2. The surface-biotinylatable fraction of newly inserted NKCC2 in the plasma membrane was reduced by SCAMP2, demonstrating that SCAMP2-induced decrease in surface NKCC2 is due to decreased exocytotic trafficking. Finally, a single amino acid mutation, cysteine 201 to alanine, within the conserved cytoplasmic E peptide of SCAMP2, which is believed to regulate exocytosis, abolished SCAMP2-mediated down-regulation of the co-transporter. Taken together, these data are consistent with a model whereby SCAMP2 regulates NKCC2 transit through recycling endosomes and limits the cell surface targeting of the co-transporter by interfering with its exocytotic trafficking. The renal-specific Na-K-2Cl co-transporter, NKCC2, plays a pivotal role in regulating body salt levels and blood pressure. NKCC2 mutations lead to type I Bartter syndrome, a life-threatening kidney disease. Regulation of NKCC2 trafficking behavior serves as a major mechanism in controlling NKCC2 activity across the plasma membrane. However, the identities of the protein partners involved in cell surface targeting of NKCC2 are largely unknown. To gain insight into these processes, we used a yeast two-hybrid system to screen a kidney cDNA library for proteins that interact with the NKCC2 C terminus. One binding partner we identified was SCAMP2 (secretory carrier membrane protein 2). Microscopic confocal imaging and co-immunoprecipitation assays confirmed NKCC2-SCAMP2 interaction in renal cells. SCAMP2 associated also with the structurally related co-transporter NCC, suggesting that the interaction with SCAMP2 is a common feature of sodium-dependent chloride co-transporters. Heterologous expression of SCAMP2 specifically decreased cell surface abundance as well as transport activity of NKCC2 across the plasma membrane. Co-immunolocalization experiments revealed that intracellularly retained NKCC2 co-localizes with SCAMP2 in recycling endosomes. The rate of NKCC2 endocytic retrieval, assessed by the sodium 2-mercaptoethane sulfonate cleavage assay, was not affected by SCAMP2. The surface-biotinylatable fraction of newly inserted NKCC2 in the plasma membrane was reduced by SCAMP2, demonstrating that SCAMP2-induced decrease in surface NKCC2 is due to decreased exocytotic trafficking. Finally, a single amino acid mutation, cysteine 201 to alanine, within the conserved cytoplasmic E peptide of SCAMP2, which is believed to regulate exocytosis, abolished SCAMP2-mediated down-regulation of the co-transporter. Taken together, these data are consistent with a model whereby SCAMP2 regulates NKCC2 transit through recycling endosomes and limits the cell surface targeting of the co-transporter by interfering with its exocytotic trafficking. IntroductionNKCC2 is an Na-K-2Cl co-transporter protein expressed exclusively in the mammalian kidney, where it provides the major route for sodium/chloride transport across the apical plasma membrane of the thick ascending limb (1Russell J.M. Physiol. Rev. 2000; 80: 211-276Crossref PubMed Scopus (728) Google Scholar). NKCC2 is therefore a pivotal protein in renal function, and exquisitely tight regulation of the apical co-transporter activity is paramount for maintaining extracellular fluid volume and acid-base homeostasis (2Gamba G. Physiol. Rev. 2005; 85: 423-493Crossref PubMed Scopus (615) Google Scholar). It is the target of loop-diuretics extensively used in the treatment of edematous states and hypertension (3Gamba G. Kidney Int. 1999; 56: 1606-1622Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Furthermore, recent work implicates NKCC2 in the dysfunction of blood pressure regulation, raising new hypotheses regarding the underlying mechanisms behind some types of essential hypertension (4Ashton N. J. Hypertens. 2006; 24: 2355-2356Crossref PubMed Scopus (1) Google Scholar, 5Richardson C. Alessi D.R. J. Cell Sci. 2008; 121: 3293-3304Crossref PubMed Scopus (224) Google Scholar, 6Gamba G. Am. J. Physiol. Renal. Physiol. 2005; 288: F245-F252Crossref PubMed Scopus (104) Google Scholar, 7Flatman P.W. Curr. Opin. Nephrol. Hypertens. 2008; 17: 186-192Crossref PubMed Scopus (51) Google Scholar). Most importantly, inactivating mutations of the NKCC2 gene in humans causes Bartter syndrome type 1, a life-threatening kidney disease (8Simon D.B. Lifton R.P. Am. J. Physiol. 1996; 271: F961-F966PubMed Google Scholar). However, despite this importance, little is known about NKCC2 regulation in renal cells, mainly because of the difficulty in expressing the co-transporter protein in mammalian cells (9Mount D.B. Am. J. Physiol. Renal. Physiol. 2006; 290: F606-F607Crossref PubMed Scopus (10) Google Scholar). As a consequence, although several studies have addressed various aspects of NKCC2 regulation (2Gamba G. Physiol. Rev. 2005; 85: 423-493Crossref PubMed Scopus (615) Google Scholar, 7Flatman P.W. Curr. Opin. Nephrol. Hypertens. 2008; 17: 186-192Crossref PubMed Scopus (51) Google Scholar, 9Mount D.B. Am. J. Physiol. Renal. Physiol. 2006; 290: F606-F607Crossref PubMed Scopus (10) Google Scholar, 10Carmosino M. Giménez I. Caplan M. Forbush B. Mol. Biol. Cell. 2008; 19: 4341-4351Crossref PubMed Scopus (66) Google Scholar, 11Zaarour N. Demaretz S. Defontaine N. Mordasini D. Laghmani K. J. Biol. Chem. 2009; 284: 21752-21764Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 12Hannemann A. Christie J.K. Flatman P.W. J. Biol. Chem. 2009; 284: 35348-35358Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar), our knowledge of the molecular mechanisms underlying membrane trafficking of NKCC2 proteins in mammalian cells, in particular its regulation by protein-protein interactions, remain poor. Identifying and functionally characterizing a key and/or global protein interaction(s) and pathway(s) involved in the regulation of NKCC2 trafficking is important to understand its different physiological functions and dysfunctions.NKCC2 belongs to the cation-chloride co-transporter (CCC) 3The abbreviations used are: CCC, cation-chloride co-transporter; TAL, thick ascending limb; MTAL, medullary TAL; CTAL, cortical TAL; EGFP, enhanced GFP; OKP, opossum kidney; aa, amino acid(s); NCC, NaCl co-transporter; HEK, human embryonic kidney; NHS, N-hydroxysuccinimide; NHE, Na+-H+ exchanger; MesNA, sodium 2-mercaptoethane sulfonate; TGN, trans-Golgi network. family, which comprises two principal branches of membrane proteins (1Russell J.M. Physiol. Rev. 2000; 80: 211-276Crossref PubMed Scopus (728) Google Scholar). One branch includes the Na+-dependent chloride co-transporters composed of the Na+-K+-2Cl− co-transporters (NKCC1 and NKCC2) and the Na+-Cl− co-transporter (NCC). The second branch includes the Na+-independent K+-Cl− co-transporters. Members within this family are very homologous to one another, possessing 12 transmembrane-spanning domains, an N terminus of variable length, and a long cytoplasmic C terminus (2Gamba G. Physiol. Rev. 2005; 85: 423-493Crossref PubMed Scopus (615) Google Scholar). Given that the C-terminal domain of NKCC2 is the predominant cytoplasmic region, it is likely to be a major factor in the trafficking of the NKCC2 protein. In support of this notion, we recently demonstrated that a highly conserved motif at the COOH terminus dictates endoplasmic reticulum exit and cell surface expression of NKCC2 (11Zaarour N. Demaretz S. Defontaine N. Mordasini D. Laghmani K. J. Biol. Chem. 2009; 284: 21752-21764Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Moreover, there have been several reports demonstrating that the trafficking to the apical membrane of several ion transport systems depend upon protein-protein interactions involving their extreme C terminus (13Caplan M.J. Am. J. Physiol. 1997; 272: F425-F429Crossref PubMed Google Scholar). For instance, deleting the last three residues of the cystic fibrosis transmembrane regulator chloride channel C terminus prevents its interactions with CAL, CAP70, and NHERF proteins and results in aberrant basolateral accumulation of the mutant protein (14Cheng J. Moyer B.D. Milewski M. Loffing J. Ikeda M. Mickle J.E. Cutting G.R. Li M. Stanton B.A. Guggino W.B. J. Biol. Chem. 2002; 277: 3520-3529Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 15Wang S. Raab R.W. Schatz P.J. Guggino W.B. Li M. FEBS Lett. 1998; 427: 103-108Crossref PubMed Scopus (249) Google Scholar, 16Wang S. Yue H. Derin R.B. Guggino W.B. Li M. Cell. 2000; 103: 169-179Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar). The interaction of NHERF proteins with the NHE-3 C terminus regulates the trafficking of the exchanger protein to the apical surface of proximal tubule cells (17Donowitz M. Cha B. Zachos N.C. Brett C.L. Sharma A. Tse C.M. Li X. J. Physiol. 2005; 567: 3-11Crossref PubMed Scopus (182) Google Scholar). PDZ interactions mediated through the C-terminal tail of the Na-Pi co-transporter control its correct targeting to the renal proximal tubular brush border (18Hernando N. Déliot N. Gisler S.M. Lederer E. Weinman E.J. Biber J. Murer H. Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 11957-11962Crossref PubMed Scopus (162) Google Scholar). Similar to NHE-3, cystic fibrosis transmembrane regulator chloride channel, and Na-Pi, NKCC2 surface expression is also subject to regulation by intracellular protein trafficking (19Giménez I. Forbush B. J. Biol. Chem. 2003; 278: 26946-26951Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 20Ortiz P.A. Am. J. Physiol. Renal. Physiol. 2006; 290: F608-F616Crossref PubMed Scopus (99) Google Scholar, 21Caceres P.S. Ares G.R. Ortiz P.A. J. Biol. Chem. 2009; 284: 24965-24971Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). However, very little is known about the protein-binding partners that control membrane sorting of NKCC2. More specifically, the identities of the protein partners of NKCC2 that orchestrate its exocytic insertion, endocytic retrieval, and recycling to and from the plasma membrane are scarcely known. Accordingly, identifying proteins that interact with the C-terminal tail of NKCC2 should help to further determine the mechanisms of regulated NKCC2 trafficking. In this report, we describe a novel protein-protein interaction between the C-terminal tail of NKCC2 and SCAMP2. SCAMP2 belongs to a family of evolutionarily conserved tetraspanning integral membrane proteins (22Hubbard C. Singleton D. Rauch M. Jayasinghe S. Cafiso D. Castle D. Mol. Biol. Cell. 2000; 11: 2933-2947Crossref PubMed Scopus (42) Google Scholar). To date, five isoforms of SCAMP (SCAMP1 to -5) have been identified in mammals and have been shown to be predominantly associated with recycling rather than degradation pathways (23Castle A. Castle D. J. Cell Sci. 2005; 118: 3769-3780Crossref PubMed Scopus (52) Google Scholar, 24Han C. Chen T. Yang M. Li N. Liu H. Cao X. J. Immunol. 2009; 182: 2986-2996Crossref PubMed Scopus (34) Google Scholar, 25Liao H. Zhang J. Shestopal S. Szabo G. Castle A. Castle D. Am. J. Physiol. Cell Physiol. 2008; 294: C797-C809Crossref PubMed Scopus (20) Google Scholar, 26Fernández-Chacón R. Achiriloaie M. Janz R. Albanesi J.P. Südhof T.C. J. Biol. Chem. 2000; 275: 12752-12756Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Indeed, SCAMPs are found mainly in the trans-Golgi network (TGN) and recycling endosomes and have been shown to play a role in endocytosis (26Fernández-Chacón R. Achiriloaie M. Janz R. Albanesi J.P. Südhof T.C. J. Biol. Chem. 2000; 275: 12752-12756Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar) and exocytosis (27Guo Z. Liu L. Cafiso D. Castle D. J. Biol. Chem. 2002; 277: 35357-35363Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 28Liao H. Ellena J. Liu L. Szabo G. Cafiso D. Castle D. Biochemistry. 2007; 46: 10909-10920Crossref PubMed Scopus (32) Google Scholar). Here, we show that SCAMP2 regulates the cell surface targeting of NKCC2 by controlling its exocytosis, revealing therefore a novel regulatory mechanism governing the trafficking of the co-transporter to the apical membrane.DISCUSSIONIn this study, we have identified SCAMP2 as a novel interacting partner of NKCC2 through yeast two-hybrid screening of a human kidney cDNA library. Our results indicate that SCAMP2 interaction with NKCC2 is specific and depends on the first 108 aa of the co-transporter C terminus. SCAMP2 binds also to the structurally related co-transporter NCC (2Gamba G. Physiol. Rev. 2005; 85: 423-493Crossref PubMed Scopus (615) Google Scholar), suggesting that the interaction with SCAMP2 is a common feature of Na+-dependent Cl− co-transporters. Functional studies demonstrated that co-expression of SCAMP2 specifically decreases NKCC2 transport activity by promoting its intracellular retention and therefore reducing its abundance at the cell surface. SCAMP2-induced decrease in surface NKCC2 is due to decreased exocytic insertion.SCAMP2 is a member of the evolutionarily conserved tetraspanning integral membrane proteins, which function as a carrier to the cell surface in post-Golgi recycling pathways (23Castle A. Castle D. J. Cell Sci. 2005; 118: 3769-3780Crossref PubMed Scopus (52) Google Scholar, 57Fernández-Chacón R. Südhof T.C. J. Neurosci. 2000; 20: 7941-7950Crossref PubMed Google Scholar). In this report, we showed evidence for the specific binding of SCAMP2 to NKCC2. Using co-immunoprecipitation, we were able to detect the interaction in vivo between full-length NKCC2 and endogenous SCAMP2 protein in renal cells. Using yeast two-hybrid analysis, we observed that NKCC2 interaction with SCAMP2 is specific to the proximal region of the NKCC2 C terminus. This proximal region is highly conserved at the C termini of the Na+-coupled Cl−-co-transporters NKCC1, NKCC2, and NCC. Accordingly, we were also able to show that SCAMP2 interacts with NCC. In contrast, ClC-5 and pendrin, two kidney chloride transporters (34Steinmeyer K. Schwappach B. Bens M. Vandewalle A. Jentsch T.J. J. Biol. Chem. 1995; 270: 31172-31177Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar, 35Mount D.B. Romero M.F. Pflugers Arch. 2004; 447: 710-721Crossref PubMed Scopus (441) Google Scholar) that are not structurally related to the CCC family, failed to co-immunoprecipitate with SCAMP2, further supporting the specificity of the interaction of SCAMP2 with NCC and NKCC2 in renal cells. To identify the subcellular site of interaction of SCAMP2 with NKCC2, we visualized the subcellular distribution of the two proteins using confocal microscopy. NKCC2 and SCAMP2 showed considerable co-localization with endogenous Rab11, a commonly used marker of recycling endosomes (41Green E.G. Ramm E. Riley N.M. Spiro D.J. Goldenring J.R. Wessling-Resnick M. Biochem. Biophys. Res. Commun. 1997; 239: 612-616Crossref PubMed Scopus (88) Google Scholar, 42Bartz R. Benzing C. Ullrich O. Biochem. Biophys. Res. Commun. 2003; 312: 663-669Crossref PubMed Scopus (13) Google Scholar, 43Ren M. Xu G. Zeng J. De Lemos-Chiarandini C. Adesnik M. Sabatini D.D. Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 6187-6192Crossref PubMed Scopus (389) Google Scholar, 44Wang X. Kumar R. Navarre J. Casanova J.E. Goldenring J.R. J. Biol. Chem. 2000; 275: 29138-29146Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar, 45Ward E.S. Martinez C. Vaccaro C. Zhou J. Tang Q. Ober R.J. Mol. Biol. Cell. 2005; 16: 2028-2038Crossref PubMed Scopus (150) Google Scholar, 46Hoekstra D. Tyteca D. van IJzendoorn S.C. J. Cell Sci. 2004; 117: 2183-2192Crossref PubMed Scopus (122) Google Scholar). However, Rab11 has also been found to be associated with the TGN and post-Golgi vesicles (48Urbé S. Huber L.A. Zerial M. Tooze S.A. Parton R.G. FEBS Lett. 1993; 334: 175-182Crossref PubMed Scopus (186) Google Scholar, 58Satoh A.K. O'Tousa J.E. Ozaki K. Ready D.F. Development. 2005; 132: 1487-1497Crossref PubMed Scopus (216) Google Scholar), indicating that the site of interaction between SCAMP2 and NKCC2 could be identified as recycling endosomes and/or TGN membranes. Because SCAMP2 and NKCC2 also co-localized with internalized transferrin, our data are consistent with the view that NKCC2 interacts with SCAMP2 in recycling endosomes. They are in agreement with a previous study showing that the internalized transferrin-containing vesicles are fused with a pre-existing internal pool of SCAMP-positive membranes and then accumulate in the SCAMP-rich perinuclear region corresponding to the recycling endosomal compartment (23Castle A. Castle D. J. Cell Sci. 2005; 118: 3769-3780Crossref PubMed Scopus (52) Google Scholar). Also, a recent study by Diering and co-workers (59Diering G.H. Church J. Numata M. J. Biol. Chem. 2009; 284: 13892-13903Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar) reported considerable co-localization of SCAMP2 with Rab11 in recycling endosomes. Likewise, Müller and co-workers (60Müller H.K. Wiborg O. Haase J. J. Biol. Chem. 2006; 281: 28901-28909Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar) demonstrated that SCAMP2-positive compartments contain the lipid raft marker follitin, supporting the idea that these structures could be also recycling endosomes. Thus, these findings together with our own suggest strongly that SCAMP2 interacts with NKCC2 in recycling endosomes. Of note, the co-immunoprecipitation experiments revealed that both immature and mature forms of NKCC2 interact with SCAMP2, which may serve as an indication that in addition to recycling endosomes, NKCC2 may also interact with SCAMP2 in other intracellular compartments, such as the endoplasmic reticulum. This possibility is further supported by previous observations reporting partial co-localization of NKCC2 (11Zaarour N. Demaretz S. Defontaine N. Mordasini D. Laghmani K. J. Biol. Chem. 2009; 284: 21752-21764Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar) and SCAMP2 (33Lin P.J. Williams W.P. Luu Y. Molday R.S. Orlowski J. Numata M. J. Cell Sci. 2005; 118: 1885-1897Crossref PubMed Scopus (53) Google Scholar) with endoplasmic reticulum markers. However, some high mannose glycosylated proteins (immature) that are trafficked to the cell surface traverse the Golgi apparatus without further processing (61Vagin O. Kraut J.A. Sachs G. Am. J. Physiol. Renal Physiol. 2009; 296: F459-F469Crossref PubMed Scopus (141) Google Scholar). As a consequence, for several membrane proteins, both immature and mature forms were detected at the plasma membranes (60Müller H.K. Wiborg O. Haase J. J. Biol. Chem. 2006; 281: 28901-28909Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 62Johns T.G. Mellman I. Cartwright G.A. Ritter G. Old L.J. Burgess A.W. Scott A.M. FASEB J. 2005; 19: 780-782Crossref PubMed Scopus (63) Google Scholar, 63Watanabe I. Wang H.G. Sutachan J.J. Zhu J. Recio-Pinto E. Thornhill W.B. J. Physiol. 2003; 550: 51-66Crossref PubMed Scopus (71) Google Scholar, 64Zhu J. Watanabe I. Gomez B. Thornhill W.B. Biochem. J. 2003; 375: 761-768Crossref PubMed Scopus (30) Google Scholar). Although only the mature form of NKCC2 is able to reach the cell surface, it is conceivable that, at least when using protein overexpression (62Johns T.G. Mellman I. Cartwright G.A. Ritter G. Old L.J. Burgess A.W. Scott A.M. FASEB J. 2005; 19: 780-782Crossref PubMed Scopus (63) Google Scholar), a portion of immature wild type NKCC2 is delivered to post-Golgi compartments, including the TGN and recycling endosomes. Indeed, several reports provided evidence that recycling endosomes play a direct role in the biosynthetic pathway by serving as an intermediate during transport from the Golgi to cell plasma membranes (65Ang A.L. Taguchi T. Francis S. Fölsch H. Murrells L.J. Pypaert M. Warren G. Mellman I. J. Cell Biol. 2004; 167: 531-543Crossref PubMed Scopus (335) Google Scholar, 66Cresawn K.O. Potter B.A. Oztan A. Guerriero C.J. Ihrke G. Goldenring J.R. Apodaca G. Weisz O.A. EMBO J. 2007; 26: 3737-3748Crossref PubMed Scopus (99) Google Scholar, 67Ellis M.A. Potter B.A. Cresawn K.O. Weisz O.A. Am. J. Physiol. Renal. Physiol. 2006; 291: F707-F713Crossref PubMed Scopus (40) Google Scholar). These observations therefore raise the possibility of an interaction between the immature form of NKCC2 and SCAMP2 in the recycling endosomes and/or the TGN. Nevertheless, regardless of the site of such an interaction, we did not observe any effect of SCAMP2 co-expression on immature NKCC2 protein. In contrast, we showed evidence that SCAMP2 is specifically involved in the regulation of the intracellular trafficking of the mature form of the co-transporter protein.In support of trafficking as a regulator of NKCC2, previous immunolocalization experiments revealed that NKCC2 is expressed not only at the cell surface but also in a population of subapical vesicles (55Nielsen S. Maunsbach A.B. Ecelbarger C.A. Knepper M.A. Am. J. Physiol. 1998; 275: F885-F893Crossref PubMed Google Scholar). In the present study, using immunocytochemistry, we showed a similar subcellular localization of NKCC2 proteins in cultured renal cells. When expressed alone, NKCC2 was detected at the cell surface and in intracellular compartments. In contrast, in cells co-expressing SCAMP2, we observed enhanced intracellular accumulation of NKCC2 and a consequent reduction in the expression of the co-transporter at the cell surface. Indeed, upon SCAMP2 co-expression, a significant fraction of NKCC2 disappeared from the cell membrane and clustered in an intracellular location, where it exhibited excellent co-localization with SCAMP2. These data suggested that SCAMP2 binding alters NKCC2 trafficking to the cell surface. To corroborate this observation, we used surface biotinylation and showed that the co-transporter expression at the cell surface was reduced in a dose-dependent fashion by SCAMP2 in HEK and OKP cells. To investigate whether a general disruption of vesicular trafficking in our cultured renal cells may underlie the SCAMP2 effect, we studied the effect of SCAMP2 on ClC-5, because this kidney chloride transporter is also expressed at the plasma membrane and in recycling endosomes (50Thakker R.V. Kidney Int. 2000; 57: 787-793Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 51Mohammad-Panah R. Harrison R. Dhani S. Ackerley C. Huan L.J. Wang Y. Bear C.E. J. Biol. Chem. 2003; 278: 29267-29277Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 52Hryciw D.H. Ekberg J. Pollock C.A. Poronnik P. Int. J. Biochem. Cell Biol. 2006; 38: 1036-1042Crossref PubMed Scopus (40) Google Scholar, 53Hryciw D.H. Ekberg J. Lee A. Lensink I.L. Kumar S. Guggino W.B. Cook D.I. Pollock C.A. Poronnik P. J. Biol. Chem. 2004; 279: 54996-55007Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). In contrast to NKCC2, overexpressed SCAMP2 protein did not interact with ClC-5 and had no effect on ClC-5 surface expression. Hence, these data suggest that the SCAMP2-V5 construct does not generally disturb trafficking of membrane proteins to and from the plasma membrane. Instead, it appears to specifically alter NKCC2 shuttling by interfering with its transit through recycling endosomes. Additional evidence for the specificity of SCAMP2 action on NKCC2 stems from the observation that SCAMP2 did not influence the distribution of Rab11 and internalized transferrin. Moreover, the specificity of SCAMP2-induced down-regulation of NKCC2 surface expression is also supported by a previous report demonstrating that NHE-5 interaction with overexpressed SCAMP2 in recycling endosomes promotes the cell surface targeting of the exchanger protein (59Diering G.H. Church J. Numata M. J. Biol. Chem. 2009; 284: 13892-13903Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Taken together, these findings suggest strongly that SCAMP2 specifically regulates NKCC2 transit through recycling endosomes to inhibit its cell surface targeting.SCAMP2-induced down-regulation of NKCC2 membrane abundance could arise from a decrease in NKCC2 exocytosis to the plasma membrane and/or to an increase of NKCC2 endocytosis. To measure NKCC2 endocytic internalization, we prelabeled NKCC2 with sulfo-NHS-SS-biotin and then measured the ability of SCAMP2 to cause NKCC2 to shift to a location where the disulfide bond would be protected from MesNa. The results showed that 36 and 58% of surface NKCC2 is internalized over 15 and 30 min, respectively, and that SCAMP2 co-expression had no effect on the process. Hence, these studies suggest that the SCAMP2 effect on the amount of NKCC2 at the cell surface is not due to increased endocytotic internalization. To measure exocytic insertion of NKCC2, we preblocked surface proteins and then measured the ability to label new surface NKCC2 with biotin in the presence or absence of SCAMP2. The results showed that SCAMP2 induced a decrease in the exocytic insertion of NKCC2 into the plasma membrane. Studies by other investigators have shown that the function of SCAMP2 in regulated exocytosis involves the E peptide, a highly conserved segment lying at the cytosolic surface of the molecule and linking the second and the third transmembrane domains (22Hubbard C. Singleton D. Rauch M. Jayasinghe S. Cafiso D. Castle D. Mol. Biol. Cell. 2000; 11: 2933-2947Crossref PubMed Scopus (42) Google Scholar). In particular, two residues within the E peptide, Cys201 and Trp202, were found to play a crucial role in this process (27Guo Z. Liu L. Cafiso D. Castle D. J. Biol. Chem. 2002; 277: 35357-35363Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 56Liu L. Guo Z. Tieu Q. Castle A. Castle D. Mol. Biol. Cell. 2002; 13: 4266-4278Crossref PubMed Scopus (40) Google Scholar). In the present work, individual mutation of cysteine 201 to alanine reversed SCAMP2-mediated down-regulation of NKCC2. Remarkably, SCAMP2 mutant was able to co-immunoprecipitate with NKCC2 protein, implying that SCAMP2 interaction with NKCC2, per se, is not sufficient to alter the co-transporter surface expression. A similar mechanism has been reported in the regulation of SERT by SCAMP2 (60Müller H.K. Wiborg O. Haase J. J. Biol. Chem. 2006; 281: 28901-28909Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Indeed, the authors of the latter study demonstrated that a point mutation in Cys201 abolishes SCAMP2-mediated reduction in the cell surface expression and activity of SERT without disturbing the interactions between the proteins. The precise molecular determinants of NKCC2 interaction with SCAMP2 remain to be resolved. Nevertheless, regardless of the mechanisms involved in such interactions, our data clearly indicate that SCAMP2 decreases exocytotic trafficking of NKCC2, thereby down-regulating the co-transporter abundance in the plasma membrane, an effect for which the SCAMP2 cysteine 201 residue is required.Our findings implicate a role for SCAMP2 in the regulation of NKCC2 trafficking. However, very few if any proteins work in isolation. Appropriately, one may reasonably postulate that SCAMP2 works in concert with other NKCC2-binding proteins to create a synergetic effect on the co-transporter expression. In support of this idea, functional expression in Xenopus laevis oocytes showed that the C-terminal truncated isoform of NKCC2 reduced the co-transporter activity by preventing its arrival to the plasma membrane (68Meade P. Hoover R.S. Plata C. Vázquez N. Bobadilla N.A. Gamba G. Hebert S.C. Am. J. Physiol. Renal. Physiol. 2003; 284: F1145-F1154Crossref PubMed Scopus (59) Google Scholar). Furthermore, we previously obtained evidence that aldolase binding to the proximal region of NKCC2 C terminus reduces NKCC2 surface expression (29Benziane B. Demaretz S. Defontaine N. Zaarour N. Cheval L. Bourgeois S. Klein C. Froissart M. Blanchard A. Paillard M. Gamba G. Houillier P. Laghmani K. J. Biol. Chem. 2007; 282: 33817-33830Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Additionally, the detection of NKCC2 proteins in Rab11-positive compartments, described here for the first time, may open new and important possibilities for studying the endocytic recycling and/or biosynthetic exocytotic membrane traffic of the co-transporter. Rab11, a small GTPase, is known to facilitate the recycling of membrane proteins from recycling endosomes to the plasma membrane (47Jones M.C. Caswell P.T. Norman J.C. Curr. Opin. Cell Biol. 2006; 18: 549-557Crossref PubMed Scopus (233) Google Scholar). In addition to its role in recycling, several reports suggested a role for Rab11 in biosynthetic exocytotic membrane traffic by being directly involved in sorting from the TGN to the plasma membrane (58Satoh A.K. O'Tousa J.E. Ozaki K. Ready D.F. Development. 2005; 132: 1487-1497Crossref PubMed Scopus (216) Google Scholar, 69Lock J.G. Stow J.L. Mol. Biol. Cell. 2005; 16: 1744-1755Crossref PubMed Scopus (296) Google Scholar). More specifically, Rab11 has been shown to remain associated with the exocytic vesicle during fusion with the plasma membrane, indicating that it is directly involved in exocytosis (45Ward E.S. Martinez C. Vaccaro C. Zhou J. Tang Q. Ober R.J. Mol. Biol. Cell. 2005; 16: 2028-2038Crossref PubMed Scopus (150) Google Scholar). Interestingly, a recent study demonstrated that Rab11 recycling of aquaporin 2 is an integral part of cAMP/vasopressin-induced trafficking of the channel to the plasma membrane (70Takata K. Matsuzaki T. Tajika Y. Ablimit A. Hasegawa T. Histochem. Cell Biol. 2008; 130: 197-209Crossref PubMed Scopus (93) Google Scholar). Based upon the above findings, it is conceivable that, in the basal state, NKCC2 accesses the cell surface from the recycling endosomes via a Rab11-dependent pathway. According to this view, SCAMP2 would regulate NKCC2 transit through the recycling endosomes and would inhibit exocytosis to the apical membrane by interfering with Rab11-dependent recycling and/or biosynthetic exocytotic membrane traffic. Moreover, similar to AQP2, in vivo and in vitro studies provided compelling evidence that cAMP/vasopressin induces the shuttling of NKCC2-containing vesicles to the cell membrane, thus leading to an increase in the surface expression of NKCC2 and its activity (19Giménez I. Forbush B. J. Biol. Chem. 2003; 278: 26946-26951Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 20Ortiz P.A. Am. J. Physiol. Renal. Physiol. 2006; 290: F608-F616Crossref PubMed Scopus (99) Google Scholar, 21Caceres P.S. Ares G.R. Ortiz P.A. J. Biol. Chem. 2009; 284: 24965-24971Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). This effect is mediated by enhanced exocytic insertion of the co-transporter into the apical membrane of TAL cells. Hence, it is tempting to speculate that the SCAMP2/Rab11-dependent pathway is also an integral part in the regulation of NKCC2 by cAMP/vasopressin. Consistent with this hypothesis, previous reports documented that NKCC2 (71Welker P. Böhlick A. Mutig K. Salanova M. Kahl T. Schlüter H. Blottner D. Ponce-Coria J. Gamba G. Bachmann S. Am. J. Physiol. Renal. Physiol. 2008; 295: F789-F802Crossref PubMed Scopus (64) Google Scholar) and SCAMP2 (60Müller H.K. Wiborg O. Haase J. J. Biol. Chem. 2006; 281: 28901-28909Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar) proteins distribute in lipid rafts and that lipid rafts mediate cAMP/vasopressin-induced trafficking of the co-transporter (71Welker P. Böhlick A. Mutig K. Salanova M. Kahl T. Schlüter H. Blottner D. Ponce-Coria J. Gamba G. Bachmann S. Am. J. Physiol. Renal. Physiol. 2008; 295: F789-F802Crossref PubMed Scopus (64) Google Scholar). Obviously, these issues need to be examined directly.In summary, we found that NKCC2 interacts specifically with SCAMP2, a post-Golgi-associated protein that is known to be involved in the regulation of vesicle transport. The interaction was confirmed by co-immunoprecipitation in renal cells. Co-immunofluorescence and biotinylation assays revealed that SCAMP2 interaction decreases NKCC2 exocytosis, therefore leading to retention and accumulation of the co-transporter in the cytoplasm. Identifying proteins that interact with kidney transporters and thereby regulate their expression is important to understand their differential physiological functions. To the best of our knowledge, this is the first study identifying a protein partner of NKCC2 that plays a role in the co-transporter exocytotic trafficking. Consequently, these findings may open up new avenues in studying the regulation of the spatial distribution of kidney transporters in general and in particular of NKCC2 and NCC, proteins necessary for normal blood pressure homeostasis. IntroductionNKCC2 is an Na-K-2Cl co-transporter protein expressed exclusively in the mammalian kidney, where it provides the major route for sodium/chloride transport across the apical plasma membrane of the thick ascending limb (1Russell J.M. Physiol. Rev. 2000; 80: 211-276Crossref PubMed Scopus (728) Google Scholar). NKCC2 is therefore a pivotal protein in renal function, and exquisitely tight regulation of the apical co-transporter activity is paramount for maintaining extracellular fluid volume and acid-base homeostasis (2Gamba G. Physiol. Rev. 2005; 85: 423-493Crossref PubMed Scopus (615) Google Scholar). It is the target of loop-diuretics extensively used in the treatment of edematous states and hypertension (3Gamba G. Kidney Int. 1999; 56: 1606-1622Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Furthermore, recent work implicates NKCC2 in the dysfunction of blood pressure regulation, raising new hypotheses regarding the underlying mechanisms behind some types of essential hypertension (4Ashton N. J. Hypertens. 2006; 24: 2355-2356Crossref PubMed Scopus (1) Google Scholar, 5Richardson C. Alessi D.R. J. Cell Sci. 2008; 121: 3293-3304Crossref PubMed Scopus (224) Google Scholar, 6Gamba G. Am. J. Physiol. Renal. Physiol. 2005; 288: F245-F252Crossref PubMed Scopus (104) Google Scholar, 7Flatman P.W. Curr. Opin. Nephrol. Hypertens. 2008; 17: 186-192Crossref PubMed Scopus (51) Google Scholar). Most importantly, inactivating mutations of the NKCC2 gene in humans causes Bartter syndrome type 1, a life-threatening kidney disease (8Simon D.B. Lifton R.P. Am. J. Physiol. 1996; 271: F961-F966PubMed Google Scholar). However, despite this importance, little is known about NKCC2 regulation in renal cells, mainly because of the difficulty in expressing the co-transporter protein in mammalian cells (9Mount D.B. Am. J. Physiol. Renal. Physiol. 2006; 290: F606-F607Crossref PubMed Scopus (10) Google Scholar). As a consequence, although several studies have addressed various aspects of NKCC2 regulation (2Gamba G. Physiol. Rev. 2005; 85: 423-493Crossref PubMed Scopus (615) Google Scholar, 7Flatman P.W. Curr. Opin. Nephrol. Hypertens. 2008; 17: 186-192Crossref PubMed Scopus (51) Google Scholar, 9Mount D.B. Am. J. Physiol. Renal. Physiol. 2006; 290: F606-F607Crossref PubMed Scopus (10) Google Scholar, 10Carmosino M. Giménez I. Caplan M. Forbush B. Mol. Biol. Cell. 2008; 19: 4341-4351Crossref PubMed Scopus (66) Google Scholar, 11Zaarour N. Demaretz S. Defontaine N. Mordasini D. Laghmani K. J. Biol. Chem. 2009; 284: 21752-21764Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 12Hannemann A. Christie J.K. Flatman P.W. J. Biol. Chem. 2009; 284: 35348-35358Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar), our knowledge of the molecular mechanisms underlying membrane trafficking of NKCC2 proteins in mammalian cells, in particular its regulation by protein-protein interactions, remain poor. Identifying and functionally characterizing a key and/or global protein interaction(s) and pathway(s) involved in the regulation of NKCC2 trafficking is important to understand its different physiological functions and dysfunctions.NKCC2 belongs to the cation-chloride co-transporter (CCC) 3The abbreviations used are: CCC, cation-chloride co-transporter; TAL, thick ascending limb; MTAL, medullary TAL; CTAL, cortical TAL; EGFP, enhanced GFP; OKP, opossum kidney; aa, amino acid(s); NCC, NaCl co-transporter; HEK, human embryonic kidney; NHS, N-hydroxysuccinimide; NHE, Na+-H+ exchanger; MesNA, sodium 2-mercaptoethane sulfonate; TGN, trans-Golgi network. family, which comprises two principal branches of membrane proteins (1Russell J.M. Physiol. Rev. 2000; 80: 211-276Crossref PubMed Scopus (728) Google Scholar). One branch includes the Na+-dependent chloride co-transporters composed of the Na+-K+-2Cl− co-transporters (NKCC1 and NKCC2) and the Na+-Cl− co-transporter (NCC). The second branch includes the Na+-independent K+-Cl− co-transporters. Members within this family are very homologous to one another, possessing 12 transmembrane-spanning domains, an N terminus of variable length, and a long cytoplasmic C terminus (2Gamba G. Physiol. Rev. 2005; 85: 423-493Crossref PubMed Scopus (615) Google Scholar). Given that the C-terminal domain of NKCC2 is the predominant cytoplasmic region, it is likely to be a major factor in the trafficking of the NKCC2 protein. In support of this notion, we recently demonstrated that a highly conserved motif at the COOH terminus dictates endoplasmic reticulum exit and cell surface expression of NKCC2 (11Zaarour N. Demaretz S. Defontaine N. Mordasini D. Laghmani K. J. Biol. Chem. 2009; 284: 21752-21764Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Moreover, there have been several reports demonstrating that the trafficking to the apical membrane of several ion transport systems depend upon protein-protein interactions involving their extreme C terminus (13Caplan M.J. Am. J. Physiol. 1997; 272: F425-F429Crossref PubMed Google Scholar). For instance, deleting the last three residues of the cystic fibrosis transmembrane regulator chloride channel C terminus prevents its interactions with CAL, CAP70, and NHERF proteins and results in aberrant basolateral accumulation of the mutant protein (14Cheng J. Moyer B.D. Milewski M. Loffing J. Ikeda M. Mickle J.E. Cutting G.R. Li M. Stanton B.A. Guggino W.B. J. Biol. Chem. 2002; 277: 3520-3529Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 15Wang S. Raab R.W. Schatz P.J. Guggino W.B. Li M. FEBS Lett. 1998; 427: 103-108Crossref PubMed Scopus (249) Google Scholar, 16Wang S. Yue H. Derin R.B. Guggino W.B. Li M. Cell. 2000; 103: 169-179Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar). The interaction of NHERF proteins with the NHE-3 C terminus regulates the trafficking of the exchanger protein to the apical surface of proximal tubule cells (17Donowitz M. Cha B. Zachos N.C. Brett C.L. Sharma A. Tse C.M. Li X. J. Physiol. 2005; 567: 3-11Crossref PubMed Scopus (182) Google Scholar). PDZ interactions mediated through the C-terminal tail of the Na-Pi co-transporter control its correct targeting to the renal proximal tubular brush border (18Hernando N. Déliot N. Gisler S.M. Lederer E. Weinman E.J. Biber J. Murer H. Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 11957-11962Crossref PubMed Scopus (162) Google Scholar). Similar to NHE-3, cystic fibrosis transmembrane regulator chloride channel, and Na-Pi, NKCC2 surface expression is also subject to regulation by intracellular protein trafficking (19Giménez I. Forbush B. J. Biol. Chem. 2003; 278: 26946-26951Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 20Ortiz P.A. Am. J. Physiol. Renal. Physiol. 2006; 290: F608-F616Crossref PubMed Scopus (99) Google Scholar, 21Caceres P.S. Ares G.R. Ortiz P.A. J. Biol. Chem. 2009; 284: 24965-24971Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). However, very little is known about the protein-binding partners that control membrane sorting of NKCC2. More specifically, the identities of the protein partners of NKCC2 that orchestrate its exocytic insertion, endocytic retrieval, and recycling to and from the plasma membrane are scarcely known. Accordingly, identifying proteins that interact with the C-terminal tail of NKCC2 should help to further determine the mechanisms of regulated NKCC2 trafficking. In this report, we describe a novel protein-protein interaction between the C-terminal tail of NKCC2 and SCAMP2. SCAMP2 belongs to a family of evolutionarily conserved tetraspanning integral membrane proteins (22Hubbard C. Singleton D. Rauch M. Jayasinghe S. Cafiso D. Castle D. Mol. Biol. Cell. 2000; 11: 2933-2947Crossref PubMed Scopus (42) Google Scholar). To date, five isoforms of SCAMP (SCAMP1 to -5) have been identified in mammals and have been shown to be predominantly associated with recycling rather than degradation pathways (23Castle A. Castle D. J. Cell Sci. 2005; 118: 3769-3780Crossref PubMed Scopus (52) Google Scholar, 24Han C. Chen T. Yang M. Li N. Liu H. Cao X. J. Immunol. 2009; 182: 2986-2996Crossref PubMed Scopus (34) Google Scholar, 25Liao H. Zhang J. Shestopal S. Szabo G. Castle A. Castle D. Am. J. Physiol. Cell Physiol. 2008; 294: C797-C809Crossref PubMed Scopus (20) Google Scholar, 26Fernández-Chacón R. Achiriloaie M. Janz R. Albanesi J.P. Südhof T.C. J. Biol. Chem. 2000; 275: 12752-12756Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Indeed, SCAMPs are found mainly in the trans-Golgi network (TGN) and recycling endosomes and have been shown to play a role in endocytosis (26Fernández-Chacón R. Achiriloaie M. Janz R. Albanesi J.P. Südhof T.C. J. Biol. Chem. 2000; 275: 12752-12756Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar) and exocytosis (27Guo Z. Liu L. Cafiso D. Castle D. J. Biol. Chem. 2002; 277: 35357-35363Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 28Liao H. Ellena J. Liu L. Szabo G. Cafiso D. Castle D. Biochemistry. 2007; 46: 10909-10920Crossref PubMed Scopus (32) Google Scholar). Here, we show that SCAMP2 regulates the cell surface targeting of NKCC2 by controlling its exocytosis, revealing therefore a novel regulatory mechanism governing the trafficking of the co-transporter to the apical membrane.Keywords:
Cell membrane
Immunoprecipitation
Internalization
Transport protein
Immunoprecipitation
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Immunoprecipitation
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Molecules travel through the yeast endocytic pathway from the cell surface to the lysosome-like vacuole by passing through two sequential intermediates. Immunofluorescent detection of an endocytosed pheromone receptor was used to morphologically identify these intermediates, the early and late endosomes. The early endosome is a peripheral organelle that is heterogeneous in appearance, whereas the late endosome is a large perivacuolar compartment that corresponds to the prevacuolar compartment previously shown to be an endocytic intermediate. We demonstrate that inhibiting transport through the early secretory pathway in sec mutants quickly impedes transport from the early endosome. Treatment of sensitive cells with brefeldin A also blocks transport from this compartment. We provide evidence that Sec18p/N-ethylmaleimide-sensitive fusion protein, a protein required for membrane fusion, is directly required in vivo for forward transport early in the endocytic pathway. Inhibiting protein synthesis does not affect transport from the early endosome but causes endocytosed proteins to accumulate in the late endosome. As newly synthesized proteins and the late steps of secretion are not required for early to late endosome transport, but endoplasmic reticulum through Golgi traffic is, we propose that efficient forward transport in the early endocytic pathway requires delivery of lipid from secretory organelles to endosomes.
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Immunoprecipitation (IP) is a method to pull down a protein out of solution using an antibody that specifically binds to that particular protein. Immunoprecipitation is a powerful technique to isolate and concentrate a particular protein from a sample containing many thousands of different proteins, to test protein-protein interactions, and to pull multiple members of a complex out of solution by latching onto one member with an antibody. This protocol describes a general immunoprecipitation strategy using cell cultures as starting material.
Immunoprecipitation
Protein detection
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One of the most commonly used methods for determining whether two proteins can interact is co-immunoprecipitation. Co-immunoprecipitation relies on the ability of an antibody to stably and specifically bind complexes containing a bait protein. The antibody provides a means of immobilizing these complexes on a solid matrix, which in the protocol presented here is accomplished through interaction with Protein A, so that irrelevant proteins can be washed away. The presence of target proteins in the bait complexes is determined by Western blot. Because of the biochemical diversity of protein-protein interactions, it is not possible to describe a single set of conditions that will work for every immunoprecipitation experiment. Instead, the goal of this chapter is to provide practical starting conditions for co-immunoprecipitation assays and to describe potential modifications to the procedure so that conditions can be optimized.
Immunoprecipitation
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GLUT4
Cell membrane
Transport protein
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Immunoprecipitation
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Cell membrane
Bulk endocytosis
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Immunoprecipitation
Protein detection
Chromatin immunoprecipitation
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Immunoprecipitation
Chromatin immunoprecipitation
Cleavage (geology)
Precipitin
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