Within any given cell many G protein-coupled receptors are expressed in the presence of multiple G proteins, yet most receptors couple to a specific subset of G proteins to elicit their programmed response. Numerous studies demonstrate that the carboxyl-terminal five amino acids of the Gα subunits are a major determinant of specificity, however the receptor determinants of specificity are less clear. We have used a collection of 133 functional mutants of the C5a receptor obtained in a mutagenesis screen targeting the intracellular loops and the carboxyl terminus (Matsumoto, M. L., Narzinski, K., Kiser, P. D., Nikiforovich, G. V., and Baranski, T. J. (2007) J. Biol. Chem. 282, 3105–3121) to investigate how specificity is encoded. Each mutant, originally selected for its ability to signal through a nearly full-length Gαi in yeast, was tested to see whether it could activate three versions of chimeric Gα subunits consisting of Gpa1 fused to the carboxyl-terminal five amino acids of Gαi, Gαq, or Gαs in yeast. Surprisingly the carboxyl-terminal tail of the C5a receptor is the most important specificity determinant in that nearly all mutants in this region showed a gain in coupling to Gαq and/or Gαs. More than half of the receptors mutated in the second intracellular loop also demonstrated broadened G protein coupling. Given a lack of selective advantage for this broadened signaling in the initial screen, we propose a model in which the carboxyl-terminal tail acts together with the intracellular loops to generate a specificity filter for receptor-G protein interactions that functions primarily to restrict access of incorrect G proteins to the receptor. Within any given cell many G protein-coupled receptors are expressed in the presence of multiple G proteins, yet most receptors couple to a specific subset of G proteins to elicit their programmed response. Numerous studies demonstrate that the carboxyl-terminal five amino acids of the Gα subunits are a major determinant of specificity, however the receptor determinants of specificity are less clear. We have used a collection of 133 functional mutants of the C5a receptor obtained in a mutagenesis screen targeting the intracellular loops and the carboxyl terminus (Matsumoto, M. L., Narzinski, K., Kiser, P. D., Nikiforovich, G. V., and Baranski, T. J. (2007) J. Biol. Chem. 282, 3105–3121) to investigate how specificity is encoded. Each mutant, originally selected for its ability to signal through a nearly full-length Gαi in yeast, was tested to see whether it could activate three versions of chimeric Gα subunits consisting of Gpa1 fused to the carboxyl-terminal five amino acids of Gαi, Gαq, or Gαs in yeast. Surprisingly the carboxyl-terminal tail of the C5a receptor is the most important specificity determinant in that nearly all mutants in this region showed a gain in coupling to Gαq and/or Gαs. More than half of the receptors mutated in the second intracellular loop also demonstrated broadened G protein coupling. Given a lack of selective advantage for this broadened signaling in the initial screen, we propose a model in which the carboxyl-terminal tail acts together with the intracellular loops to generate a specificity filter for receptor-G protein interactions that functions primarily to restrict access of incorrect G proteins to the receptor. G protein-coupled receptors (GPCRs) 2The abbreviations used are: GPCR, G protein-coupled receptor; 3AT, 3-aminotriazole; C5a, complement factor 5a; C5aR, complement factor 5a receptor; CT, carboxyl terminus; CT1, first half of the carboxyl terminus; CT2, second half of the carboxyl terminus; CT2stop323, truncated C5aR with a stop codon at position 323; Endo-Hf, endo-β-N-acetylglucosaminidase H; ER, endoplasmic reticulum; IC1, intracellular loop 1; IC2, intracellular loop 2; IC3, intracellular loop 3; IP3, inositol 1,4,5-triphosphate; r.m.s., root mean square deviation; RSM, random saturation mutagenesis; W5Cha, hexapeptide agonist of the C5a receptor. are seven transmembrane-spanning receptors that constitute one of the largest families of proteins, encoded by more than 1% of human genes (1Miller K.J. Murphy B.J. Pelleymounter M.A. Curr. Drug Targets CNS Neurol. Disord. 2004; 3: 357-377Crossref PubMed Scopus (15) Google Scholar). GPCRs are necessary for transmission of signals across cell membranes to orchestrate essential processes ranging from cell fusion in yeast to the ability to mobilize leukocytes to sites of infection in humans. The central role of GPCRs in nearly all physiologic processes is underscored by the fact that they are targets of ∼30% of all available pharmaceutical drugs (1Miller K.J. Murphy B.J. Pelleymounter M.A. Curr. Drug Targets CNS Neurol. Disord. 2004; 3: 357-377Crossref PubMed Scopus (15) Google Scholar). GPCRs are named for their ability to bind and activate heterotrimeric G proteins made up of a Gα, Gβ, and Gγ subunit. Activation occurs by transmitting a conformational change in the receptor, triggered by ligand binding, to the G protein. This catalyzes the release of GDP from Gα allowing GTP to bind and trigger dissociation of this subunit from Gβγ. Gα and Gβγ are then free to signal to their downstream targets to begin the signaling response. In humans there are 17 Gα, 5 Gβ, and 12 Gγ subunits (2Wettschureck N. Offermanns S. Physiol. Rev. 2005; 85: 1159-1204Crossref PubMed Scopus (868) Google Scholar). The Gα subunits are classified by the type of downstream effectors that they signal to and the responses they evoke and can divided into four families: Gαs, Gαi/o, Gαq/11, and Gα12/13 (3Pierce K.L. Premont R.T. Lefkowitz R.J. Nat. Rev. Mol. Cell. Biol. 2002; 3: 639-650Crossref PubMed Scopus (2163) Google Scholar, 4Cabrera-Vera T.M. Vanhauwe J. Thomas T.O. Medkova M. Preininger A. Mazzoni M.R. Hamm H.E. Endocr. Rev. 2003; 24: 765-781Crossref PubMed Scopus (524) Google Scholar). Gαs subunits stimulate adenylyl cyclase, whereas Gαi subunits inhibit adenylyl cyclase. Gαq subunits signal through phospholipase C, and Gα12/13 activates various Rho guanine nucleotide exchange factors. Given that a single cell may express many different G proteins and GPCRs, the G proteins to which the receptors couple to must be tightly controlled in order for a GPCR to activate the correct downstream response. Each GPCR signals through only a subset of Gα subunits creating a specificity profile for the receptor. For example, the GPCR that we study, the complement-derived C5a receptor (C5aR), couples to Gαi and the promiscuous Gα16 subunit of the Gαq family, but not Gαq itself, nor Gαs. Despite the ability of many receptors to couple to the same G protein, there is little homology among their intracellular loops, making it difficult to predict which G protein(s) a receptor will signal through based upon primary sequence alone. The nature of G protein specificity has been previously investigated using chimeric GPCRs created by swapping intracellular loop regions between receptors that couple to different Gα subunits. These experiments demonstrate that the second (5Liu J. Wess J. J. 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Specific points of contact between the G protein and receptor have been identified, and these include the amino terminus (residues 8–23), the α4–β6 loop (residues 311–328), and the last 11 amino acids (residues 340–450) of the Gα subunit (25Hamm H.E. Deretic D. Arendt A. Hargrave P.A. Koenig B. Hofmann K.P. Science. 1988; 241: 832-835Crossref PubMed Scopus (421) Google Scholar, 26Onrust R. Herzmark P. Chi P. Garcia P.D. Lichtarge O. Kingsley C. Bourne H.R. Science. 1997; 275: 381-384Crossref PubMed Scopus (198) Google Scholar, 27Cai K. Itoh Y. Khorana H.G. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4877-4882Crossref PubMed Scopus (138) Google Scholar, 28Itoh Y. Cai K. Khorana H.G. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4883-4887Crossref PubMed Scopus (104) Google Scholar). The receptor has also been demonstrated to interact with the carboxyl-terminal tail of the Gγ subunit (29Kisselev O. Gautam N. J. Biol. 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Yeast. 2000; 16: 11-22Crossref PubMed Scopus (152) Google Scholar). We have used a collection of 133 functional C5aR mutants obtained by random saturation mutagenesis (RSM) of each intracellular region (Matsumoto et al., Ref. 67Matsumoto M.L. Narzinski K. Kiser P.D. Nikiforovich G.V. Baranski T.J. J. Biol. Chem. 2007; 282: 3105-3121Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar) to identify residues that interact with the last five amino acids of Gα to confer specificity. We found that all of the carboxyl-terminal mutants and more than half of the IC2 mutants demonstrated a gain in coupling to Gαq and/or Gαs chimeras. Based on the wide variety of mutations and truncations observed that allowed coupling to multiple chimeric Gα subunits, we propose that the carboxyl terminus and the intracellular loops create a specificity filter that acts to restrict access of incorrect G proteins to the receptor. Yeast Strains and Plasmids—Yeast strains BY1142 (Gαi3), BY1173 (Gαi3), BY1172 (Gαq), BY1401 (Gαs), and BY1404 (Gαs) have been previously described (33Brown A.J. Dyos S.L. Whiteway M.S. White J.H. Watson M.A. Marzioch M. Clare J.J. Cousens D.J. Paddon C. Plumpton C. Romanos M.A. Dowell S.J. Yeast. 2000; 16: 11-22Crossref PubMed Scopus (152) Google Scholar, 34Baranski T.J. Herzmark P. Lichtarge O. Gerber B.O. Trueheart J. Meng E.C. Iiri T. Sheikh S.P. Bourne H.R. J. Biol. Chem. 1999; 274: 15757-15765Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). Briefly, BY1142 has the genotype MATα far1Δ1442 tbt1-1 fus1Δ::PFUS1-HIS3 can1 ste14::trp1::LYS2 ste3Δ1156 gpa1 (41)-Gαi3 lys2 ura3 leu2 trp1 his3 ade2. BY1142 contains a fusion of the amino-terminal 41 amino acids of the yeast Gα protein, Gpa1, followed by residues 34–354 of the human Gαi3. The BY1143 strain was created by transforming the BY1142 strain with a URA3 plasmid encoding C5a (pBN444) as previously described (34Baranski T.J. Herzmark P. Lichtarge O. Gerber B.O. Trueheart J. Meng E.C. Iiri T. Sheikh S.P. Bourne H.R. J. Biol. Chem. 1999; 274: 15757-15765Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 35Klco J.M. Wiegand C.B. Narzinski K. Baranski T.J. Nat. Struct. Mol. Biol. 2005; 12: 320-326Crossref PubMed Scopus (139) Google Scholar). BY1173 has the genotype MATa ura3 leu2 trp1 his3 can1 gpa1Δ::ade2Δ::3XHA far1Δ::ura3Δ fus1Δ::PFUS1-HIS3 LEU2::PFUS1-lacZ sst2Δ::ura3Δ ste2Δ::G418R trp1::GPA1/Gαi3. BY1172 has the genotype MATa ura3 leu2 trp1 his3 can1 gpa1Δ:: ade2Δ::3XHA far1Δ::ura3Δ fus1Δ::PFUS1-HIS3 LEU2::PFUS1-lacZ sst2Δ::ura3Δ ste2Δ::G418R trp1::GPA1/Gαq. BY1401 has the genotype MATa ura3 leu2 trp1 his3 can1 gpa1Δ::ade2Δ::3XHA far1Δ::ura3Δ fus1Δ::PFUS1-HIS sst2Δ::ura Δtrp1::GPA1/Gαs. BY1404 has the genotype MATa ura3 leu2 trp1 his3 can1 gpa1Δ::ade2Δ::3XHA far1Δ::ura3Δ fus1Δ::PFUS1-HIS3 LEU2::PFUS1-lacZ sst2Δ::ura3Δ ste2ΔG418R lys2ΔΔtrp1::GPA1/Gαs. BY1173, BY1172, BY1401, and BY1404 contain a fusion of amino acids 1–467 of the yeast Gα protein, Gpa1, followed by the last 5 amino acids of human Gαi3, Gαq, Gαs, and Gαs, respectively. These strains were transformed with either a URA3 plasmid expressing C5a (pBN444) or an empty URA3 vector (pBN443) and an ADE2 plasmid expressing the C5aR that were previously described (34Baranski T.J. Herzmark P. Lichtarge O. Gerber B.O. Trueheart J. Meng E.C. Iiri T. Sheikh S.P. Bourne H.R. J. Biol. Chem. 1999; 274: 15757-15765Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 35Klco J.M. Wiegand C.B. Narzinski K. Baranski T.J. Nat. Struct. Mol. Biol. 2005; 12: 320-326Crossref PubMed Scopus (139) Google Scholar). Activation of the C5aR expressed from a plasmid in all strains used leads to signaling through the mitogen-activated protein kinase cascade and expression of the PFUS1-HIS3 reporter gene, allowing the yeast to grow in the absence of histidine. In addition, BY1173, BY1172, and BY1404 contain a PFUS1-β-galactosidase reporter gene. The CT2stop323 truncation was generated by mutating codon 323 of the C5aR to a stop codon using Pfu turbo mutagenesis (Stratagene). The RGS4 plasmid was a gift from Dr. Maurine Linder. Mutants M1–M4 were made by designing complementary oligonucleotides encoding the desired mutation(s), and a two-step PCR strategy was used to introduce the mutation(s) into the wild-type C5aR coding sequence in a pcDNA3.1(+) (Invitrogen) mammalian expression vector. All mutations were verified by sequencing at the Washington University Protein and Nucleic Acid Chemistry Laboratory. Yeast Transformation and Receptor Signaling Assays—Yeast transformations were done according to standard lithium acetate protocols. Relative signaling abilities of mutant receptors were assayed by restreaking three transformants of each mutant onto histidine-deficient medium containing varying amounts of 3-amino-1,2,4-triazole (3AT) (Sigma) (0, 1, 5, 10, 20, and 50 mm). Signaling levels were compared with wild-type C5aR expressed from an ADE2 plasmid, pBN482, and a non-functional mutant C5aR containing a stop codon in transmembrane helix 3, pBN483, which were previously described (34Baranski T.J. Herzmark P. Lichtarge O. Gerber B.O. Trueheart J. Meng E.C. Iiri T. Sheikh S.P. Bourne H.R. J. Biol. Chem. 1999; 274: 15757-15765Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 36Geva A. Lassere T.B. Lichtarge O. Pollitt S.K. Baranski T.J. J. Biol. Chem. 2000; 275: 35393-35401Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Growth in the absence of histidine was inferred to be dependent on C5aR signaling based on colony color (red colonies lack the C5aR ADE2 plasmid). β-Galactosidase assays were done by treating BY1173, BY1172, and BY1404 transformed with pBN482 with a range of the C5aR hexapeptide agonist W5Cha (GenScript) from 10-10 m to 10-5 m, and the assay was carried out as previously described (35Klco J.M. Wiegand C.B. Narzinski K. Baranski T.J. Nat. Struct. Mol. Biol. 2005; 12: 320-326Crossref PubMed Scopus (139) Google Scholar). Endo-β-N-acetylglucosaminidase Treatment and Western Blots —RSM receptors were subcloned into a pIRES vector (Clontech) with Gαq (University of Missouri-Rolla cDNA Resource Center) and transiently transfected into HEK293 cells by standard calcium phosphate methods. Cells were lysed 2 days after transfection in 250 μl of a 1 × sample buffer (50 mm Tris-Cl, pH 6.8, 2% SDS, 10% glycerol) supplemented with 2% β-mercaptoethanol, 1 μg/ml leupeptin, 1 μg/ml aprotinin, and 500 μm phenylmethylsulfonyl fluoride by shearing through a 27G1/2 syringe. Lysates were heated for 5 min at 50 °C. 27 μl of each lysate was treated with 1000 units of endo-β-N-acetylglucosaminidase H-maltose-binding protein fusion (Endo-Hf, New England Biolabs) at 37 °C for 3 h. Samples were heated for 5 min at 50 °C, resolved on a 12% SDS-PAGE gel, transferred to polyvinylidene difluoride, and immunoblotted with a rabbit polyclonal anti-C5aR antibody raised against residues 9–29 of the amino terminus. Western blots of yeast lysates were done as previously described. 3M. L. Matsumoto, K. Narzinski, P. D. Kiser, G. V. Nikiforovich, and T. J. Baranski, unpublished observation. Inositol 1,4,5-Triphosphate Accumulation—For Gαq signaling, the RSM receptors with Gαq in the pIRES vector were transiently transfected into HEK293 cells by standard calcium phosphate methods. The m1 muscarinic acetylcholine receptor (University of Missouri-Rolla cDNA Resource Center) was used as a positive control. For Gα16 signaling, RSM receptors and Gα16 were subcloned into pcDNA3.1(+) (Invitrogen) and transiently co-transfected into HEK293 cells. IP3 levels were measured as previously described (35Klco J.M. Wiegand C.B. Narzinski K. Baranski T.J. Nat. Struct. Mol. Biol. 2005; 12: 320-326Crossref PubMed Scopus (139) Google Scholar) where cells expressing the C5aR were treated with 1 μm W5Cha (GenScript) and cells expressing the m1 muscarinic acetylcholine receptor were treated with 10-4 m carbachol (Sigma). Molecular Modeling—Molecular modeling procedures for restoring low energy backbone conformations of the intracellular loops in the mutant C5a receptors were exactly as those described earlier for the WT, including employment of the ECEPP/2 force field, and mounting the loops on the three-dimensional structure of the transmembrane region of C5aR (67Matsumoto M.L. Narzinski K. Kiser P.D. Nikiforovich G.V. Baranski T.J. J. Biol. Chem. 2007; 282: 3105-3121Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). All possible low energy conformations for IC1 (fragment 63–71), IC2-(138–150), IC3-(224–236), and the C-terminal fragment 300–310, as well as for the “package” of IC1+IC2+IC3+fragment 300–310 were determined by subsequent application of geometrical sampling followed by energy minimization (67Matsumoto M.L. Narzinski K. Kiser P.D. Nikiforovich G.V. Baranski T.J. J. Biol. Chem. 2007; 282: 3105-3121Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). Final selection of low energy conformations for the package was based on the energy cut-off of 30 kcal/mol and yielded 56 low energy structures for WT (see Matsumoto et al., Ref. 67Matsumoto M.L. Narzinski K. Kiser P.D. Nikiforovich G.V. Baranski T.J. J. Biol. Chem. 2007; 282: 3105-3121Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar), 22 for R35, 27 for R91, 33 for R89, 35 for R40, 28 for R58, 44 for R111, 50 for R52, 45 for M1, 47 for M2, 46 for M3, and 28 for M4. The obtained low energy structures for different mutant receptors were compared with each other by overlapping spatial positions of residues comprising the stems of transmembrane helices (residues 135–138 and 150–153 for IC2) and calculating the so-called “global” r.m.s. values for all heavy backbone atoms of the IC2 loop (residues 139–149). Determining G Protein Coupling of Intracellular Loop Mutants—The 133 functional intracellular loop C5aR mutants were originally selected by their ability to signal in the yeast strain BY1143, which contains a chimera of residues 1–41 of the yeast Gα, Gpa1, followed by residues 34–354 of human Gαi3 (67Matsumoto M.L. Narzinski K. Kiser P.D. Nikiforovich G.V. Baranski T.J. J. Biol. Chem. 2007; 282: 3105-3121Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). All of these mutants were then tested for signaling in three different strains carrying chimeras of amino acids 1–467 of Gpa1 followed by the last five amino acids of human Gαi3 (strain BY1173), Gαq (BY1172), or Gαs (BY1401) (33Brown A.J. Dyos S.L. Whiteway M.S. White J.H. Watson M.A. Marzioch M. Clare J.J. Cousens D.J. Paddon C. Plumpton C. Romanos M.A. Dowell S.J. Yeast. 2000; 16: 11-22Crossref PubMed Scopus (152) Google Scholar) to elucidate receptor determinants of specificity (Fig. 1a). The ability to express a single G protein chimera of interest in the presence of a single human GPCR provides a powerful tool for studying G protein-coupling specificity. In the isolated environment of the yeast cell, a single human GPCR can be studied in the absence of other receptors competing for G protein binding, and the readout of receptor activation is clear, because only a single G protein chimera is expressed. All yeast strains used in this study have been engineered so that receptor signaling leads to activation of the yeast-mating pathway resulting in expression of a PFUS1-HIS3 or PFUS1-β-galactosidase reporter gene (33Brown A.J. Dyos S.L. Whiteway M.S. White J.H. Watson M.A. Marzioch M. Clare J.J. Cousens D.J. Paddon C. Plumpton C. Romanos M.A. Dowell S.J. Yeast. 2000; 16: 11-22Crossref PubMed Scopus (152) Google Scholar). Thus, if a receptor activates the particular G protein expressed, it confers the ability of the yeast to grow on histidine-deficient medium or express the β-galactosidase enzyme. To quantify the relative signaling strength of mutant receptors a β-galactosidase assay can be performed or the yeast can be grown in the presence of increasing amounts of 3AT, a competitive inhibitor of His3. The wild-type C5aR receptor was tested for signaling in the BY1173 (Gαi3), BY1172 (Gαq), and BY1404 (Gαs) strains by β-galactosidase assay (Fig. 1b). The wild-type C5aR demonstrated dose-dependent β-galactosidase activity when treated with increasing amounts of the C5aR hexapeptide agonist W5Cha in the presence of the Gαi chimera but not in the presence of the Gαq or Gαs chimeras. This specificity profile was confirmed by the growth assay. BY1173 (Gαi3), BY1172 (Gαq), and BY1401 (Gαs) transformants of wild-type C5aR in the presence of the C5a ligand were assayed for signaling by testing their ability to grow on histidine-deficient medium in the presence of 0, 1, 5, 10, 20, and 50 mm 3AT. The wild-type receptor demonstrated signaling in the presence of up to 50 mm 3AT with the Gαi chimera but showed no signaling with the Gαq or Gαs chimeras (Figs. 2, 3, 4, 5, 6). Thus, the chimeric Gα subunits demonstrate the natural specificity profile of the C5aR in mammalian cells. Each of the 133 receptors in our functional mutant collection were tested for the ability to signal through the chimeric Gα subunits in the presence of the C5a ligand in strains BY1173 (Gαi3), BY1172 (Gαq), and BY1401 (Gαs) using the growth assay. All 133 mutants that were originally selected for signaling in BY1143 signaled as well as the wild-type C5aR in the BY1173 strain (Figs. 2, 3, 4, 5, 6). Thus, there was no difference in the ability of the mutants to couple to either a more human Gαi3-like (BY1143) or a more Gpa1-like Gα subunit (BY1173).FIGURE 3Signaling of IC2 mutants via Gαi, Gαq, and Gαs chimeras. The wild-type sequence of the region targeted for mutagenesis in IC2 is given (top in bold) with the residue numbers marked. The amino acid sequences of the functional mutant receptors obtained (designated R and numbered, left) are indicated (dots, unchanged amino acid compared with wild-type) and annotated as in Fig. 2.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 4Signaling of IC3 mutants via Gαi, Gαq, and Gαs chimeras. The wild-type sequence of the region targeted for mutagenesis in IC3 is given (top in bold) with the residue numbers marked. The amino acid sequences of the functional mutant receptors obtained (designated R and numbered, left) are indicated (dots, unchanged amino acid compared with wild-type) and annotated as in Fig. 2.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 5Signaling of CT1 mutants via Gαi, Gαq, and Gαs chimeras. The wild-type sequence of the region targeted for mutagenesis in CT1 is given (top in bold) with the residue numbers marked. The amino acid sequences of the functional mutant receptors obtained (designated R and numbered, left) are indicated (dots, unchanged amino acid compared with wild-type) and annotated as in Fig. 2. @, stop codon.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 6Signaling of CT2 mutants via Gαi, Gαq, and Gαs chimeras. The wild-type sequence of the region targeted for mutagenesis in CT2 is given (top in bold) with the residue numbers marked. The amino acid sequences of the functional mutant receptors obtained (designated R and numbered, left) are indicated (dots, unchanged amino acid compared with wild-type) and annotated as in Fig. 2. @, stop codon.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Twenty-eight intracellular loop 1 (IC1) mutants were tested, and only nine of these coupled to the Gαq chimera, whereas none of the mutants gained the ability to couple to the Gαs chimera (Fig. 2). Of note, the IC1 mutants that show signaling through the Gαq chimera do not signal as strongly as many of the second intracellular loop (IC2) or CT mutants that showed a gain in coupling to the Gαq chimera (see below). Comparison of the sequences of mutant receptors that are able to couple to the Gαq chimera does not reveal any trend in amino acid substitutions associated with a gain in coupling. Of the 30 IC2 mutant receptors tested, 20 were able to signal through Gαq, and three of these were also able to signal through Gαs (Fig. 3). Analysis of the sequences of mutant IC2 receptors that show broadened coupling reveals that a single point mutation, Q145R, is sufficient to allow signaling through Gαq in yeast (Fig. 3). Comparison of IC2 mutant sequences that couple to Gαq show that nine of these receptors contain the Q145R mutation, whereas this mutation is not observed in any of the receptors that couple exclusively to Gαi. Interestingly, a positive charge at this position is not sufficient to allow Gαq coupling, because several recepto
Background: Patients with acute lymphocytic leukemia (ALL) and those with lymphoblastic lymphoma (LBL) have overlapping clinical and immunophenotypic features and they have been treated with the same or very similar chemotherapy regimens. The goal of this multi-institutional phase II trial was to evaluate the therapeutic efficacy of a short-term, six-drug chemotherapy regimen for adult patients with untreated ALL or LBL.
IgA antibodies have broad potential as a novel therapeutic platform based on their superior receptor-mediated cytotoxic activity, potent neutralization of pathogens, and ability to transcytose across mucosal barriers via polymeric immunoglobulin receptor (pIgR)-mediated transport, compared to traditional IgG-based drugs. However, the transition of IgA into clinical development has been challenged by complex expression and characterization, as well as rapid serum clearance that is thought to be mediated by glycan receptor scavenging of recombinantly produced IgA monomer bearing incompletely sialylated N-linked glycans. Here, we present a comprehensive biochemical, biophysical, and structural characterization of recombinantly produced monomeric, dimeric and polymeric human IgA. We further explore two strategies to overcome the rapid serum clearance of polymeric IgA: removal of all N-linked glycosylation sites creating an aglycosylated polymeric IgA and engineering in FcRn binding with the generation of a polymeric IgG-IgA Fc fusion. While previous reports and the results presented in this study indicate that glycan-mediated clearance plays a major role for monomeric IgA, systemic clearance of polymeric IgA in mice is predominantly controlled by mechanisms other than glycan receptor clearance, such as pIgR-mediated transcytosis. The developed IgA platform now provides the potential to specifically target pIgR expressing tissues, while maintaining low systemic exposure.
The 150-kD oxygen-regulated protein (ORP150) was initially characterized based on its selective expression in astrocytes subjected to oxygen deprivation (Kuwabara, K., M. Matsumoto, J. Ikeda, O. Hori, S. Ogawa, Y. Maeda, K. Kitagawa, N. Imuta, K. Kinoshita, D.M. Stern, et al. 1996. J. Biol. Chem. 279:5025-5032). We have found that exposure of cultured human aortic smooth muscle cells and mononuclear phagocytes (MPs) to hypoxia (pO2 approximately 12-14 torr) induces ORP150 transcripts and production of the antigen, whereas incubation with either hydrogen peroxide, sodium arsenite, heat shock, or 2-deoxyglucose was without effect. Tissue extracts prepared from human atherosclerotic lesions demonstrated expression of ORP150 mRNA and antigen, vs lack of ORP150 in samples from nonatherosclerotic areas. In situ hybridization using ORP150 riboprobes showed the mRNA to be predominantly [correction of predominately] present in macrophages in in atherosclerotic plaques. Furthermore, autoantibody to ORP150 was demonstrated in the serum of patients with severe atherosclerosis, consistent with inducible in vivo expression of ORP150. Introduction of antisense oligonucleotide for ORP150 selectively diminished hypoxia-mediated induction of ORP150 antigen and reduced the viability of hypoxic MPs, especially in the presence of modified (oxidized/acetylated) LDL. In support of a role for ORP150 in the MPs' response to the microenvironment of an atheroma, the presence of oxidized LDL enhanced by approximately 10-fold ORP150 expression in hypoxic cultures. These data indicate that cells of the atherosclerotic vessel wall express ORP150 as part of a protective mechanism, potentially triggered by local hypoxia/hypoxemia and augmented by modified lipoproteins. The presence of antibody to ORP150 in sera of patients with severe atherosclerosis emphasizes the possibility that ORP150 may be a marker of vascular pathology.
A major glycosphingolipid in rat bone marrow cells was purified, and its structure was studied.The glycolipid was found to exhibit blood group B activity by the hemagglutination inhibition test.The structure was determined to be Gal(al-3)Gal(gl-3)GalNAc~l-4)Gal(~l-4)Glc(~l-l)ceramide 2 I Fuca 1 by studies of nuclear magnetic resonance, sequential hydrolysis by exoglycosidases, linkage analysis of methylated sugars by gas chromatography-mass spectrometry, and immunological tests.The blood group B active glycolipid was detected not only in the bone marrow cells but also in spleen, thymus, and rat ascites hepatoma AH 7974F cells.Besides the glycolipid, gangliotriaosylceramide, gangliotetraosylceramide, and fucogangliotetraosylceramide were commonly detected in these cells.The similarity between the glycolipid species on the cell surfaces of the immunocytes and the tumor cells is discussed with the respect to an escape mechanism of the tumor cells from the immunosurveillance system.In previous papers, the presence of asialogangliosides and G M ~~~ in rat bone marrow cells was described together with ganglioside biosynthesis via asialogangliosides in the cells (1, 2).These asialogangliosides have been demonstrated to be cell surface markers useful for discriminating various kinds of cells in the bone marrow using antibodies against the lipids (3).
