The Structural Evolution of a P2Y-like G-protein-coupled Receptor
2003; Elsevier BV; Volume: 278; Issue: 37 Linguagem: Inglês
10.1074/jbc.m303346200
ISSN1083-351X
AutoresAngela Schulz, Torsten Schöneberg,
Tópico(s)Neuroscience and Neuropharmacology Research
ResumoBased on the now available crystallographic data of the G-protein-coupled receptor (GPCR) prototype rhodopsin, many studies have been undertaken to build or verify models of other GPCRs. Here, we mined evolution as an additional source of structural information that may guide GPCR model generation as well as mutagenesis studies. The sequence information of 61 cloned orthologs of a P2Y-like receptor (GPR34) enabled us to identify motifs and residues that are important for maintaining the receptor function. The sequence data were compared with available sequences of 77 rhodopsin orthologs. Under a negative selection mode, only 17% of amino acid residues were preserved during 450 million years of GPR34 evolution. On the contrary, in rhodopsin evolution ∼43% residues were absolutely conserved between fish and mammals. Despite major differences in their structural conservation, a comparison of structural data suggests that the global arrangement of the transmembrane core of GPR34 orthologs is similar to rhodopsin. The evolutionary approach was further applied to functionally analyze the relevance of common scaffold residues and motifs found in most of the rhodopsin-like GPCRs. Our analysis indicates that, in contrast to other GPCRs, maintaining the unique function of rhodopsin requires a more stringent network of relevant intramolecular constrains. Based on the now available crystallographic data of the G-protein-coupled receptor (GPCR) prototype rhodopsin, many studies have been undertaken to build or verify models of other GPCRs. Here, we mined evolution as an additional source of structural information that may guide GPCR model generation as well as mutagenesis studies. The sequence information of 61 cloned orthologs of a P2Y-like receptor (GPR34) enabled us to identify motifs and residues that are important for maintaining the receptor function. The sequence data were compared with available sequences of 77 rhodopsin orthologs. Under a negative selection mode, only 17% of amino acid residues were preserved during 450 million years of GPR34 evolution. On the contrary, in rhodopsin evolution ∼43% residues were absolutely conserved between fish and mammals. Despite major differences in their structural conservation, a comparison of structural data suggests that the global arrangement of the transmembrane core of GPR34 orthologs is similar to rhodopsin. The evolutionary approach was further applied to functionally analyze the relevance of common scaffold residues and motifs found in most of the rhodopsin-like GPCRs. Our analysis indicates that, in contrast to other GPCRs, maintaining the unique function of rhodopsin requires a more stringent network of relevant intramolecular constrains. Among the different families of transmembrane receptors, G-protein-coupled receptors (GPCRs) 1The abbreviations used are: GPCR, G-protein-coupled receptor; dN, non-synonymous substitution; dS, synonymous substitution; EL, extracellular loop; GFP, green fluorescent protein; IL, intracellular loop; IP, inositol phosphate; TMD, transmembrane domain.1The abbreviations used are: GPCR, G-protein-coupled receptor; dN, non-synonymous substitution; dS, synonymous substitution; EL, extracellular loop; GFP, green fluorescent protein; IL, intracellular loop; IP, inositol phosphate; TMD, transmembrane domain. form the largest superfamily. Molecular cloning studies and genome data analyses have revealed ∼1200–1300 members of the GPCR superfamily in mammalian genomes (1Schöneberg T. Schulz A. Gudermann T. Rev. Physiol. Biochem. Pharmacol. 2002; 144: 143-227PubMed Google Scholar). To predict and understand ligand binding and signal transduction as well as the consequences of structural changes (e.g. disease-causing mutations) within a receptor molecule, detailed information about the native receptor structure in its inactive and active conformations is required. Currently, a high-resolution structure is available only for bovine rhodopsin (2Palczewski K. Kumasaka T. Hori T. Behnke C.A. Motoshima H. Fox B.A. Le Trong I. Teller D.C. Okada T. Stenkamp R.E. Yamamoto M. Miyano M. Science. 2000; 289: 739-745Crossref PubMed Scopus (4992) Google Scholar), which now provides the basis to generate models of other GPCRs (3Vaidehi N. Floriano W.B. Trabanino R. Hall S.E. Freddolino P. Choi E.J. Zamanakos G. Goddard III, W.A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 12622-12627Crossref PubMed Scopus (252) Google Scholar). However, recent studies point out that even with a crystal structure in hand, construction of reliable receptor models still requires time-consuming refinements based on data from mutagenesis, cross-linking, and NMR studies (4Archer E. Maigret B. Escrieut C. Pradayrol L. Fourmy D. Trends Pharmacol. Sci. 2003; 24: 36-40Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar).Herein, we mined evolution as an additional source of structural information that may direct GPCR modeling and mutagenesis studies. This idea has been successfully applied in an early stage of GPCR structure/function analysis. The sequences of >200 different members of the GPCR family were used to predict the approximate arrangements of the seven transmembrane helices (5Baldwin J.M. Curr. Opin. Cell Biol. 1994; 6: 180-190Crossref PubMed Scopus (340) Google Scholar). To determine more distinct structural determinants that participate in ligand recognition and signal transduction, sequence analysis has to be focused on a single receptor subtype. The structural diversity of a given receptor among different species is the result of a long evolutionary process characterized by a continuous accumulation of mutations. However, the maintenance of vital functions in an organism strictly requires enough structural conservation to ensure the functionality of the receptor protein. Studying the structural diversity of a single GPCR will help to determine evolutionary preserved elements and may disclose the "spatial freedom" of each amino acid position.For a valid evolutionary analysis, the chosen GPCR should meet the following requirements. First, the GPCR should be "evolutionarily old" to allow for a broad structural variety during evolution. Second, GPCRs with structurally indistinguishable subtypes or pseudogenes should be avoided, because the interpretation of data will be more difficult. Third, the physiological agonist should not be a peptide or a protein because of problems in separating co-evolutionary processes. Fourth, the coding region of the GPCR gene should contain no or only small introns to allow for an easy amplification from genomic DNA.Among the GPCRs of family 1, only a few receptors meet these requirements. The group of ADP receptor-like GPCRs includes P2Y12, P2Y13, GPR105, GPR87, and GPR34. In an initial study we have shown that GPR34 is an evolutionarily old single-copy gene and has clear structural and functional features that allows its differentiation from other members of the group of ADP-like GPCRs (6Schöneberg T. Schulz A. Grosse R. Schade R. Henklein P. Schultz G. Gudermann T. Biochim. Biophys. Acta. 1999; 1446: 57-70Crossref PubMed Scopus (30) Google Scholar). Therefore, GPR34 was chosen to identify structural and functional determinants by an evolutionary approach. Our analysis includes 61 full-length or partial sequences of GPR34 and GPR34-like GPCRs that were cloned from 52 species, including mammals, birds, reptiles, amphibians, teleost fish, and sharks. This large set of sequence information enabled us to identify motifs and residues that are important for maintaining the receptor function. The sequence data were compared with sequences of 77 rhodopsin orthologs. Despite the fact that GPR34 orthologs and vertebrate rhodopsins share less than 7% identical amino acid residues, the gross global structure appears to have been maintained between both receptor groups as judged by analyzing the periodicity of conserved residues and hydrophobicity. During 450 million years of evolution, only 17% of all amino acid residues remained unchanged in GPR34 orthologs. In contrast, in rhodopsin evolution ∼43% of residues were absolutely conserved in fish and mammalian orthologs. This indicates that maintaining the unique function of rhodopsin requires a more stringent network of relevant determinants.EXPERIMENTAL PROCEDURESCloning of GPR34-like and ADP-like Receptor Orthologs and Generation of GPR34 Mutants—To identify GPR34 sequence in other vertebrates, genomic DNA samples were prepared from tissue or peripheral mononuclear blood cells of various species or were kindly provided by several other labs (supplemental Table S1, available in the on-line version of this article). Tissue samples were digested in lysis buffer (50 mm Tris/HCl, 100 mm EDTA, 100 mm NaCl, 1% SDS, and 0.5 mg/ml proteinase K) and incubated at 55 °C for 18 h. DNA was purified by phenol/chloroform extraction and ethanol precipitation. Based on the sequence of the human, mouse, and carp GPR34 sequences (6Schöneberg T. Schulz A. Grosse R. Schade R. Henklein P. Schultz G. Gudermann T. Biochim. Biophys. Acta. 1999; 1446: 57-70Crossref PubMed Scopus (30) Google Scholar), sets of degenerated primer pairs (supplemental Table S3, available in the on-line version of this article) were tested for their ability to amplify GPR34-specific sequences. Standard PCR reaction was performed with Taq polymerase under the following conditions (35 cycles): 1 min at 94 °C; 1 min at 56–62 °C; and 2 min at 72 °C. Specific fragments were directly sequenced and/or subcloned into the pCR2.1-TOPO vector (Invitrogen), and at least three different clones were sequenced.For identification of complete GPR34 open reading frames, a 5′- and 3′-rapid amplification of cDNA ends (RACE) strategy was used. Thus, 1 μg of genomic DNA per reaction was digested with various blunt end cutting enzymes (EcoRV, HincII, MscI, and DraI; New England Biolabs). Then, an amidated oligonucleotide adapter (Marathon cDNA amplification kit, Clontech) was linked to the digested DNA. 5′- and 3′-RACE PCR reactions were performed with the AP1 primer (Clontech) and species-specific primers using the tagged genomic DNA as a template in the Expand™ high fidelity PCR system (Roche Applied Science). PCR reactions were carried out under the following conditions (35 cycles): 1 min at 94 °C; 1 min at 62 °C; and 3 min at 68 °C. After treatment with Taq polymerase for 10 min at 72 °C, PCR products were subcloned into the pCR2.1-TOPO vector and sequenced with Thermo-Sequenase and dye-labeled terminator chemistry by an automated sequencer (Applied Biosystems, Foster City, CA).Comparison of the 5′-untranslated region of human and rodent GPR34 genes revealed high sequence similarity, allowing the use of a primer derived from the sequence upstream of the coding sequence for ortholog amplification from mammalian genomic DNA (see supplemental Table S3, available in the on-line version of this article). Thus, sequences encoding the N termini of GPR34 orthologs were identified from several mammals.Full-length GPR34 sequences were inserted into the mammalian expression vector pcDps. GPR34 mutations (N137A and A265Y) were introduced into the hemagglutinin-tagged version of the human GPR34 (6Schöneberg T. Schulz A. Grosse R. Schade R. Henklein P. Schultz G. Gudermann T. Biochim. Biophys. Acta. 1999; 1446: 57-70Crossref PubMed Scopus (30) Google Scholar) using a PCR-based site-directed mutagenesis and restriction fragment replacement strategy. The identity of the various constructs and the correctness of all PCR-derived sequences were confirmed by restriction analysis and sequencing.Based on the mRNA sequence of the human P2Y12 (AF313449), primers were designed, and the coding region was amplified from genomic DNA. The product was subcloned into the eucaryotic expression vector pcDps and verified by sequencing.Cell Culture, Transfection, and Functional Assays—COS-7 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin at 37 °C in a humidified 7% CO2 incubator. For transient transfection of COS-7 cells, a calcium phosphate co-precipitation method (7Hitt M. Bett A.J. Addison C.L. Prevec L. Graham F.L. Adolph K.W. Viral Gene Techniques. Academic Press, San Diego, CA1995: 13-30Google Scholar) was applied. Thus, cells were split into 12-well plates (2 × 105 cells/well) and transfected with a total amount of 5 μg of plasmid DNA per well. For cyclic AMP accumulation assays, cells were prelabeled with 2 μCi/ml [3H]adenine (31.7 Ci/mmol; PerkinElmer Life Sciences) 48 h after transfection and incubated overnight. Then, transfected cells were washed once in serum-free Dulbecco's modified Eagle's medium containing 1 mm 3-isobutyl-1-methylxanthine (Sigma) followed by incubation with or without the indicated substances for 30 min at 37 °C. Reactions were terminated by aspiration of the medium and the addition of 1 ml of 5% trichloracetic acid. The cAMP content of cell extracts was determined after chromatography as described (8Salomon Y. Londos C. Rodbell M. Anal. Biochem. 1974; 58: 541-548Crossref PubMed Scopus (3368) Google Scholar).To measure inositol phosphate (IP) formation, transfected COS-7 cells were incubated with 2 μCi/ml myo-[3H]inositol (18.6 Ci/mmol, PerkinElmer Life Sciences) for 18 h. Thereafter, cells were washed once with serum-free Dulbecco's modified Eagle's medium containing 10 mm LiCl followed by incubation for1hat37 °C. Intracellular IP levels were determined by anion-exchange chromatography as described (9Berridge M.J. Biochem. J. 1983; 212: 849-858Crossref PubMed Scopus (765) Google Scholar). To estimate cell surface expression of receptors carrying an N-terminal hemagglutinin tag, we used an indirect cellular enzyme-linked immunosorbent assay (ELISA) (10Schöneberg T. Sandig V. Wess J. Gudermann T. Schultz G. J. Clin. Invest. 1997; 100: 1547-1556Crossref PubMed Scopus (56) Google Scholar).Sequence Analyses and Model Generation—Nucleotide and amino acid sequence alignments were made with Clustal X (11Thompson J.D. Gibson T.J. Plewniak F. Jeanmougin F. Higgins D.G. Nucleic Acids Res. 1997; 24: 4876-4882Crossref Scopus (35214) Google Scholar) and the PHYLIP software package (12Felsenstein J. PHYLIP: Phylogeny Inference Package, Version 3.6a2.1. Dept. of Genetics, University of Washington, Seattle2001Google Scholar) with visual adjustments. The conservation of all positions in the receptor protein was determined using the algorithm and the PAM matrices implemented in the Clustal X software package. Phylogenetic trees were reconstructed using the Neighbor-Joining method (13Saitou N. Nei M. Mol. Biol. Evol. 1987; 4: 406-425PubMed Google Scholar), the PHYLIP software package (12Felsenstein J. PHYLIP: Phylogeny Inference Package, Version 3.6a2.1. Dept. of Genetics, University of Washington, Seattle2001Google Scholar), and maximum likelihood analysis (14Yang Z. Nielsen R. J. Mol. Evol. 1998; 46: 409-418Crossref PubMed Scopus (457) Google Scholar), and 1,000 bootstrap replications were conducted to evaluate the reliability of the trees. The PAML software package was used to estimate synonymous and non-synonymous substitution rates in GPR34 and rhodopsin sequences (15Yang Z. Nielsen R. Mol. Biol. Evol. 2000; 17: 32-43Crossref PubMed Scopus (1193) Google Scholar). The primer-encoded regions as well as those sites that contain gaps in the alignment were not used in phylogenetic analyses.To visualize the position of conserved amino acid residues in a GPR34 receptor model, crystal structure data of the bovine rhodopsin (1L9H, molecule A) were taken as template. The homology modeling algorithms implemented in Deep View Swiss-PDB Viewer 3.7 (16Guex N. Peitsch M.C. Electrophoresis. 1997; 18: 2714-2723Crossref PubMed Scopus (9474) Google Scholar) was used to generate a rough model of the human GPR34. For refinement, the rough model was submitted to www.expasy.org/spdbv/. The final model was checked for clashes. For clarity, the model was generated to visualize the relative position of conserved GPR34 residues within the rhodopsin crystal structure and highlight molecule portions that are probably similar or different between rhodopsin and GPR34 receptors. Therefore, proline-induced distortions of helices in the rhodopsin molecules were kept in the receptor model.RESULTS AND DISCUSSIONGPR34 Has Existed for More than 450 Million Years—A long phylogenetic history is a prerequisite to making structural changes during evolution significant. To address this point, we set out to identify GPR34 orthologs from all eight vertebrate classes. Degenerate primers derived from human, mouse, and carp GPR34 (6Schöneberg T. Schulz A. Grosse R. Schade R. Henklein P. Schultz G. Gudermann T. Biochim. Biophys. Acta. 1999; 1446: 57-70Crossref PubMed Scopus (30) Google Scholar) were used to amplify partial GPR34 sequences from various vertebrate species including mammals, marsupials, monotremes, birds, reptiles, amphibians, and teleost and cartilage fish (sequences were deposited in GenBank™). As shown for selected GPR34 orthologs (Fig. 1; for all cloned orthologs and full-length receptors see supplemental Fig. S1, available in the on-line version of this article), analysis revealed the expected vertebrate ancestry. The largest variety of GPR34 sequences, including subtypes and intron-containing genes, were found in teleost fish (see below). We were also successful in cloning GPR34 orthologs from sharks, which are common ancestors of ray-finned fish and tetrapods. This indicates that GPR34 may have existed for at least 450 million years. All attempts to identify GPR34-like sequences in primitive chordate species such as lamprey and hagfish have failed so far, although primer pairs yielded partial sequences of other related GPCRs (data not shown). Similarly, sequence analyses of the known genomes of Caenorhabditis elegans, Drosophila melanogaster, and Anopheles gambiae (NCBI data base) as well as cloning attempts from other insects (Spodoptera frugiperda and Melolontha melolontha) revealed no GPR34-like sequences.Our phylogenetic analysis revealed that, in all tetrapod species investigated, GPR34 coding regions are obviously intronless and lack any subtypes in their genomes. This picture completely changed when phylogenetic analysis was extended to teleost fish. First, up to three structurally distinct GPR34 subtypes were identified in various fish species (e.g. carp) (Fig. 2A). Second, several fish species (e.g. tilapia) present a single intron in phase one (inserted after codon position one) within the coding region of EL2, which was identified by sequence comparison with other orthologs and intron/exon prediction tools.Fig. 2Phylogenetic diversity of GPR34 in fish. A, degenerated primers were designed to amplify genomic fragments encoding the sequence from TMD1 or TMD3 to TMD7. Most amplification attempts led to more than one GPR34-like ortholog. The open reading frame of several ortholog sequences was disrupted by a small intron sequence (subgroup 2). The resulting amino acid sequences between the relative codon positions 3.52 to 7.48 were aligned. The phylogenetic tree was reconstructed by using the Neighbor-Joining method (13Saitou N. Nei M. Mol. Biol. Evol. 1987; 4: 406-425PubMed Google Scholar), and 1,000 bootstrap replications were conducted to evaluate the reliability of the tree. Bootstrap values >750 are shown on the tree branches. B, several fish species (cod, tilapia, platyfish, red perch, and salmon) presented a single intron within the coding region of EL2. The relative position within the coding sequence is conserved, and an almost identical splice acceptor site indicates a common evolutionary origin (shaded boxes). The intron size between species and receptor subtypes varies only marginally (for exact intron sizes see panel A).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Sequence comparison, duplicated genes, and appearance/loss of introns provide a valid basis for unveiling the evolutionary history of a protein. Based on our genomic and amino acid sequence analyses, at least two subgroups of GPR34 sequences in teleost fish can be distinguished already in evolutionarily old teleost fish (Anguillidae and Cyprinidae). The first intronless receptor subgroup, which includes e.g. tilapia type 1 and GPR34 receptors from fugu and tetraodon (see Fig. 2A), displays some unique structural features such as a conserved Trp residue at the C-terminal end of IL2 (relative position 4.43) and a second Pro residue in TMD6 (relative position 6.56) (supplemental Fig. S2, available in the on-line version of this article). The second subgroup clusters intron-containing GPR34 genes. Some species present more than one ortholog of the second subgroup (cod and carp). This receptor divergence may be the result of an additional gene or even genome duplication. However, the proposed sub-grouping is probably restricted to the evolutionarily younger Acanthopteryii (e.g. fugu and tilapia) and cannot be applied to more ancient orders such as Anguillidae (eel), Cyprinidae (carp and zebrafish), and Siluridae (catfish). It is interesting to note that in some Acanthopteryii GPR34 is only a single-copy gene (fugu and tetraodon) as judged by PCR and data base search. Tetraodontidae are phylogenetically young, and it is therefore reasonable to assume that the second gene was lost again during evolution. Similarly, all attempts to amplify a second GPR34 subtype from two cartilaginous fish and two sturgeon species failed, indicating the existence of only one GPR34 ortholog at the very beginning of fish evolution. Low stringency genomic PCR yielded only a new and structurally distinguishable intermediate group of GPR34-like/ADP-like receptors (see Fig. 1).Another interesting marker of GPR34 evolution in fish is the presence of an intron in some orthologs (subgroup 2 in Fig. 2A). The consensus sequence flanking the introns is highly preserved (Fig. 2B). The intron size between species and receptor subtypes varies only marginally. The relative position within the coding sequence is conserved, and almost identical splice acceptor sites indicate a common evolutionary origin. No other obvious introns were found within the coding region between TMD1 and TMD7 as analyzed in cod GPR34 receptors. Gain and loss of spliceosomal introns in a lineage is a unique event that occurs at a specific point in its evolution. Two hypotheses to explain the origin of spliceosomal introns could be proposed. The "intron early" hypothesis would suggest that introns were ancient and were lost during evolution, whereas the "intron late" hypothesis would suggest that introns were inserted into the gene later in evolution (17de Souza S.J. Long M. Klein R.J. Roy S. Lin S. Gilbert W. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5094-5099Crossref PubMed Scopus (159) Google Scholar). The obvious absence of introns in GPR34 genes in cartilaginous fish and evolutionarily old teleost fish (eel, carp, and zebrafish) and a relative uniform intron size suggest that the intron was an evolutionarily new invention after gene duplication. Additional support for an intron late introduction in GPR34 genes comes from the ADP receptor evolution. Some ADP receptors (fugu and tetraodon P2Y12) also contain a spliceosomal intron. 2K. Zierau, A. Schulz, and T. Schöneberg, unpublished results. However, the position within the coding region and the size are different, excluding a common ancestral origin of the introns found in GPR34 and ADP-like receptors.Taken together, the evolution of GPR34 probably started in the Cambrian period more than 450 million years ago as suggested by its presence in cartilaginous and ray-finned fish genomes. The evolutionary split between GPR34 and ADP-like receptors is already found in teleost fish, indicating their coexistence for at least 450 million years. Identification of a clear, distinguishable ADP-like receptor from cartilaginous fish may set this evolutionary point even earlier. Furthermore, our data suggest that a GPR34 gene duplication took place in early teleost fish evolution. Then, a single intron was introduced in one of the two GPR34 genes before the divergence of Acanthopteryii. GPR34 diversification was further increased by gene or genome duplication in some teleost fish species.GPR34 and ADP-like Receptors Have a Common Evolutionary Origin—While this study was being conducted, P2Y12 and P2Y13 receptors were cloned, and ADP was found to be the agonist for both receptors (18Hollopeter G. Jantzen H.M. Vincent D. Li G. England L. Ramakrishnan V. Yang R.B. Nurden P. Nurden A. Julius D. Conley P.B. Nature. 2001; 409: 202-207Crossref PubMed Scopus (1276) Google Scholar, 19Communi D. Gonzalez N.S. Detheux M. Brezillon S. Lannoy V. Parmentier M. Boeynaems J.M. J. Biol. Chem. 2001; 276: 41479-41485Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar). A high overall amino acid sequence homology already implicated a common ancestral origin of GPR34- and ADP-like receptors (20Abbracchio M.P. Boeynaems J.M. Barnard E.A. Boyer J.L. Kennedy C. Miras-Portugal M.T. King B.F. Gachet C. Jacobson K.A. Weisman G.A. Burnstock G. Trends Pharmacol. Sci. 2003; 24: 52-55Abstract Full Text Full Text PDF PubMed Scopus (366) Google Scholar, 21Joost P. Methner A. Genome Biol. 2002; 3: 63Crossref Google Scholar). Further evidence for a close structural relation between GPR34- and ADP-like receptors came from our genomic PCR experiments. Using the same sets of degenerate primers, we amplified several other GPCR fragments from fish genomic DNA (carp, salmon, pike, zebrafish, sturgeon, dog, and shark), which are closely related to both the GPR34- and the ADP-like receptors. Comparative structural analyses revealed that two carp receptors (carp ADP-like type 1 and type 2; see Fig. 1) and one receptor from salmon and pike (not shown) were more closely related to ADP-like receptors, whereas zebrafish GPR34-like, sturgeon GPR34-like, and dog shark GPR34-like receptors combine structural features of GPR34 and ADP-like receptors, thus forming an intermediate subgroup (see Fig. 1).To support a common evolutionary origin of GPR34- and ADP-like receptors by functional data, we initiated studies to determine the signal transduction and agonist specificity of the GPR34 receptors. P2Y12 and P2Y13 are coupled to Gi/o proteins, which inhibit adenylyl cyclases and, therefore, decrease intracellular cAMP levels. According to the current model of GPCR function (22Lefkowitz R.J. Nature. 1993; 365: 603-604Crossref PubMed Scopus (87) Google Scholar), receptor overexpression can result in a constitutive activation of signaling pathways. Thus, the coupling abilities of several receptors, including "orphan" receptors, have been characterized by overexpression in the absence of an agonist. For example, the wild-type ACCA, an orphan GPCR, stimulates the Gs/adenylyl cyclase system to some extent when expressed in COS-7 cells (23Eggerickx D. Denef J.F. Labbe O. Hayashi Y. Refetoff S. Vassart G. Parmentier M. Libert F. Biochem. J. 1995; 309: 837-843Crossref PubMed Scopus (99) Google Scholar). It has been demonstrated that replacement of the four or five C-terminal amino acids of Gαq with the corresponding Gαi residues (referred to as Gαqi4) confers the ability to stimulate the PLC-β pathway onto Gi-coupled receptors (24Conklin B.R. Farfel Z. Lustig K.D. Julius D. Bourne H.R. Nature. 1993; 363: 274-276Crossref PubMed Scopus (601) Google Scholar). As expected, ADP stimulation (10 μm ADP) of P2Y12 and Gαqi4-transfected COS-7 cells resulted in a robust increase in intracellular IP levels (Fig. 3A). COS-7 cells co-transfected with GFP and Gαqi4 responded to ADP stimulation with only a 1.7-fold increase in IP levels because of endogenous ADP receptor expression. However, cells expressing the human GPR34 and Gαqi4 did not show an effect above the endogenous response following ADP application (see Fig. 3A). Interestingly, P2Y12 and human GPR34 displayed some degree of basal activities when compared with GFP-transfected cells. To verify the basal activation of the Gi pathway, we measured the inhibition of forskolin-induced cAMP formation. Indeed, the forskolin-induced cAMP formation was significantly reduced in cells transfected with the human GPR34 (77.2 ± 8.8%), the murine GPR34 (72.7 ± 4.8%), and the human P2Y12 receptor (73.4 ± 5.5%) when compared with GFP-transfected COS-7 cells (100%).Fig. 3High basal activity is preserved among GPR34 receptors. To evaluate signaling specificity of GPR34 and ADP receptors, COS-7 cells were co-transfected with the indicated receptor construct and the chimeric Gα protein (see "Experimental Procedures"). IP assays were performed 72 h after transfection as described under "Experimental Procedures." A, plasmids encoding GFP (control), the human GPR34, and the human ADP receptor were co-transfected with Gαqi4. IP formation under basal conditions (light gray bars) and in the presence of 10 μm ADP (dark gray bars) was determined. Data are presented as means ± S.E. (cpm/well) of three independent experiments, each carried out in triplicate. B, to verify the constitutive activity of GPR34 and ADP receptors, COS-7 cells were co-transfected with the designated GPR34 ortholog constructs and the chimeric GαΔ6qi4myr plasmid (see "Experimental Procedures"). Basal IP formation is expressed as fold over basal levels of GFP-transfected cells (404 ± 167 cpm/well). Data are presented as means ± S.E. of three independent experiments, each carried out in triplicate.View Large Image Figure ViewerD
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