Oligomerization of G-protein-coupled Receptors Shown by Selective Co-immunoprecipitation

Abstract

Recent studies have shown that G-protein-coupled receptors (GPCRs) can assemble as high molecular weight homo- and hetero-oligomeric complexes. This can result in altered receptor-ligand binding, signaling, or intracellular trafficking. We have co-transfected HEK-293 cells with differentially epitope-tagged GPCRs from different subfamilies and determined whether oligomeric complexes were formed by co-immunoprecipitation and immunoblot analysis. This gave the surprising result that the 5HT1A receptor was capable of forming hetero-oligomers with all GPCRs tested including the 5HT1B, 5HT1D, EDG1, EDG3, GPR26, and GABAB2 receptors. The testing of other GPCR combinations showed similar results with hetero-oligomer formation occurring for the 5HT1D with the 5HT1B and EDG1 receptor. Control studies showed that these complexes were present in co-transfected cells before the time of lysis and that the hetero-oligomers were comprised of GPCRs at discrete stoichiometries. These findings suggest that GPCRs have a natural tendency to form oligomers when co-transfected into cells. Future studies should therefore investigate the presence and physiological role of GPCR hetero-oligomers in cells in which they are endogenously expressed. Recent studies have shown that G-protein-coupled receptors (GPCRs) can assemble as high molecular weight homo- and hetero-oligomeric complexes. This can result in altered receptor-ligand binding, signaling, or intracellular trafficking. We have co-transfected HEK-293 cells with differentially epitope-tagged GPCRs from different subfamilies and determined whether oligomeric complexes were formed by co-immunoprecipitation and immunoblot analysis. This gave the surprising result that the 5HT1A receptor was capable of forming hetero-oligomers with all GPCRs tested including the 5HT1B, 5HT1D, EDG1, EDG3, GPR26, and GABAB2 receptors. The testing of other GPCR combinations showed similar results with hetero-oligomer formation occurring for the 5HT1D with the 5HT1B and EDG1 receptor. Control studies showed that these complexes were present in co-transfected cells before the time of lysis and that the hetero-oligomers were comprised of GPCRs at discrete stoichiometries. These findings suggest that GPCRs have a natural tendency to form oligomers when co-transfected into cells. Future studies should therefore investigate the presence and physiological role of GPCR hetero-oligomers in cells in which they are endogenously expressed. G-protein-coupled receptor Recent studies have shown that G-protein-coupled receptors (GPCRs)1 may form dimers or higher order oligomers (1Maggio R. Vogel Z. Wess J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3103-3107Crossref PubMed Scopus (302) Google Scholar, 2Maggio R. Vogel Z. Wess J. FEBS Lett. 1993; 319: 195-200Crossref PubMed Scopus (118) Google Scholar, 3Hebert T.E. Moffett S. 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Co-expression studies with chimeric α2C-adrenergic/M3 muscarinic receptor constructs have shown that intermolecular interactions can occur between different GPCR subtypes (1Maggio R. Vogel Z. Wess J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3103-3107Crossref PubMed Scopus (302) Google Scholar, 2Maggio R. Vogel Z. Wess J. FEBS Lett. 1993; 319: 195-200Crossref PubMed Scopus (118) Google Scholar). More direct studies have shown that the GABABR1 and GABABR2 receptors are not functional as separate units and can only form a functional receptor complex with the correct pharmacological properties and plasma membrane expression when they are co-expressed (8Jones K.A. Borowsky B. Tamm J.A. Craig D.A. Durkin M.M. Dai M. Yao W.J. Johnson M. Gunwaldsen C. Huang L.Y. Tang C. Shen Q. Salon J.A. Morse K. Laz T. Smith K.E. Nagarathnam D. Noble S.A. Branchek T.A. Gerald C. Nature. 1998; 396: 674-679Crossref PubMed Scopus (925) Google Scholar, 9Kaupmann K. Malitschek B. Schuler V. Heid J. Froestl W. 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The finding that co-transfection of the κ and δ opioid receptors results in the formation of heterodimers with ligand binding and functional properties that are distinct from singly transfected receptors suggests that heterodimerization may be involved in modulation of GPCR function (13Jordan B.A. Devi L.A. Nature. 1999; 399: 697-700Crossref PubMed Scopus (978) Google Scholar). Also, Western blot studies have shown that the 5HT1D and 5HT1B receptors form homodimers when expressed alone although they preferentially form heterodimers when co-expressed (14Xie Z. Lee S.P. O'Dowd B.F. George S.R. FEBS Lett. 1999; 456: 63-67Crossref PubMed Scopus (109) Google Scholar). In the present study, we have directly examined whether 5HT1 receptors are capable of forming oligomeric complexes with a variety of GPCRs by specific immunoprecipitation of each receptor followed by identification of the co-precipitated proteins using immunoblot analysis. The main objective was to determine the specificity of the interactions across different GPCR subfamilies to gain further insight into the mechanism of GPCR hetero-oligomerization. Restriction endonucleases and other DNA-modifying enzymes were from New England Biolabs (Beverly, MA) or Amersham Biosciences. Dulbecco's modified Eagle's medium and fetal calf serum were from Invitrogen. All other biochemicals were obtained from Sigma unless specified otherwise. The c-Myc epitope (EQKLISEEDL) was inserted at the amino termini of 5HT1A and 5HT1Dreceptors by PCR mutagenesis using a Stratagene Robocycler with Hot-Top assembly (Amsterdam, Holland) as described by Nelson and Long (15Nelson R.M. Long G.L. Anal. Biochem. 1989; 180: 147-151Crossref PubMed Scopus (294) Google Scholar). The FLAG epitope (DYKDDDDK) was inserted by a similar procedure into the amino termini of 5HT1A, 5HT1B, 5HT1D, EDG1, and EDG2 receptors. FLAG-tagged EDG3, GPR26, and GABAB2receptors were a gift from Dr. Kevin Lynch (University of Virginia, Charlottesville, VA). PCR products were cloned into the eucaryotic expression vector pCDNA3.1+ (Invitrogen) using standard techniques (16Davis L.G. Kuehl W.M. Battey J.F. Basic Methods in Molecular Biology. Appleton & Lange, Norwalk, CT1994Google Scholar). Inserts were sequenced with an ABI Prism dye terminator cycle sequencing kit and analyzed on an Applied Biosystems 373A stretch DNA sequencer. Transient transfection of HEK-293 cells with the epitope-tagged constructs was performed by calcium phosphate-mediated gene transfer as described previously (17Chen C.A. Okayama H. BioTechniques. 1988; 6: 632-638Crossref PubMed Scopus (28) Google Scholar). Forty-eight hours after transfection cells were washed three times in phosphate-buffered saline, harvested by scraping, and centrifuged for 5 min at 500 × g. The pelleted cells were homogenized at 4 °C by drawing up and down through a 20-gauge syringe needle in 1 ml of cell lysis buffer (Sigma). The homogenates were centrifuged for 20 min at 14,000 ×g at 4 °C, and the supernatants were combined with 12.5 μl (packed gel) of either anti-c-Myc or anti-FLAG M2 affinity agarose (Sigma) and mixed overnight at 4 °C. The immunoadsorbents were recovered by centrifugation for 5 min at 700 × g and washed three times by resuspension and centrifugation (5 min at 700 × g) in cell lysis buffer and two times in 50 mm Tris (pH 7.5) containing 0.1% (w/v) SDS and 150 mm NaCl. The samples were eluted into 60 μl of SDS loading buffer (Sigma). Eluted samples were heated for 3 min at 100 °C and subjected to SDS-PAGE on slab gels (180 × 160 × 1.5 mm) polymerized from 10% (w/v) acrylamide and 0.1%N,N′-methylenebisacrylamide in Tris/glycine/SDS buffer (Novex, San Diego, CA) using the discontinuous buffer system of Laemmli (18Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207231) Google Scholar). The electrophoresed proteins were subjected to semi-dry electrophoretic transfer onto polyvinylidene difluoride membranes as described previously (19Guest P.C. Ravazzola M. Davidson H.W. Orci L. Hutton J.C. Endocrinology. 1991; 129: 734-740Crossref PubMed Scopus (35) Google Scholar). The membranes were incubated with either mouse c-Myc antibodies (1:1000) or mouse FLAG antibodies (1:1000) and then with peroxidase-conjugated sheep anti-mouse serum (1:1000) for detection of immunoreactive bands by enhanced chemiluminescence (Amersham Biosciences). Molecular size calibration was achieved with the MultiMark standards (Novex). HEK-293 cells transfected with either FLAG-tagged 5HT1A, 5HT1B, or 5HT1Dreceptor constructs were immunoprecipitated with anti-FLAG-agarose, and immunoblot analysis of the precipitates revealed the presence of 46, 44, and 42-kDa FLAG-immunoreactive bands, respectively (Fig.1A). Higher molecular weight immunoreactive forms were also present, which might represent oligomeric forms of the 5HT1 receptors. An immunoreactive band that migrated at a size predicted for a FLAG-tagged 5HT1A receptor dimer (92 kDa) showed a strong increase in immunostaining with the reciprocal decrease of the 46-kDa monomer band when the disulfide bond-reducing agent dithiothreitol was omitted from the sample loading buffer (Fig. 1B). An immunoreactive band that migrated at a size predicted for a FLAG-tagged 5HT1Breceptor dimer (88 kDa) showed increased immunostaining with the loss of the 44-kDa monomer band when the samples were electrophoresed in the absence of dithiothreitol (Fig. 1B). Similarly, a possible FLAG-tagged 5HT1D receptor dimer (82 kDa) showed an increase in immunostaining on non-reducing gels with the reciprocal disappearance of the 41-kDa monomer band (Fig. 1B). Similar results have been shown previously for the 5HT1B and 5HT1D receptors, which led to the suggestion that they form homodimers (14Xie Z. Lee S.P. O'Dowd B.F. George S.R. FEBS Lett. 1999; 456: 63-67Crossref PubMed Scopus (109) Google Scholar). The mechanism of 5HT1B receptor homodimerization appeared to differ from that of the 5HT1Aand 5HT1D receptors because a high proportion 5HT1B receptor homodimer was observed even when gels were run under disulfide bond-reducing conditions (Fig. 1A). To directly test for hetero-oligomerization of different GPCR combinations we used sequential co-immunoprecipitation and immunoblot analyses of cells co-transfected with differentially epitope-tagged receptors. This showed that the c-Myc-tagged 5HT1A receptor co-precipitated with FLAG-tagged versions of all receptors tested including the 5HT1A, 5HT1B, 5HT1D, EDG1, EDG3, GPR26, and GABAB2 receptors (Fig. 2,A and B). The same finding was observed for the c-Myc-tagged 5HT1D receptor, which was co-precipitated with FLAG-tagged versions of the 5HT1B and EDG1receptors (Fig. 2, A and B). Previous studies have also shown that the 5HT1B and 5HT1Dreceptors form heterodimers (14Xie Z. Lee S.P. O'Dowd B.F. George S.R. FEBS Lett. 1999; 456: 63-67Crossref PubMed Scopus (109) Google Scholar). The finding that the FLAG- and c-Myc-tagged versions of the 5HT1A receptor were co-precipitated provides direct evidence that this GPCR forms a homo-oligomer. This was also found for the 5HT1D receptor using the c-Myc- and FLAG-tagged versions of this GPCR (data not shown). Co-precipitation of high molecular weight FLAG-immunoreactive 5HT1B, 5HT1D, EDG1, and EDG3 species with the c-Myc-5HT1A receptor (Fig. 2A) suggests that these GPCRs might be present as homodimers and trimers in higher order hetero-oligomeric complexes comprised of at least one c-Myc-5HT1A receptor and two or more of the FLAG-tagged constructs. To rule out the possibility that the observed hetero-oligomerizations were caused by nonspecific aggregation of receptors, extracts from cells separately expressing the c-Myc-tagged 5HT1A and FLAG-tagged 5HT1Breceptors were mixed, immunoprecipitated with anti-c-Myc agarose, and subjected to immunoblot analysis with FLAG antibodies. In this case, the FLAG-5HT1B receptor was not detected in the precipitated material (Fig.3A). Similarly, immunoprecipitation of the mixed extract with anti-FLAG agarose followed by immunoblot analysis with c-Myc antibodies did not detect the c-Myc-5HT1A receptor (Fig. 3B). This confirmed that successful immunoprecipitation of 5HT1A/5HT1B hetero-oligomers requires co-expression within the same cells before the time of lysis. Thus, the oligomerization of the c-Myc-tagged 5HT1A and FLAG-tagged 5HT1B receptors in the present study is not likely to be caused by nonspecific aggregation. The same results were found for all of the other GPCR/GPCR hetero-oligomers tested in this study (data not shown). To determine whether the hetero-oligomers observed in this study are comprised of GPCRs at discrete protein/protein ratios, HEK-293 cells were co-transfected with a fixed amount (25 μg) of the c-Myc-tagged 5HT1A receptor and varying amounts (25, 5, 1, and 0.2 μg) of the FLAG-tagged EDG1 receptor. The relative amounts of the c-Myc-5HT1A receptor that were associated with the decreasing levels of the FLAG-EDG1 receptor were determined by sequential immunoprecipitation and immunoblot analysis. Immunoprecipitation of the FLAG-EDG1 receptor with anti-FLAG-agarose resulted in co-precipitation of similar decreasing levels of c-Myc-5HT1A receptor (Fig.4B) as shown by immunoblot analysis with c-Myc antibodies. Similarly, immunoprecipitation of the c-Myc-5HT1A receptor resulted in co-precipitation of decreasing levels of the FLAG-EDG1 receptor (Fig.4D). Sequential immunoprecipitation/immunoblot analysis of the lysates with c-Myc/c-Myc or FLAG/FLAG confirmed that the c-Myc-5HT1A receptor was actually expressed in the cells at relatively constant levels and that expression of the FLAG-EDG1 receptor decreased accordingly with the amounts of cDNA transfected into the cells (Fig. 4, Aand C). Similar results were obtained for all of the other receptors under study (data not shown). We have used a co-immunoprecipitation approach to provide direct evidence that GPCRs from different subfamilies are capable of forming hetero-oligomers when co-expressed in HEK-293 cells. Cells were co-transfected with GPCR constructs containing different epitope tags followed by immunoprecipitation of one receptor and detection of co-precipitated receptors by immunoblot analysis. The 5HT1Areceptor formed a homo-oligomer when expressed alone and formed hetero-oligomers when co-expressed with the closely related 5HT1B and 5HT1D receptors (∼36% amino acid homology) and also with the more distantly related EDG1, EDG3, GPR26, and GABAB2 receptors (4–16% amino acid homology). Interestingly, hetero-oligomer formation was shown for all other GPCR combinations tested including the 5HT1D receptor with the 5HT1B and EDG1 receptors. The heterodimerization of 5HT1Band 5HT1D receptors has been shown previously in insect SF9 cells using a viral expression system followed by Western blot analysis (14Xie Z. Lee S.P. O'Dowd B.F. George S.R. FEBS Lett. 1999; 456: 63-67Crossref PubMed Scopus (109) Google Scholar). The results of this study suggest that the receptor complexes were pre-existing in cells at the time of lysis and were not caused by nonspecific aggregation. Oligomerization was observed only in lysates from co-transfected cells and not in mixed lysates from singly transfected cells. Also, the observed receptor/receptor interactions appeared to occur at defined stoichiometries in co-transfected cells because decreased expression of one receptor resulted in a similar incremental decrease in the amount of the second receptor immunoprecipitated. The appearance of high molecular weight immunoreactive bands corresponding in size to 5HT1A and 5HT1D receptor homo-oligomers appeared to require intact disulfide bonds because these were only detected when gels were run under non-reducing conditions. Little or no 5HT1A and 5HT1D receptor homo-oligomers could be detected when gels were run under reducing conditions. In contrast, a significant proportion of the 5HT1B receptor was detected as high molecular weight oligomers when gels were run under reducing conditions with increased levels of the oligomer and reciprocal loss of the monomer under non-reducing conditions. This indicates that the mechanism of 5HT1B receptor homo-oligomer formation differs from that of the 5HT1A and 5HT1D receptors. Also, the observed immuno-isolation of the putative 5HT1Breceptor dimer by immunoprecipitation of the 5HT1A receptor indicates that these receptors may be present as higher order hetero-oligomers such as trimers or tetramers. A major prerequisite for the physiological assembly of hetero-oligomeric GPCRs is co-expression in the same cells. To demonstrate this would require detailed immunological or specific binding experiments that localize the receptors under study to the same cells combined with co-precipitation studies using receptor-specific antibodies or ligands. The GPCRs tested in this study show a wide range of tissue distributions in the brain. Within the 5HT receptor subfamily 5HT1A, 5HT1B, and 5HT1D receptors all show pre- and postsynaptic localizations and can be found together in some of the same brain regions including the cortex and dorsal raphe nucleus (20Fink K. Zentner J. Gothert M. Naunyn-Schmiedeberg's Arch. Pharmacol. 1995; 352: 451-454PubMed Google Scholar, 21Bonaventure P. Voorn P. Luyten W.H. Jurzak M. Schotte A. Leysen J.E. Neuroscience. 1998; 82: 469-484Crossref PubMed Scopus (109) Google Scholar, 22Barnes N.M. Sharp T. Neuropharmacology. 1999; 38: 1083-1152Crossref PubMed Scopus (2724) Google Scholar). The 5HT1B receptor shows co-expression with the 5HT1A receptor in the hippocampus and with the 5HT1D receptor in the olfactory tubercle although the 5HT1B receptor is expressed separately in the anterior caudate putamen, hypothalamus, and thalamus, and the 5HT1Dreceptor is localized separately in the trigeminal nucleus and in parts of the cerebellum (21Bonaventure P. Voorn P. Luyten W.H. Jurzak M. Schotte A. Leysen J.E. Neuroscience. 1998; 82: 469-484Crossref PubMed Scopus (109) Google Scholar). Hetero-oligomerization of the 5HT1Awith the GABAB2 and GPR26 receptors may be of interest because all of these receptors are widely distributed throughout the cortex and may therefore share some regions of overlap (22Barnes N.M. Sharp T. Neuropharmacology. 1999; 38: 1083-1152Crossref PubMed Scopus (2724) Google Scholar, 23Durkin M.M. Gunwaldsen C.A. Borowsky B. Jones K.A. Branchek T.A. Mol. Brain Res. 1999; 71: 185-200Crossref PubMed Scopus (83) Google Scholar, 24Lee D.K. Lynch K.R. Im T. Nguyen D.S. Cheng R. Saldivia V.R. Liu Y. Liu I.S. Heng H.H. Seeman P. George S.R. O'Dowd B.F. Marchese A. Biochim. Biophys. Acta. 2000; 149: 311-323Crossref Scopus (40) Google Scholar). The physiological relevance of the hetero-oligomerization of 5HT1A receptor with the EDG1 and EDG3 receptors is not clear because no detailed studies have been carried out on the distribution of the latter receptors within the brain. However, the interactions observed between the 5HT1A and EDG3 receptors are not likely to occur in vivo because the relative expression levels of the latter are relatively low in the brain with the highest levels observed in peripheral tissues such as lung and heart (25Yamaguchi F. Tokuda M. Hatase O. Brenner S. Biochem. Biophys. Res. Commun. 1996; 227: 608-614Crossref PubMed Scopus (106) Google Scholar, 26Zhang G. Contos J.J. Weiner J.A. Fukushima N. Chun J. Gene (AMST.). 1999; 227: 89-99Crossref PubMed Scopus (136) Google Scholar). Supporting evidence for oligomerization of endogenously expressed GPCRs comes from Western blot studies of brain tissues revealing the presence of high molecular weight immunoreactive forms of the dopamine D2 receptor (27Ng G.Y. O'Dowd B.F. Lee S.P. Chung H.T. Brann M.R. Seeman P. George S.R. Biochem. Biophys. Res. Commun. 1996; 227: 200-204Crossref PubMed Scopus (246) Google Scholar) and the A1 adenosine receptor (28Ciruela F. Casado V. Mallol J. C. R. J. Res. 1995; PubMed Scopus Google Scholar). More an study showed that and receptors are and closely on and in of and cells S. J. J.A. J. J.E. L. L. M.J. J. PubMed Scopus Google Scholar). The evidence comes from studies that GABABR1 and GABABR2 receptors could be from cortex K. Malitschek B. Schuler V. Heid J. Froestl W. Beck P. Mosbacher J. Bischoff S. Kulik A. Shigemoto R. Karschin A. Bettler B. Nature. 1998; 396: 683-687Crossref PubMed Scopus (1017) Google Scholar). This is the study in which the of GPCR oligomerization has been using a of receptor Interestingly, all of the receptors tested were capable of forming hetero-oligomers when co-expressed in tissue cells that this might be a of these receptors. These findings the for further studies to investigate the of GPCR hetero-oligomerization in tissues and cells they are endogenously expressed. studies to the physiological role of such complexes in GPCR function.