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• W2056556818 abstract "G-protein-coupled receptors (GPCRs) represent the largest and most diverse family of cell surface receptors. Several GPCRs have been documented to dimerize with resulting changes in pharmacology. We have previously reported by means of photobleaching fluorescence resonance energy transfer (pbFRET) microscopy and fluorescence correlation spectroscopic (FCS) analysis in live cells, that human somatostatin receptor (hSSTR) 5 could both homodimerize and heterodimerize with hSSTR1 in the presence of the agonist SST-14. In contrast, hSSTR1 remained monomeric when expressed alone regardless of agonist exposure in live cells. In an effort to elucidate the role of ligand and receptor subtypes in heterodimerization, we have employed both pb-FRET microscopy and Western blot on cells stably co-expressing hSSTR1 and hSSTR5 treated with subtype-specific agonists. Here we provide evidence that activation of hSSTR5 but not hSSTR1 is necessary for heterodimeric assembly. This property was also reflected in signaling as shown by increases in adenylyl cyclase coupling efficiencies. Furthermore, receptor C-tail chimeras allowed for the identification of the C-tail as a determinant for dimerization. Finally, we demonstrate that heterodimerization is subtype-selective involving ligand-induced conformational changes in hSSTR5 but not hSSTR1 and could be attributed to molecular events occurring at the C-tail. Understanding the mechanisms by which GPCRs dimerize holds promise for improvements in drug design and efficacy. G-protein-coupled receptors (GPCRs) represent the largest and most diverse family of cell surface receptors. Several GPCRs have been documented to dimerize with resulting changes in pharmacology. We have previously reported by means of photobleaching fluorescence resonance energy transfer (pbFRET) microscopy and fluorescence correlation spectroscopic (FCS) analysis in live cells, that human somatostatin receptor (hSSTR) 5 could both homodimerize and heterodimerize with hSSTR1 in the presence of the agonist SST-14. In contrast, hSSTR1 remained monomeric when expressed alone regardless of agonist exposure in live cells. In an effort to elucidate the role of ligand and receptor subtypes in heterodimerization, we have employed both pb-FRET microscopy and Western blot on cells stably co-expressing hSSTR1 and hSSTR5 treated with subtype-specific agonists. Here we provide evidence that activation of hSSTR5 but not hSSTR1 is necessary for heterodimeric assembly. This property was also reflected in signaling as shown by increases in adenylyl cyclase coupling efficiencies. Furthermore, receptor C-tail chimeras allowed for the identification of the C-tail as a determinant for dimerization. Finally, we demonstrate that heterodimerization is subtype-selective involving ligand-induced conformational changes in hSSTR5 but not hSSTR1 and could be attributed to molecular events occurring at the C-tail. Understanding the mechanisms by which GPCRs dimerize holds promise for improvements in drug design and efficacy. In recent years, G-protein-coupled receptors (GPCRs), 1The abbreviations used are: GPCR(s), G-protein-coupled receptor(s); hSSTR, human somatostatin receptor; SST, somatostatin; HA, hemagglutinin; CHO, Chinese hamster ovary; pbFRET, photobleaching fluorescence resonance energy transfer; FITC, fluorescein isothiocyanate; TR, Texas red; FCS, fluorescence correlation spectroscopy; TM, transmembrane domain; GABA, γ-aminobutyric acid. once believed to exist at the plasma membrane as monomers, have been shown to assemble on the membrane as functional homo- and heterodimers (1Agnati L.F. Ferré S. LLuis C. Franco R. Fuxe K. Pharm. Rev. 2003; 55: 509-550Crossref PubMed Scopus (298) Google Scholar, 2Angers S. Salahpour A. Bouvier M. Annu. Rev. Pharmacol. Toxicol. 2002; 42: 409-435Crossref PubMed Scopus (517) Google Scholar). Dimerization 2The terms dimerization and oligomerization are used interchangeably. of GPCRs has been shown to affect a multitude of receptor functions including ligand binding, signaling, receptor desensitization, and receptor trafficking (1Agnati L.F. Ferré S. LLuis C. Franco R. Fuxe K. Pharm. Rev. 2003; 55: 509-550Crossref PubMed Scopus (298) Google Scholar, 2Angers S. Salahpour A. Bouvier M. Annu. Rev. Pharmacol. Toxicol. 2002; 42: 409-435Crossref PubMed Scopus (517) Google Scholar). The influence of GPCR dimerization was shown to include cellular immunity, neurotransmission (1Agnati L.F. Ferré S. LLuis C. Franco R. Fuxe K. Pharm. Rev. 2003; 55: 509-550Crossref PubMed Scopus (298) Google Scholar), taste (3Nelson G. Hoon M.A. Chandrashekar J. Zhang Y. Ryba N.J.P. Zuker C.S. Cell. 2001; 106: 381-390Abstract Full Text Full Text PDF PubMed Scopus (1407) Google Scholar, 4Nelson G. Chandrashekar J. Hoon M.A. Feng L. Zhao G. Ryba N.J.P. Zuker C.S. Nature. 2002; 416: 199-202Crossref PubMed Scopus (1168) Google Scholar, 5Li X. Staszewski L. Xu H. Durick K. Zoller M. Adler E. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 4692-4696Crossref PubMed Scopus (1135) Google Scholar), and disease (6Abdalla S. Lother H. Massiery A.E. Quitterer U. Nat. Med. 2001; 7: 1003-1009Crossref PubMed Scopus (417) Google Scholar). Although the mechanism by which GPCR dimerization occurs remains obscure, one model suggests that ligand binding of cell surface receptors induces a conformational change that favors dimer formation; while the other suggests that dimerization is an exclusive event occurring early on during receptor biogenesis most probably in the ER and is a necessary event for proper receptor trafficking and function. This latter model has been suggested for members of the class C subfamily of GPCRs, which include the GABAergic receptors (7White J.H. Wise A. Main M.J. Green A. Fraser N.J. Disney G.H. Barnes A.A. Emson P. Foord S.M. Marshall F.M. Nature. 1998; 396: 679-682Crossref PubMed Scopus (1019) Google Scholar, 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 (929) Google Scholar, 9Kaupmann 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 (1018) Google Scholar), calcium-sensing receptor (10Bai M. Trivedi S. Brown E.M. J. Biol. Chem. 1998; 273: 23605-23610Abstract Full Text Full Text PDF PubMed Scopus (347) Google Scholar, 11Jensen A.A. Hansen J.L. Sheikh S.P. Bräuner-Osborne H. Eur. J. Biochem. 2002; 269: 5076-5087Crossref PubMed Scopus (82) Google Scholar), the metabotropic glutamate receptor (12Romano C. Yang W.L. O'Malley K.L. J. Biol. Chem. 1996; 271: 28612-28616Abstract Full Text Full Text PDF PubMed Scopus (449) Google Scholar), and the sweet taste receptors (3Nelson G. Hoon M.A. Chandrashekar J. Zhang Y. Ryba N.J.P. Zuker C.S. Cell. 2001; 106: 381-390Abstract Full Text Full Text PDF PubMed Scopus (1407) Google Scholar, 4Nelson G. Chandrashekar J. Hoon M.A. Feng L. Zhao G. Ryba N.J.P. Zuker C.S. Nature. 2002; 416: 199-202Crossref PubMed Scopus (1168) Google Scholar, 5Li X. Staszewski L. Xu H. Durick K. Zoller M. Adler E. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 4692-4696Crossref PubMed Scopus (1135) Google Scholar). However, this paradigm of GPCR assembly is not consistent among the class A/rhodopsin-like family of GPCRs. Several reports have shown that agonist plays an active role in GPCR dimerization at the plasma membrane, suggesting an equilibrium between GPCR dimers/monomers that can be regulated by ligand occupancy. These receptors include the human somatostatin receptors (hSSTRs) (13Rocheville M. Lange D.C. Kumar U. Sasi R. Patel R.C. Patel Y.C. J. Biol. Chem. 2000; 275: 7862-7869Abstract Full Text Full Text PDF PubMed Scopus (470) Google Scholar, 14Patel R.C. Kumar U. Lamb D.C. Eid J.S. Rocheville M. Grant M. Rani A. Hazlett T. Patel S.C. Gratton E. Patel Y.C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 3294-3299Crossref PubMed Scopus (178) Google Scholar), dopamine D2 receptor (15Wurch T. Matsumoto A. Pauwels P.J. FEBS Lett. 2001; 507: 109-113Crossref PubMed Scopus (39) Google Scholar), gonadotrophin-releasing hormone receptor (16Cornea A. Janovick J.A. Maya-Nunez G. Conn P.M. J. Biol. Chem. 2000; 276: 2153-2158Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 17Horvat R.D. Roess D.A. Nelson S.E. Barisas B.G. Clay C.M. Mol. Endocrinol. 2001; 15: 695-703Crossref PubMed Scopus (55) Google Scholar), luteinizing hormone/chorionic gonadotrophin hormone receptor (18Roess D.A. Horvat R.D. Munnelly H. Barisas B.G. Endocrinology. 2000; 141: 4518-4523Crossref PubMed Scopus (67) Google Scholar), bradykinin B2 receptor (19Abdalla S. Zaki E. Lother H. Quitterer U. J. Biol. Chem. 1999; 274: 26079-26084Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar), thyrotropin-releasing hormone receptor (20Kroeger K.M. Hanyaloglu A.C. Seeber R.M. Miles L.E.C. Eidne K.A. J. Biol. Chem. 2001; 276: 12736-12743Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar), cholecystokinin receptor (21Cheng Z-J. Miller L.J. J. Biol. Chem. 2001; 276: 48040-48047Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar), thyrotropin receptor (22Latif R. Graves P. Davies T.F. J. Biol. Chem. 2002; 277: 45059-45067Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar), and the chemokine receptors (23Rodriguez-Frade J.M. Vila-Coro A.J. de Ana A.M. Albar J.P. Martinez-A C. Mellado M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3628-3633Crossref PubMed Scopus (204) Google Scholar, 24Vila-Coro A.J. Mellado M. de Ana A.M. Lucas P. del Real G. Martinez A.-C. Rodriguez-Frade J.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3388-3393Crossref PubMed Scopus (130) Google Scholar, 25Vila-Coro A.J. Rodriguez-Frade J.M. de Ana A.M. Moreno-Ortiz M.C. Martinez-A C. Mellado M. FASEB J. 1999; 13: 1699-1710Crossref PubMed Scopus (443) Google Scholar, 26Mellado M. Rodriguez-Frade J.M. Vila-Coro A.J. Fernandez S. Martin de Ana A. Jones D.R. Toran J.L. Martinez A.-C. EMBO J. 2001; 20: 2497-2507Crossref PubMed Scopus (384) Google Scholar). We have previously reported that hSSTRs, known to modulate neurotransmission, cell secretion, and cell proliferation (27Moller L.S. Stidsen C.E. Hartmann B. Holst J.J. Biochim. Biophys. Acta. 2003; 1616: 1-84Crossref PubMed Scopus (306) Google Scholar, 28Patel Y.C. Front. Neuroendocrinol. 1999; 20: 157-198Crossref PubMed Scopus (1418) Google Scholar) are capable of undergoing both homo- and heterodimerization at the cell membrane (13Rocheville M. Lange D.C. Kumar U. Sasi R. Patel R.C. Patel Y.C. J. Biol. Chem. 2000; 275: 7862-7869Abstract Full Text Full Text PDF PubMed Scopus (470) Google Scholar, 14Patel R.C. Kumar U. Lamb D.C. Eid J.S. Rocheville M. Grant M. Rani A. Hazlett T. Patel S.C. Gratton E. Patel Y.C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 3294-3299Crossref PubMed Scopus (178) Google Scholar, 29Rocheville M. Lange D.C. Kumar U. Patel S.C. Patel R.C. Patel Y.C. Science. 2000; 288: 154-157Crossref PubMed Scopus (755) Google Scholar). Recently, we have demonstrated ligand-dependent homo- and heterodimers on the plasma membrane in live cells in both a homogeneous and heterogeneous receptor expressing cell line, using both single and two photon dual color fluorescence correlation spectroscopy (FCS) with cross-correlation analysis (a method that discriminates based upon molecular size, number density, and average brightness/particle in femtoliter confocal volumes) (14Patel R.C. Kumar U. Lamb D.C. Eid J.S. Rocheville M. Grant M. Rani A. Hazlett T. Patel S.C. Gratton E. Patel Y.C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 3294-3299Crossref PubMed Scopus (178) Google Scholar). One of the receptor subtypes, hSSTR1, did not form homodimers in either the absence or presence of ligand. In contrast, hSSTR5 showed robust dimerization upon agonist exposure. When both receptors were co-expressed in the same cell, we were able to observe two populations of dimers, hSSTR5 homodimers and hSSTR1/hSSTR5 heterodimers (14Patel R.C. Kumar U. Lamb D.C. Eid J.S. Rocheville M. Grant M. Rani A. Hazlett T. Patel S.C. Gratton E. Patel Y.C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 3294-3299Crossref PubMed Scopus (178) Google Scholar). However, it remains unclear as to whether one or both receptor subtypes are capable of promoting heterodimerization, and which receptor motifs may be attributed to this behavior. In the present study, using subtype-specific agonists and both photobleaching fluorescence resonance energy transfer (pbFRET) and Western blot analysis, we demonstrate that ligand-bound hSSTR5 but not hSSTR1 can promote the heterodimerization of hSSTR1/hSSTR5. Moreover, using receptor C-tail chimeras, we were able to abrogate the homodimerization of hSSTR5 and induce the formation of hSSTR1 homodimers. The hSSTR5 subtype-specific analog of somatostatin, SMS 201-995, displayed a relatively poor signaling profile for hSSTR5 expressed alone despite having nanomolar binding affinity. Accordingly, co-expression with hSSTR1 resulted in a robust signaling efficiency by SMS 201-995 that correlated in part with its ability to induce heterodimerization. Finally, we demonstrate that not all agonists can induce heterodimerization, which was dependent upon ligand occupancy of a specific receptor subtype that can lead to alterations in pharmacology. Materials and Antisera—The peptides SST-14, d-Trp-SST-14, SST-28, and [Leu (8)-d-Trp-22, Tyr-25]-SST-28 (LTT-SST-28) were purchased from Bachem, Torrance, CA; Octreotide [SMS (201-995)] was given by Sandoz, Basel, Switzerland and des-AA1,2,5-[d-Trp8IAmp9]SS (SCH-275) was a gift from Dr. J. Rivier, Salk Institute. Fluorescein- and rhodamine-conjugated and unconjugated mouse monoclonal antibodies against hemagglutinin (HA) (12CA5) were purchased from Roche Applied Science. Anti-c-Myc monoclonal antibody was purchased from Sigma-Aldrich, Inc. Rabbit polyclonal antibodies directed against the N-terminal segment of hSSTR1 was generated and characterized as described (30Kumar U. Sasi R. Suresh S. Patel A. Thangaraju M. Metrakos P. Patel S.C. Patel Y.C. Diabetes. 1999; 48: 77-85Crossref PubMed Scopus (247) Google Scholar). Protein A/G-agarose beads were purchased from Oncogene Research Products, La Jolla, CA. SSTR Constructs and Expressing Cell Lines—Stable transfections of CHO-K1 cells expressing hSSTR5, hSSTR1, and both HA-tagged hSSTR5 and hSSTR1 and c-Myc-tagged hSSTR5 were prepared by LipofectAMINE transfection reagent as previously described (13Rocheville M. Lange D.C. Kumar U. Sasi R. Patel R.C. Patel Y.C. J. Biol. Chem. 2000; 275: 7862-7869Abstract Full Text Full Text PDF PubMed Scopus (470) Google Scholar). Chimeric receptors R1CR5 and R5CR1 were constructed by interchanging the C-tail of each receptor with one another. R1CR5 was created by adding the C-tail of hSSTR5, the last 46 residues, to hSSTR1 after residue 331. Similarly, R5CR1 includes the remaining 60 residues of hSSTR1 joined to hSSTR5 following residue 318 (31Hukovic N. Rocheville M. Kumar U. Sasi R. Khare S. Patel Y.C. J. Biol. Chem. 1999; 274: 24550-24558Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Clones were selected and maintained in CHO-K1 medium containing Hams F12 with 10% fetal bovine serum and 700 μg/ml neomycin. Stable transfections of CHO-K1 and HEK-293 cells co-expressing hSSTR5 and hSSTR1 were made using the vectors pCDNA3.1/Neo (neomycin resistance) and pCDNA3.1/Hygro (hygromycin resistance) such that hSSTR5 was cloned into pCDNA3.1/Hygro and hSSTR1 was cloned into pCDNA3.1/Neo. Stable transfections were selected in CHO-K1 medium containing 700 μg/ml of neomycin with 500 μg/ml of hygromycin or, HEK-293 medium containing 700 μg/ml of neomycin and 400 μg/ml of hygromycin. Fluorescent SST Ligands—Fluorescent-labeled SST ligands were prepared by N-terminal conjugation of d-Trp-SST-14 to fluorescein by the use of fluorescein isothiocyanate (SST-FITC) and SST-14 to Texas Red by use of Texas Red succinimidyl ester (SST-TR). The reaction of the dye with the ligand was performed in 0.2 m sodium bicarbonate, pH 7.5for 4 h at 4 °C in the absence of light. The reaction was stopped with 1.5 m hydroxylamine followed by HPLC separation as previously described (32Nouel D. Gaudriault G. Houle M. Reisine T. Vincent J.P. Mazella J. Beaudet A. Endocrinology. 1997; 138: 296-306Crossref PubMed Scopus (103) Google Scholar). Binding Assays—Cells were harvested, homogenized using a glass homogenizer, and membranes were prepared by centrifugation as previously described (13Rocheville M. Lange D.C. Kumar U. Sasi R. Patel R.C. Patel Y.C. J. Biol. Chem. 2000; 275: 7862-7869Abstract Full Text Full Text PDF PubMed Scopus (470) Google Scholar, 31Hukovic N. Rocheville M. Kumar U. Sasi R. Khare S. Patel Y.C. J. Biol. Chem. 1999; 274: 24550-24558Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Binding studies were performed with 20–40 μg of membrane protein collected from CHO-K1 cells stably expressing the receptor constructs, and 125I-labeled LTT-SST-28 radioligand (∼60 pm) in 50 mm HEPES, pH 7.5, 2 mm CaCl2, 5 mm MgCl2, 0.5% bovine serum albumin, 0.02% phenylmethylsulfonyl fluoride, and 0.02% bacitracin (binding buffer) for 30 min at 37 °C. Incubations were terminated by the addition of ice-cold binding buffer. Membrane pellets were quantified for radioactivity using a LKB gamma counter (LKB-Wallach, Turku, Finland). Binding data were analyzed with Prism 3.0 (Graph Pad Software, San Diego, CA) by non-linear regression analysis. Coupling to Adenylyl Cyclase—Cells were grown in 6-multiwell plates and tested for receptor coupling to adenylyl cyclase by incubation for 30 min with 20 μm forskolin and 0.5 mm 3-isobutyl-1-methylxanthine with or without agonists (10–11–10–6m) at 37 °C as previously described (31Hukovic N. Rocheville M. Kumar U. Sasi R. Khare S. Patel Y.C. J. Biol. Chem. 1999; 274: 24550-24558Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Cells were then scraped in 0.1 n HCl and quantified for cAMP by radioimmunoassay using a cAMP Kit (Inter Medico, Markham, ON) following the manufacturer's guidelines. Data were analyzed by non-linear regression analysis using Prism 3.0 (Graph Pad Software). S.E. are representative of at least three independent experiments. PbFRET Microscopy and Immunocytochemistry—PbFRET experiments were performed on CHO-K1 cells as previously described (13Rocheville M. Lange D.C. Kumar U. Sasi R. Patel R.C. Patel Y.C. J. Biol. Chem. 2000; 275: 7862-7869Abstract Full Text Full Text PDF PubMed Scopus (470) Google Scholar, 14Patel R.C. Kumar U. Lamb D.C. Eid J.S. Rocheville M. Grant M. Rani A. Hazlett T. Patel S.C. Gratton E. Patel Y.C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 3294-3299Crossref PubMed Scopus (178) Google Scholar, 29Rocheville M. Lange D.C. Kumar U. Patel S.C. Patel R.C. Patel Y.C. Science. 2000; 288: 154-157Crossref PubMed Scopus (755) Google Scholar, 33Patel R.C. Lange D.C. Patel Y.C. Methods. 2002; 27: 240-248Crossref Scopus (44) Google Scholar) stably co-expressing HA-hSSTR5 and hSSTR1, and mono-expressing hSSTR5, hSSTR1, and the receptor chimeras. The effective FRET efficiency (E) was calculated in terms of a percent based upon the photobleaching (pb) time constants of the donor taken in the absence (D–A) and presence (D + A) of acceptor according to E = 1 – (τD – A/τD+A) × 100. CHO-K1 cells expressing HA-hSSTR5 and hSSTR1 were grown on glass coverslips for 24 h, treated with different concentrations of agonist for 15 min at 37 °C and fixed with 4% paraformaldehyde for 20 min on ice and processed for immunocytochemistry. Antibodies used were mouse monoclonal anti-HA antibody conjugated to Rhodamine directed to HA-hSSTR5 and rabbit primary antibody followed by secondary anti-rabbit IgG antibody conjugated to fluorescein-directed to hSSTR1. pbFRET in CHO-K1 cells expressing hSSTR1, hSSTR5 and the chimera receptors was performed using fluorescently labeled SST ligands. Cells were grown on coverslips as mentioned above, treated with either 20 nm SST-FITC or 20 nm SST-FITC and 20 nm SST-TR. Both reactions, either antibody or ligand resulted in specific staining at the plasma membrane. The plasma membrane region was used to analyze the photobleaching decay on a pixel-by-pixel basis as described earlier (13Rocheville M. Lange D.C. Kumar U. Sasi R. Patel R.C. Patel Y.C. J. Biol. Chem. 2000; 275: 7862-7869Abstract Full Text Full Text PDF PubMed Scopus (470) Google Scholar, 33Patel R.C. Lange D.C. Patel Y.C. Methods. 2002; 27: 240-248Crossref Scopus (44) Google Scholar). Co-immunoprecipitation and Western Blot—Membranes from HA-hSSTR1, HA-SSTR5, and HA-hSSTR1/c-Myc-hSSTR5 stably transfected in HEK-293 cells were prepared using a glass homogenizer in 20 mm Tris-HCl, 2.5 mm dithiothreitol, pH 7.5 as previously described (13Rocheville M. Lange D.C. Kumar U. Sasi R. Patel R.C. Patel Y.C. J. Biol. Chem. 2000; 275: 7862-7869Abstract Full Text Full Text PDF PubMed Scopus (470) Google Scholar). The membrane pellet was washed and resuspended in 20 mm Tris-HCl, pH 7.5 in the absence of dithiothreitol. Membrane protein (300 μg) was treated with SST-14 (0 and 10–6m) in binding buffer (50 mm Hepes, 2 mm CaCl2, 5 mm MgCl2, pH 7.5) for 30 min at 37 °C. Following treatment membrane protein was solubilized in 1 ml of radioimmune precipitation assay buffer (150 mm NaCl, 50 mm Tris-HCL, 1% Nonidet P-40, 0.1% SDS, 0.5% sodium deoxycholate, pH 8.0) for 1 h at 4 °C. Samples were centrifuged, and lysate was collected and incubated with 1 μg of anti-HA antibody overnight at 4 °C. Antibody was immunoprecipitated with 20 μl of protein A/G-agarose beads for 2 h at 4 °C. Beads were then washed three times in radioimmune precipitation assay buffer before being solubilized in Laemmli sample buffer containing 62.5 mm Tris-HCl (pH 6.8), 25% glycerol, 2% SDS, 0.01% bromphenol blue, and 710 mm 2-mercaptoethanol (Bio-Rad). The sample was heated at 85 °C for 5 min before being fractionated by electrophoresis on a 7% SDS-polyacrylamide gel. The fractionated proteins were transferred by electrophoresis to a 0.2 μm nitrocellulose membrane (Trans-Blot Transfer Medium, Bio-Rad) in transfer buffer consisting of 25 mm Tris, 192 mm glycine, and 20% methanol. Membrane was blotted with anti-HA antibody (dilution 1:5000) for detection of HA-hSSTR1 and HA-hSSTR5 from single expressions, and anti-c-Myc antibody (1:5000) for detection of c-Myc-hSSTR5 from co-expressions. Blocking of membrane, incubation of primary antibodies, incubation of secondary antibodies, and detection by chemiluminescence were performed following WesternBreeze® (Invitrogen Life Technologies) according to manufacturer's instructions. Images were captured using an Alpha Innotech FluorChem 8800 (Alpha Innotech Co., San Leandro, CA) gel box imager and densitometry was carried out using FluorChem software (Alpha Innotech Co.). Ligand-dependent Heterodimerization of hSSTR1 and hSSTR5 by pbFRET—To study the heterodimerization of hSSTRs, we stably expressed hSSTR5 with an N-terminal HA tag and wild-type hSSTR1 in CHO-K1 cells (Bmax 395 ± 12 fmol/mg of protein; KD, 2.3 ± 0.1 nm). Cells were treated with various concentrations of the agonists: SST-14, SST-28, endogenous agonists for both the receptors, SCH-275 (subtype-agonist for hSSTR1) and SMS 201-995 (subtype-agonist for hSSTR5) for 15 min. Treatment was terminated by putting the cells on ice, washing once with phosphate-buffered saline followed by fixing in 4% paraformaldehyde for 20 min. To determine the physical association between the two receptors, we performed pbFRET microscopy on the cells by using a primary antibody followed by a secondary antibody conjugated with fluorescein (donor) to hSSTR1 and an anti-HA monoclonal antibody conjugated with rhodamine (acceptor) to hSSTR5. A panel of images depicting the co-expression of both receptor subtypes within the same cell is shown in Fig. 1. The decrease in donor fluorescence intensity due to photobleaching during prolonged exposure to excitation light was monitored in the absence and presence of acceptor fluorophore. Delays in the photobleaching decay of the donor in the presence of the acceptor related to an increase in FRET efficiency. Because FRET occurs at distances between 10–100 Å, it is a direct measure of protein-protein interaction. By taking a series of digital photographs, we analyzed the photobleaching decay of the donor on the surface of cell membranes on a pixel-by-pixel basis (Fig. 1, B and C). Cells were treated with different concentrations of four agonists, which displayed differences in their ability to induce heterodimerization. As shown in Fig. 2, in absence of agonist, a low relative FRET efficiency (<3%) was present in each condition. Treatment of SST-14 resulted in a concentration-dependent increase in heterodimer formation as indicated by increases in FRET efficiency. A maximum of 13.0 ± 1.1% at 10–6m was achieved possibly suggesting a saturation in the response (EC50 of 3.4 ± 2.1 nm) (Fig. 2A). A similar phenomenon was observed for SST-28, which also induced a concentration-dependent increase in FRET efficiency however with greater efficacy (EC50 0.14 ± 0.04 nm) (Fig. 2B). This may indicate that SST-28 is a more potent agonist at inducing heterodimerization than is SST-14. The hSSTR5 subtype agonist SMS 201-995, although capable of promoting heterodimerization, did so at much higher concentrations as determined by its EC50 value (EC50 119 ± 16 nm) (Fig. 2C). One possible explanation for this event could be that SMS 201-995 favors the formation of hSSTR5 homodimers than heterodimers; however, further studies are required. In contrast, treatment with the hSSTR1 subtype agonist SCH-275, did not result in significant increases in FRET efficiency (Fig. 2D). These results demonstrate that hSSTR1 is unable to promote heterodimerization. To further illustrate the active contribution of hSSTR5 in heterodimerization, we performed Western blot and co-immunoprecipitation on membranes prepared from cells either individually or co-expressing the two receptors.Fig. 2Concentration-dependent increase in effective FRET efficiencies from CHO-K1 cells stably expressing HA-hSSTR5 and hSSTR1 by different agonists. Cells were treated with the indicated concentrations of each agonist and analyzed by pbFRET microscopy. The calculated FRET efficiencies (%) and EC50 values for each agonist (A) SST-14 (3.4 ± 2.1 nm), (B) SST-28 (0.14 ± 0.04 nm), (C) SMS 201-995 (119 ± 16 nm) were plotted and analyzed by a sigmoidal dose-response equation using Graph Pad Prism 3.0. D, treatment with SCH-275 did not result in a significant increase in FRET efficiency. Data were analyzed by ANOVA, posthoc Dunnett's and compared with basal conditions without treatment. Means ± S.E. are representative of three independent experiments performed in triplicate; *, p < 0.05; **, p < 0.01.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Western Blot on Ligand-activated hSSTRs—To verify the receptor subtype actively involved in the heteromeric assembly of hSSTR1 and hSSTR5, we performed co-immunoprecipitation and Western blot on membranes from HEK-293 cells mono- and co-expressing the two receptors. In the absence of SST-14, hSSTR5 was found mainly as a monomer (∼55 kDa) (Fig. 3). Treatment with SST-14 resulted in the formation of dimers (∼110 kDa) including higher order oligomers (Fig. 3). A similar phenomenon was reported for hSSTR5 transfected in CHO-K1 cells, whereby agonist induced the dimerization of the receptor (13Rocheville M. Lange D.C. Kumar U. Sasi R. Patel R.C. Patel Y.C. J. Biol. Chem. 2000; 275: 7862-7869Abstract Full Text Full Text PDF PubMed Scopus (470) Google Scholar). Unlike hSSTR5, hSSTR1 did not form dimers in response to agonist nor was it self-associated under basal conditions (Fig. 3). This is in agreement with a previous report on hSSTR1 showing that it remained monomeric even in the presence of agonist in live cells using FCS (14Patel R.C. Kumar U. Lamb D.C. Eid J.S. Rocheville M. Grant M. Rani A. Hazlett T. Patel S.C. Gratton E. Patel Y.C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 3294-3299Crossref PubMed Scopus (178) Google Scholar). Co-immunoprecipitation of membranes expressing both receptor subtypes resulted in the detection of a weak band in the absence of agonist, however, upon agonist stimulation a strong signal was detected (∼115 kDa) indicating heterodimeric interaction (Fig. 3). Taken together these results and those obtained by pbFRET (Fig. 2), suggest that hSSTR1 is not actively involved in heterodimeric assembly. Membrane Binding Analysis of the hSSTR1 and hSSTR5 Heterodimer—To determine whether heterodimerization altered the binding properties of the receptors, we compared the binding constants for each agonist. Membranes were collected from CHO-K1 cells stably expressing hSSTR1, hSSTR5, and from cells co-expressing the two receptors. Saturation analysis with the radioligand 125I-LTT-SST-28 gave a Bmax of 415 ± 14 fmol/mg of protein and a KD of 0.49 ± 0.08 nm from membranes of the co-transfectants and Bmax and KD values of 284 ± 5 fmol/mg, 1.4 ± 0.05 nm and 231 ± 25 fmol/mg, 1.1 ± 0.15 nm for membranes transfected with hSSTR5 and hSSTR1 respectively. Binding constants represented as Ki values for each of the four agonists from each receptor species are shown in Table I. Heteromeric assembly of hSSTR1/hSSTR5 did not result in changes in the Ki values for SST-14 as determined by the lack of statistical significance when compared with the individual receptors. Although th" @default.
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• W2056556818 title "The Role of Subtype-specific Ligand Binding and the C-tail Domain in Dimer Formation of Human Somatostatin Receptors" @default.
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