Unraveling G Protein-coupled Receptor Endocytosis Pathways Using Real-time Monitoring of Agonist-promoted Interaction between β-Arrestins and AP-2
2007; Elsevier BV; Volume: 282; Issue: 40 Linguagem: Inglês
10.1074/jbc.m700577200
ISSN1083-351X
AutoresFadi F. Hamdan, Moulay Driss Rochdi, Billy Breton, Delphine Fessart, Douce E. Michaud, Pascale G. Charest, Stéphane A. Laporte, Michel Bouvier,
Tópico(s)Chemokine receptors and signaling
ResumoThe most widely studied pathway underlying agonist-promoted internalization of G protein-coupled receptors (GPCRs) involves β-arrestin and clathrin-coated pits. However, both β-arrestin- and clathrin-independent processes have also been reported. Classically, the endocytic routes are characterized using pharmacological inhibitors and various dominant negative mutants, resulting sometimes in conflicting results and interpretational difficulties. Here, taking advantage of the fact that β-arrestin binding to the β2 subunit of the clathrin adaptor AP-2 (β2-adaptin) is needed for the β-arrestin-mediated targeting of GPCRs to clathrin-coated pits, we developed a bioluminescence resonance energy transfer-based approach directly assessing the molecular steps involved in the endocytosis of GPCRs in living cells. For 10 of the 12 receptors tested, including some that were previously suggested to internalize via clathrin-independent pathways, agonist stimulation promoted β-arrestin 1 and 2 interaction with β2-adaptin, indicating a β-arrestin- and clathrin-dependent endocytic process. Detailed analyses of β-arrestin interactions with both the receptor and β2-adaptin also allowed us to demonstrate that recruitment of β-arrestins to the receptor and the ensuing conformational changes are the leading events preceding AP-2 engagement and subsequent clathrin-mediated endocytosis. Among the receptors tested, only the endothelin A and B receptors failed to promote interaction between β-arrestins and β2-adaptin. However, both receptors recruited β-arrestins upon agonist stimulation, suggesting a β-arrestin-dependent but clathrin-independent route of internalization for these two receptors. In addition to providing a new tool to dissect the molecular events involved in GPCR endocytosis, the bioluminescence resonance energy transfer-based β-arrestin/β2-adaptin interaction assay represents a novel biosensor to assess receptor activation. The most widely studied pathway underlying agonist-promoted internalization of G protein-coupled receptors (GPCRs) involves β-arrestin and clathrin-coated pits. However, both β-arrestin- and clathrin-independent processes have also been reported. Classically, the endocytic routes are characterized using pharmacological inhibitors and various dominant negative mutants, resulting sometimes in conflicting results and interpretational difficulties. Here, taking advantage of the fact that β-arrestin binding to the β2 subunit of the clathrin adaptor AP-2 (β2-adaptin) is needed for the β-arrestin-mediated targeting of GPCRs to clathrin-coated pits, we developed a bioluminescence resonance energy transfer-based approach directly assessing the molecular steps involved in the endocytosis of GPCRs in living cells. For 10 of the 12 receptors tested, including some that were previously suggested to internalize via clathrin-independent pathways, agonist stimulation promoted β-arrestin 1 and 2 interaction with β2-adaptin, indicating a β-arrestin- and clathrin-dependent endocytic process. Detailed analyses of β-arrestin interactions with both the receptor and β2-adaptin also allowed us to demonstrate that recruitment of β-arrestins to the receptor and the ensuing conformational changes are the leading events preceding AP-2 engagement and subsequent clathrin-mediated endocytosis. Among the receptors tested, only the endothelin A and B receptors failed to promote interaction between β-arrestins and β2-adaptin. However, both receptors recruited β-arrestins upon agonist stimulation, suggesting a β-arrestin-dependent but clathrin-independent route of internalization for these two receptors. In addition to providing a new tool to dissect the molecular events involved in GPCR endocytosis, the bioluminescence resonance energy transfer-based β-arrestin/β2-adaptin interaction assay represents a novel biosensor to assess receptor activation. G protein-coupled receptors (GPCRs) 7The abbreviations used are:GPCRG protein-coupled receptorAVP8-arginine-vasopressinAP-21967synthetic heterodimerizer binding with high affinity to both cyclophilin FKBP and FRB fragmentsAT1aRangiotensin II receptorβ2ARβ2-adrenergic receptorB2Rbradykinin B2 receptorBRETbioluminescence resonance energy transferC5acomplement component 5aC5aRthe complement component C5a receptorCCR5CC-chemokine 5 receptorDMEMDulbecco's modified Eagle's mediumdynI(K44A)dominant negative mutant of dynamin IEP4Rprostaglandin EP4 receptorET1endothelinETARendothelin A receptorETBRendothelin B receptorFKBPcyclophilin fragmentsFRBcyclophilin fragmentG418GeneticinGFPgreen fluorescent proteinHEKhuman embryonic kidney cellsHRPhorseradish peroxidaseM2RM2 muscarinic receptorPEIpolyethyleneimineRlucRenilla reniformis luciferaseV1aRV1a vasopressin receptorV2Rvasopressin V2 receptorVIPvasoactive intestinal peptide 1VIP1Rvasoactive intestinal peptide 1 receptorEYFPenhanced yellow fluorescent proteinPBSphosphate-buffered salineELISAenzyme-linked immunosorbent assayHAhemagglutininsiRNAsmall interference RNA.7The abbreviations used are:GPCRG protein-coupled receptorAVP8-arginine-vasopressinAP-21967synthetic heterodimerizer binding with high affinity to both cyclophilin FKBP and FRB fragmentsAT1aRangiotensin II receptorβ2ARβ2-adrenergic receptorB2Rbradykinin B2 receptorBRETbioluminescence resonance energy transferC5acomplement component 5aC5aRthe complement component C5a receptorCCR5CC-chemokine 5 receptorDMEMDulbecco's modified Eagle's mediumdynI(K44A)dominant negative mutant of dynamin IEP4Rprostaglandin EP4 receptorET1endothelinETARendothelin A receptorETBRendothelin B receptorFKBPcyclophilin fragmentsFRBcyclophilin fragmentG418GeneticinGFPgreen fluorescent proteinHEKhuman embryonic kidney cellsHRPhorseradish peroxidaseM2RM2 muscarinic receptorPEIpolyethyleneimineRlucRenilla reniformis luciferaseV1aRV1a vasopressin receptorV2Rvasopressin V2 receptorVIPvasoactive intestinal peptide 1VIP1Rvasoactive intestinal peptide 1 receptorEYFPenhanced yellow fluorescent proteinPBSphosphate-buffered salineELISAenzyme-linked immunosorbent assayHAhemagglutininsiRNAsmall interference RNA. are seven transmembrane domain receptors that constitute the largest family of cell surface proteins involved in signal transduction. In humans, it is estimated that GPCRs are encoded by ∼800 distinct genes that control a variety of important physiological responses (1Foord S.M. Bonner T.I. Neubig R.R. Rosser E.M. Pin J.P. Davenport A.P. Spedding M. Harmar A.J. Pharmacol. Rev. 2005; 57: 279-288Crossref PubMed Scopus (396) Google Scholar). Following agonist binding, GPCRs undergo conformational changes that regulate the activity of downstream effector systems to mediate various cellular responses. The extent and duration of GPCR signaling is tightly regulated by mechanisms that terminate the initial signaling and later re-establish the capacity of the receptors to respond to new agonist exposure. The removal of GPCRs from the cell surface, also known as internalization or endocytosis, plays an important role in these processes (2Claing A. Laporte S.A. Caron M.G. Lefkowitz R.J. Prog. Neurobiol. 2002; 66: 61-79Crossref PubMed Scopus (447) Google Scholar, 3Marchese A. Chen C. Kim Y.M. Benovic J.L. Trends Biochem. Sci. 2003; 28: 369-376Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). For most GPCRs, rapid feedback desensitization is initiated by G protein-coupled receptor kinases that phosphorylate agonist-occupied GPCRs to create high affinity binding sites for β-arrestins, which in turn uncouple the receptor from its cognate G protein (reviewed in Ref. 4Lefkowitz R.J. Shenoy S.K. Science. 2005; 308: 512-517Crossref PubMed Scopus (1408) Google Scholar). β-Arrestins also target receptors for endocytosis by linking them to the endocytic machinery, including clathrin and the clathrin adaptor AP-2 (5Edeling M.A. Mishra S.K. Keyel P.A. Steinhauser A.L. Collins B.M. Roth R. Heuser J.E. Owen D.J. Traub L.M. Dev Cell. 2006; 10: 329-342Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 6Goodman O.B. Krupnick Jr., J.G. Santini F. Gurevich V.V. Penn R.B. Gagnon A.W. Keen J.H. Benovic J.L. Nature. 1996; 383: 447-450Crossref PubMed Scopus (1154) Google Scholar, 7Kim Y.M. Benovic J.L. J. Biol. Chem. 2002; 277: 30760-30768Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 8Laporte S.A. Miller W.E. Kim K.M. Caron M.G. J. Biol. Chem. 2002; 277: 9247-9254Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 9Laporte S.A. Oakley R.H. Holt J.A. Barak L.S. Caron M.G. J. Biol. Chem. 2000; 275: 23120-23126Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar, 10Laporte S.A. Oakley R.H. Zhang J. Holt J.A. Ferguson S.S. Caron M.G. Barak L.S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3712-3717Crossref PubMed Scopus (520) Google Scholar, 11Santini F. Gaidarov I. Keen J.H. J. Cell Biol. 2002; 156: 665-676Crossref PubMed Scopus (91) Google Scholar). By controlling receptor recycling following endocytosis, β-arrestins have also been shown to regulate the rate of receptor resensitization (12Prossnitz E.R. Life Sci. 2004; 75: 893-899Crossref PubMed Scopus (43) Google Scholar). Based on their interaction with β-arrestins, GPCRs are divided into two main classes. Class A, which includes receptors such as the β2-adrenergic (β2AR), endothelin A (ETAR), and V1a vasopressin (V1aR), interacts transiently with β-arrestins and can rapidly recycle back from the endosomes to the cell surface (13Oakley R.H. Laporte S.A. Holt J.A. Caron M.G. Barak L.S. J. Biol. Chem. 2000; 275: 17201-17210Abstract Full Text Full Text PDF PubMed Scopus (673) Google Scholar, 14Terrillon S. Barberis C. Bouvier M. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 1548-1553Crossref PubMed Scopus (134) Google Scholar). Class B receptors, such as vasopressin V2 (V2R), CC-chemokine 5 (CCR5), prostaglandin EP4 (EP4R), and vasoactive intestinal peptide 1 (VIP1R), interact more stably with β-arrestins, leading to a complex that resides for extended periods of time into endosomes. Receptors tightly associated with β-arrestins in endosomes are only poorly recycled to the cell surface and eventually targeted for lysosomal degradation (13Oakley R.H. Laporte S.A. Holt J.A. Caron M.G. Barak L.S. J. Biol. Chem. 2000; 275: 17201-17210Abstract Full Text Full Text PDF PubMed Scopus (673) Google Scholar, 14Terrillon S. Barberis C. Bouvier M. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 1548-1553Crossref PubMed Scopus (134) Google Scholar, 15Fraile-Ramos A. Kohout T.A. Waldhoer M. Marsh M. Traffic. 2003; 4: 243-253Crossref PubMed Scopus (87) Google Scholar, 16Neuschafer-Rube F. Hermosilla R. Rehwald M. Ronnstrand L. Schulein R. Wernstedt C. Puschel G.P. Biochem. J. 2004; 379: 573-585Crossref PubMed Scopus (21) Google Scholar, 17Shetzline M.A. Walker J.K. Valenzano K.J. Premont R.T. J. Biol. Chem. 2002; 277: 25519-25526Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). The two classes of receptors also differ by their binding preference toward β-arrestin1 and β-arrestin2. Indeed, whereas class A receptors binds with greater affinity to β-arrestin2, class B receptors do not show preference between the two β-arrestins (13Oakley R.H. Laporte S.A. Holt J.A. Caron M.G. Barak L.S. J. Biol. Chem. 2000; 275: 17201-17210Abstract Full Text Full Text PDF PubMed Scopus (673) Google Scholar). More recently, it was suggested that some receptors may not be easily classified in class A or B. For example, the bradykinin B2 receptor (B2R) was found to internalize with β-arrestins into endosomes but can then dissociate from β-arrestin and efficiently recycle to the plasma membrane after agonist removal (18Simaan M. Bedard-Goulet S. Fessart D. Gratton J.P. Laporte S.A. Cell Signal. 2005; 17: 1074-1083Crossref PubMed Scopus (45) Google Scholar). The authors suggested that receptors with such hybrid features could be referred to as class C.Although the roles of β-arrestins and clathrin-coated vesicles in GPCR endocytosis have been well characterized, alternative pathways involving non-coated vesicles, such as caveolae, or other non-clathrin and non-caveolae mediated routes, have also been described for several receptors (reviewed in Refs. 2Claing A. Laporte S.A. Caron M.G. Lefkowitz R.J. Prog. Neurobiol. 2002; 66: 61-79Crossref PubMed Scopus (447) Google Scholar, 3Marchese A. Chen C. Kim Y.M. Benovic J.L. Trends Biochem. Sci. 2003; 28: 369-376Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar, 19Chini B. Parenti M. J. Mol. Endocrinol. 2004; 32: 325-338Crossref PubMed Scopus (305) Google Scholar). Also, the requirement for β-arrestin does not seem to be universal, and endocytosis of some GPCRs through either clathrin-coated vesicles or caveolae was proposed to be β-arrestin-independent (20Okamoto Y. Ninomiya H. Miwa S. Masaki T. J. Biol. Chem. 2000; 275: 6439-6446Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). To characterize the various endocytic routes used by different receptors, several pharmacological and biochemical tools have been used. These include blockers that do not discriminate between clathrin-dependent or caveolae-mediated endocytosis (e.g. hypertonic sucrose, low temperature, concanavalin-A, and dominant negative mutants of dynamin) as well as inhibitors that are believed to selectively inhibit clathrin-coated vesicle (e.g. monodansylcadaverine, chlorpromazine, as well as dominant negative mutants of β-arrestin and Eps-15) or caveolae-mediated endocytosis (e.g. filpin and nystatin) (3Marchese A. Chen C. Kim Y.M. Benovic J.L. Trends Biochem. Sci. 2003; 28: 369-376Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar, 19Chini B. Parenti M. J. Mol. Endocrinol. 2004; 32: 325-338Crossref PubMed Scopus (305) Google Scholar, 21Claing A. Perry S.J. Achiriloaie M. Walker J.K. Albanesi J.P. Lefkowitz R.J. Premont R.T. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1119-1124Crossref PubMed Scopus (144) Google Scholar, 22Heuser J.E. Anderson R.G. J. Cell Biol. 1989; 108: 389-400Crossref PubMed Scopus (768) Google Scholar). Although these approaches have been useful, the interpretation of their data was sometimes difficult and generated some controversies on the exact mechanism underlying the endocytosis of certain GPCRs. For example, inhibition of endocytosis by dominant negative mutants of β-arrestins (β-arrestin1(V53D), β-arrestin1-(319–418), and β-arrestin2(ΔLIELD/F391A) (7Kim Y.M. Benovic J.L. J. Biol. Chem. 2002; 277: 30760-30768Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 23Ferguson S.S. Downey W.E. Colapietro 3rd, A.M. Barak L.S. Menard L. Caron M.G. Science. 1996; 271: 363-366Crossref PubMed Scopus (839) Google Scholar, 24Krupnick J.G. Santini F. Gagnon A.W. Keen J.H. Benovic J.L. J. Biol. Chem. 1997; 272: 32507-32512Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar)) or a dominant negative of the AP-2 interacting protein Eps15 (25Benmerah A. Bayrou M. Cerf-Bensussan N. Dautry-Varsat A. J. Cell Sci. 1999; 112: 1303-1311Crossref PubMed Google Scholar) has often been used to suggest the involvement of a clathrin-mediated process. However, in the case of the ETAR, endocytosis through caveolae was also inhibited by a dominant negative mutant of β-arrestin (20Okamoto Y. Ninomiya H. Miwa S. Masaki T. J. Biol. Chem. 2000; 275: 6439-6446Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). Similarly, inhibiting Eps15 has been recently found to prevent caveolae-mediated endocytosis of the epidermal growth factor receptor (26Sigismund S. Woelk T. Puri C. Maspero E. Tacchetti C. Transidico P. Di Fiore P.P. Polo S. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 2760-2765Crossref PubMed Scopus (653) Google Scholar). Off-target effects of dominant negative mutants can also complicate interpretation of the results. For instance, whereas it was initially believed that β-arrestins were specific regulators for GPCRs, growing evidences demonstrate its role in the regulation of non-GPCRs membrane proteins endocytosis (27Chen W. Kirkbride K.C. How T. Nelson C.D. Mo J. Frederick J.P. Wang X.F. Lefkowitz R.J. Blobe G.C. Science. 2003; 301: 1394-1397Crossref PubMed Scopus (207) Google Scholar). Although this may reflect direct interaction of β-arrestins with non-GPCR proteins (28Dalle S. Ricketts W. Imamura T. Vollenweider P. Olefsky J.M. J. Biol. Chem. 2001; 276: 15688-15695Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar), it could also result from the interference with components of the endocytic machinery that could be shared by clathrin- and caveolae-mediated routes.The above discussion illustrates the importance of developing new tools that will allow the direct assessment of the molecular steps involved in the endocytosis of specific GPCRs. To generate one such tool, we took advantage of the fact that, for internalization, the β-arrestins binding to the β2-adaptin subunit of the clathrin adaptor AP-2 are needed for the β-arrestin-mediated targeting of GPCRs to clathrin-coated pits (7Kim Y.M. Benovic J.L. J. Biol. Chem. 2002; 277: 30760-30768Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 8Laporte S.A. Miller W.E. Kim K.M. Caron M.G. J. Biol. Chem. 2002; 277: 9247-9254Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 10Laporte S.A. Oakley R.H. Zhang J. Holt J.A. Ferguson S.S. Caron M.G. Barak L.S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3712-3717Crossref PubMed Scopus (520) Google Scholar, 29Orem N.R. Xia L. Dolph P.J. J. Cell Sci. 2006; 119: 3141-3148Crossref PubMed Scopus (38) Google Scholar). Specifically, we designed a bioluminescence resonance energy transfer (BRET)-based assay that allows real-time monitoring of the interaction between β-arrestins and β2-adaptin as a biosensor for β-arrestin-promoted clathrin-mediated endocytosis. BRET is a non-radiative energy transfer that occurs between the energy donor Renilla luciferase (Rluc) and variants of the green fluorescent proteins (EYFP for BRET1 and GFP, GFP2, or Tsapphire for BRET2) as the energy acceptor only when the two proteins are within 100 Å from one another and situated in favorable orientation, making it an attractive assay for studying both inducible and constitutive protein-protein interactions (30Hamdan F.F. Percherancier Y. Breton B. Bouvier M. Curr. Prot. Neurosci.. John Wiley & Sons, Inc., New York2006Google Scholar, 31Pfleger K.D. Eidne K.A. Biochem. J. 2004; 85: 625-637Google Scholar). Thus, in response to the activation of 12 different GPCRs, previously suggested to internalize via different endocytic routes, BRET1 was assessed between β-arrestin-Rluc and β2-adaptin-EYFP and used as an indicator of clathrin-mediated endocytosis involving β-arrestins. By combining the spectrally resolved BRET1 and BRET2 technologies, we could simultaneously assess the recruitment of β-arrestin to GPCR and β-arrestin/AP-2 interaction, which allowed us to monitor the kinetics of the two events. The data obtained herein show that some of the GPCRs previously suggested to internalize via a clathrin-independent pathway were capable of promoting β-arrestin interaction with AP-2. Thus, contrary to what was deduced from indirect methods, these receptors internalize via a β-arrestin- and clathrin-dependent mechanism. Other receptors, such as ETAR and ETBR, which are capable of recruiting β-arrestins but were shown to internalize via caveolae, did not promote β-arrestin interaction with β2-adaptin, indicating a β-arrestin-dependent but clathrin-independent endocytic process. In addition to providing a new tool to dissect the molecular events involved in GPCR endocytosis, the BRET-based β-arrestin/β2-adaptin interaction assay can also be used to detect constitutive receptor internalization and quantify receptor activation in a pharmacologically relevant manner.EXPERIMENTAL PROCEDURESMaterials—Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum, Geneticin (G418), l-glutamine, and penicillin-streptomycin were purchased from Wisent. Fugene6 was obtained from Roche Diagnostics. Coelenterazine-h was from Prolume, and DeepBlueC was from Biotium. Recombinant human RANTES (regulated on activation normal T cell expressed and secreted) was from PeproTech. Poly-d-lysine, 8-arginine-vasopressin (AVP), isoproterenol, bradykinin, human recombinant C5a, prostaglandin E2, vasoactive intestinal peptide, endothelin-1 (ET1), and carbachol were from Sigma-Aldrich Canada. Antibodies against β2-adaptin, α-adaptin, and clathrin were obtained from BD Biosciences. The anti-GFP was from Molecular Probes. White opaque and clear bottom 96-well plates were from Corning. Linear polyethyleneimine (25 kDa) was from Polysciences. Plate readers used to measure BRET in this study were the Mithras LB940 from Berthold for BRET1 and a modified TopCount from Packard for BRET2.Expression Plasmids—The constructs presented herein were made using standard molecular biology techniques employing PCR and fragment replacement strategies. β-Arrestin2-Rluc and β-arrestin1-Rluc were generated by PCR amplification of the coding sequences of the rat β-arrestin2 (kindly provided by S. Marullo, Institute Cochin, Paris) and the rat β-arrestin1 (a gift from Kathryn DeFea, University of California, Riverside) without their stop codons that were ligated upstream of a humanized Renilla reniformis luciferase (Rluc, originally PCR-amplified from phRluc-N1 from PerkinElmer Life Sciences) in pcDAN3.1zeo+ (Invitrogen); both β-arrestin1 and β-arrestin2 were fused to Rluc via the same 6-amino acid linker (GSGTAT). Rluc-β-arrestin2-EYFP construct was made as described by Charest et al. (42Charest P.G. Terrillon S. Bouvier M. EMBO Rep. 2005; 6: 334-340Crossref PubMed Scopus (147) Google Scholar). β-Arrestin2-(R393E,R395E)-Rluc DNA was obtained by subcloning the mutant β-arrestin2 into pcDNA3.1. V2R-EYFP construct was generated by amplifying V2R coding sequences by PCR without its stop codon and cloned upstream of EYFP in pRK5. β2-Adaptin-EYFP was generated by subcloning the coding region, minus the stop codon, and coming from a human β2-adaptin plasmid template (10Laporte S.A. Oakley R.H. Zhang J. Holt J.A. Ferguson S.S. Caron M.G. Barak L.S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3712-3717Crossref PubMed Scopus (520) Google Scholar) into pEYFP-N1 (Clontech). To construct V2R-Tsapphire, the coding sequence of Turbo Sapphire (32Zapata-Hommer O. Griesbeck O. BMC Biotechnol. 2003; 3: 5Crossref PubMed Scopus (126) Google Scholar) was PCR-amplified and fused, in-frame, downstream of the V2R coding sequence lacking its stop codon (which was removed from V2R-VENUS-pIRESpuro3 (33Hamdan F.F. Audet M. Garneau P. Pelletier J. Bouvier M. J. Biomol. Screen. 2005; 10: 463-475Crossref PubMed Scopus (162) Google Scholar)) and ligated into pcDNA3.1/zeo(+). EP4R and the complement component C5a receptor (C5aR) in pcDNA3.1(+) were from UMR cDNA Resource Center. The angiotensin II receptor (HA-AT1aR-pRc) expression construct was a gift from Dr. Sylvain Meloche (Université de Montréal, Montréal, Canada). The V2R-FKBP and FRB-β-arrestin2-Rluc (34Terrillon S. Bouvier M. EMBO J. 2004; 23: 3950-3961Crossref PubMed Scopus (97) Google Scholar), Myc-V2R(R137H) (35Barak L.S. Oakley R.H. Laporte S.A. Caron M.G. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 93-98Crossref PubMed Scopus (196) Google Scholar), HA-β2AR (36Hebert T.E. Moffett S. Morello J.P. Loisel T.P. Bichet D.G. Barret C. Bouvier M. J. Biol. Chem. 1996; 271: 16384-16392Abstract Full Text Full Text PDF PubMed Scopus (680) Google Scholar), dynI(K44A) (37Rochdi M.D. Parent J.L. J. Biol. Chem. 2003; 278: 17827-17837Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar), Myc-V2R, Myc-V1aR (38Terrillon S. Durroux T. Mouillac B. Breit A. Ayoub M.A. Taulan M. Jockers R. Barberis C. Bouvier M. Mol. Endocrinol. 2003; 17: 677-691Crossref PubMed Scopus (274) Google Scholar), M2 muscarinic receptor (HA-M2R) (21Claing A. Perry S.J. Achiriloaie M. Walker J.K. Albanesi J.P. Lefkowitz R.J. Premont R.T. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1119-1124Crossref PubMed Scopus (144) Google Scholar), vasoactive peptide 1 receptor (FLAG-VIP1R) (21Claing A. Perry S.J. Achiriloaie M. Walker J.K. Albanesi J.P. Lefkowitz R.J. Premont R.T. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1119-1124Crossref PubMed Scopus (144) Google Scholar), endothelin receptor subtypes A and B (HA-ETAR and FLAG-ETBR (39Freedman N.J. Ament A.S. Oppermann M. Stoffel R.H. Exum S.T. Lefkowitz R.J. J. Biol. Chem. 1997; 272: 17734-17743Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar)), CCR5 (18Simaan M. Bedard-Goulet S. Fessart D. Gratton J.P. Laporte S.A. Cell Signal. 2005; 17: 1074-1083Crossref PubMed Scopus (45) Google Scholar), and bradykinin receptor subtype 2 (HA-B2R) (40Mirzabekov T. Bannert N. Farzan M. Hofmann W. Kolchinsky P. Wu L. Wyatt R. Sodroski J. J. Biol. Chem. 1999; 274: 28745-28750Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar) expression constructs were previously described. Except for CCR5, EP4R, and C5aR, all GPCRs used in this study contained an N-terminal epitope tag (e.g. Myc, HA, or FLAG) that was previously shown not to compromise the activity of these receptors. For the purpose of brevity, unless stated otherwise, GPCRs used here will be identified by their names only without indicating their N-terminal epitope tag. All GPCRs used herein were from human cDNAs, except for the VIP1R, which was from rat.Cell Culture, Plasmid Transfections, and Stable Cell Line Production—Unless otherwise stated, human embryonic kidney (HEK) 293T cells were cultured in high glucose DMEM supplemented with 10% fetal bovine serum, 100 units/ml penicillin-streptomycin, and 2 mml-glutamine at 37 °C in a humidified chamber at 95% air and 5% CO2. For transient transfections in 6-well plates, 400,000 HEK293T cells were seeded and transfected the next day using Fugene6 according to manufacturer's recommendations. For transfections in 100-mm plates, cells were seeded at a density of 2.5 million cells/plate in DMEM containing 5% fetal bovine serum and transfected the next day using polyethyleneimine (PEI, 25-kDa molecular mass, linear form prepared at 1 mg/ml in sterile distilled H2O) at a DNA:PEI ratio of 1:3. Plasmid DNA and PEI were diluted, each in separate tubes, with 500 μl of NaCl solution (150 mm). The PEI solution was then added onto the DNA solution, vortexed at maximum speed for 5 s, and incubated at room temperature for 20 min prior to addition to the cells. To generate cells stably expressing β2-adaptin-EYFP (HEK293T/β2-adaptin-EYFP), transfected cells were selected with 1 mg/ml G418. Clonal cells expressing β2-adaptin-EYFP were obtained by limited dilution and were tested for expression of β2-adaptin-EYFP by fluorescence measurements and Western blotting. HEK293T cells stably expressing MycV2R (HEK293T/V2R) were generated by selection with 0.45 mg/ml G418, and a clonal cell line (41Charest P.G. Oligny-Longpre G. Bonin H. Azzi M. Bouvier M. Cell Signal. 2006; 19: 32-41Crossref PubMed Scopus (58) Google Scholar) expressing ∼8 pmol/mg of V2R was used for transient cotransfection of β2-adaptin-EYFP and β-arrestin2-Rluc.Immunoprecipitation and Western Blotting—Immunoprecipitation experiments were carried out as described previously (65Fessart D. Simaan M. Zimmerman B. Comeau J. Hamdan F.F. Wiseman P.W. Bouvier M. Laporte S.A. J. Cell Sci. 2007; 120: 1723-1732Crossref PubMed Scopus (38) Google Scholar). Immunoprecipitated proteins were separated on 6% SDS-PAGE, whereas proteins from total cell lysates were separated on 10% SDS-PAGE before transfer onto nitrocellulose membranes. Protein immunodetection on membranes was assessed using either anti-β2-adaptin (0.2 μg/ml), anti-α-adaptin (0.25 μg/ml), anti-clathrin (0.25 μg/ml), or anti-GFP (2 μg/ml) (65Fessart D. Simaan M. Zimmerman B. Comeau J. Hamdan F.F. Wiseman P.W. Bouvier M. Laporte S.A. J. Cell Sci. 2007; 120: 1723-1732Crossref PubMed Scopus (38) Google Scholar) antibodies.Total Fluorescence Measurements—To measure total fluorescence, cells were washed once with PBS-Mg (PBS containing 0.5 mm MgCl2), gently detached by pipetting in PBS-Mg, and seeded at a density of ∼100,000 cells/well in 96-well plates (white wall with clear bottom plates). All throughout this study, cell number quantification was based on measuring the A600 of the cell suspension and correlating it to a pre-established standard curve (A600 versus cell number). The fluorescence level was measured using a FlexStation II (Molecular Devices). For measuring fluorescence of V2R-Tsapphire, transfected cells were excited at 400 nm, and the emission was recorded at 511 nm (cut-off at 490 nm), whereas measurements of β2-adaptin-EYFP fluorescence were done at an excitation wavelength of 470 nm and emission wavelength of 535 nm (cut-off at 500 nm).Monitoring Protein-Protein Interaction Using BRET1—Approximately 18 h after transfection, cells were detached by trypsinization and seeded (∼50,000 cells/well) into 96-well (white wall, clear bottom) tissue culture plates previously treated with poly-d-lysine, and re-incubated at 37 °C for another 18 h. On the day of the experiment, the culture medium was replaced by PBS-Mg and incubated with or without various concentrations of the tested agonist for the specified time at room temperature. To measure the BRET1 signal, the transparent bottom of the 96-well plate was covered with a white-backed tape adhesive (PerkinElmer Life Sciences), and the BRET1 substrate for Rluc, coelenterazine-h, was added to all wells (5 μm final concentration), followed by BRET1 measurement on the Mithras LB940 plate reader, which allows the sequential integration of signals detected in the 480 ± 20 nm and 530 ± 20 nm windows. The BRET1 signal was calculated as a ratio of the light emitted by EYFP (530 ± 20 nm) over the light emitted b
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