A β-Arrestin Binding Determinant Common to the Second Intracellular Loops of Rhodopsin Family G Protein-coupled Receptors
2005; Elsevier BV; Volume: 281; Issue: 5 Linguagem: Inglês
10.1074/jbc.m508074200
ISSN1083-351X
AutoresSébastien Marion, Robert H. Oakley, Kyeong Man Kim, Marc G. Caron, Larry S. Barak,
Tópico(s)Photoreceptor and optogenetics research
Resumoβ-Arrestins have been shown to inhibit competitively G proteindependent signaling and to mediate endocytosis for many of the hundreds of nonvisual rhodopsin family G protein-coupled receptors (GPCR). An open question of fundamental importance concerning the regulation of signal transduction of several hundred rhodopsin-like GPCRs is how these receptors of limited sequence homology, when considered in toto, can all recruit and activate the two highly conserved β-arrestin proteins as part of their signaling/desensitization process. Although the serine and threonine residues that form GPCR kinase phosphorylation sites are common β-arrestin-associated receptor determinants regulating receptor desensitization and internalization, the agonist-activated conformation of a GPCR probably reveals the most fundamental determinant mediating the GPCR and arrestin interaction. Here we identified a β-arrestin binding determinant common to the rhodopsin family GPCRs formed from the proximal 10 residues of the second intracellular loop. We demonstrated by both gain and loss of function studies for the serotonin 2C, β2-adrenergic, α2a-adrenergic, and neuropeptide Y type 2 receptorsthat the highly conserved amino acids, proline and alanine, naturally occurring in rhodopsin family receptors six residues distal to the highly conserved second loop DRY motif regulate β-arrestin binding and β-arrestin-mediated internalization. In particular, as demonstrated for the β2 AR, this occurs independently of changes in GPCR kinase phosphorylation. These results suggest that a GPCR conformation directed by the second intracellular loop, likely using the loop itself as a binding patch, may function as a switch for transitioning β-arrestin from its inactive form to its active receptor-binding state. β-Arrestins have been shown to inhibit competitively G proteindependent signaling and to mediate endocytosis for many of the hundreds of nonvisual rhodopsin family G protein-coupled receptors (GPCR). An open question of fundamental importance concerning the regulation of signal transduction of several hundred rhodopsin-like GPCRs is how these receptors of limited sequence homology, when considered in toto, can all recruit and activate the two highly conserved β-arrestin proteins as part of their signaling/desensitization process. Although the serine and threonine residues that form GPCR kinase phosphorylation sites are common β-arrestin-associated receptor determinants regulating receptor desensitization and internalization, the agonist-activated conformation of a GPCR probably reveals the most fundamental determinant mediating the GPCR and arrestin interaction. Here we identified a β-arrestin binding determinant common to the rhodopsin family GPCRs formed from the proximal 10 residues of the second intracellular loop. We demonstrated by both gain and loss of function studies for the serotonin 2C, β2-adrenergic, α2a-adrenergic, and neuropeptide Y type 2 receptorsthat the highly conserved amino acids, proline and alanine, naturally occurring in rhodopsin family receptors six residues distal to the highly conserved second loop DRY motif regulate β-arrestin binding and β-arrestin-mediated internalization. In particular, as demonstrated for the β2 AR, this occurs independently of changes in GPCR kinase phosphorylation. These results suggest that a GPCR conformation directed by the second intracellular loop, likely using the loop itself as a binding patch, may function as a switch for transitioning β-arrestin from its inactive form to its active receptor-binding state. In the eye the light-induced signaling mediated through the G protein transducin is competitively blocked by the binding of visual arrestin to rhodopsin. An analogous paradigm is repeated outside the visual system to terminate G protein-mediated signaling for rhodopsin family GPCRs 5The abbreviations used are: GPCR, G protein-coupled receptor; 5HT, 5-hydroxytryptamine;α2aAR,α2a-adrenergic receptor;β2AR,β2-adrenergic receptor; GFP, green fluorescent protein; GRK, G protein-coupled receptor kinase; NPY, neuropeptide Y; GTPγS, guanosine 5′-3-O-(thio) triphosphate; ELISA, enzyme-linked immunosorbent assay. 5The abbreviations used are: GPCR, G protein-coupled receptor; 5HT, 5-hydroxytryptamine;α2aAR,α2a-adrenergic receptor;β2AR,β2-adrenergic receptor; GFP, green fluorescent protein; GRK, G protein-coupled receptor kinase; NPY, neuropeptide Y; GTPγS, guanosine 5′-3-O-(thio) triphosphate; ELISA, enzyme-linked immunosorbent assay. except visual arrestin is replaced by β-arrestins. Variations inβ-arrestin affinity for individual receptors and between different GPCRs primarily rest on the following two factors: the agonist-induced conformation of the receptor, and the ability of G protein-coupled receptor kinases (GRK) to phosphorylate serine and threonine residues on the C-tail and third intracellular loop of a receptor (1Krupnick J.G. Gurevich V.V. Schepers T. Hamm H.E. Benovic J.L. J. Biol. Chem. 1994; 269: 3226-3232Abstract Full Text PDF PubMed Google Scholar, 2Gurevich V.V. Pals-Rylaarsdam R. Benovic J.L. Hosey M.M. Onorato J.J. J. Biol. Chem. 1997; 272: 28849-28852Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 3Oakley R.H. Laporte S.A. Holt J.A. Barak L.S. Caron M.G. J. Biol. Chem. 2001; 276: 19452-19460Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar, 4Raman D. Osawa S. Gurevich V.V. Weiss E.R. J. Neurochem. 2003; 84: 1040-1050Crossref PubMed Scopus (30) Google Scholar, 5Oakley R.H. Laporte S.A. Holt J.A. Barak L.S. Caron M.G. J. Biol. Chem. 1999; 274: 32248-32257Abstract Full Text Full Text PDF PubMed Scopus (446) Google Scholar).Receptor agonist-induced phosphorylation has long been demonstrated to be of great importance for β-arrestin binding, being initially described for visual arrestin binding of the phosphorylated MII state of light-activated rhodopsin (6Gibson S.K. Parkes J.H. Liebman P.A. Biochemistry. 2000; 39: 5738-5749Crossref PubMed Scopus (64) Google Scholar, 7Kuhn H. Hall S.W. Wilden U. FEBS Lett. 1984; 176: 473-478Crossref PubMed Scopus (273) Google Scholar, 8Schleicher A. Kuhn H. Hofmann K.P. Biochemistry. 1989; 28: 1770-1775Crossref PubMed Scopus (166) Google Scholar). More recently, the formation of stable β-arrestin complexes with agonist-activated GPCRs has been shown to require phosphorylation of serine and threonine clusters located in the C-terminal tails of the receptor (3Oakley R.H. Laporte S.A. Holt J.A. Barak L.S. Caron M.G. J. Biol. Chem. 2001; 276: 19452-19460Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar). It has been proposed that in addition to phosphorylation, high affinity arrestin/receptor binding also requires receptor determinants that are exposed only in the active receptor conformation (9Gurevich V.V. Benovic J.L. J. Biol. Chem. 1993; 268: 11628-11638Abstract Full Text PDF PubMed Google Scholar, 10Gurevich V.V. Gurevich E.V. Trends Pharmacol. Sci. 2004; 25: 105-111Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar). Supporting this alternative are observations that agonist-activated GPCRs bind β-arrestins even in the absence of GRK phosphorylation (2Gurevich V.V. Pals-Rylaarsdam R. Benovic J.L. Hosey M.M. Onorato J.J. J. Biol. Chem. 1997; 272: 28849-28852Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). This phosphorylation-independent binding suggests that determinants, resulting from conserved primary amino acid sequences or protein secondary structural motifs, exist in all GPCRs to regulate receptor/arrestin association. However, the receptor regions that would comprise these arrestin-binding motifs have not been thoroughly defined, perhaps as a result of the sequence variability occurring throughout the entire GPCR family and the absence of crystal structure data other than for rhodopsin.GPCRs are structurally similar in their seven transmembrane architecture and share behaviors that originate from commonly occurring stretches of amino acids. The (E/D) RY motif, one of the most conserved of these sequences in the large family of rhodopsin-like GPCRs, is found at the cytoplasmic/intracellular loop junction of transmembrane III. The DRY motif presumably mediates interactions with both G proteins and arrestins and serves to maintain the receptor transmembranes in an inactive conformation in the absence of ligand (11Barak L.S. Tiberi M. Freedman N.J. Kwatra M.M. Lefkowitz R.J. Caron M.G. J. Biol. Chem. 1994; 269: 2790-2795Abstract Full Text PDF PubMed Google Scholar, 12Scheer A. Costa T. Fanelli F. De Benedetti P.G. Mhaouty-Kodja S. Abuin L. Nenniger-Tosato M. Cotecchia S. Mol. Pharmacol. 2000; 57: 219-231PubMed Google Scholar, 13Rasmussen S.G. Jensen A.D. Liapakis G. Ghanouni P. Javitch J.A. Gether U. Mol. Pharmacol. 1999; 56: 175-184Crossref PubMed Scopus (193) Google Scholar, 14Li J. Huang P. Chen C. de Riel J.K. Weinstein H. Liu-Chen L.Y. Biochemistry. 2001; 40: 12039-12050Crossref PubMed Scopus (66) Google Scholar, 15Barak L.S. Wilbanks A.M. Caron M.G. Assay Drug. Dev. Technol. 2003; 1: 339-346Crossref PubMed Scopus (21) Google Scholar). Scattered residues on the first two rhodopsin intracellular loops have been identified as contributing to visual arrestin binding exclusive of the phosphorylated rhodopsin C-tail (4Raman D. Osawa S. Gurevich V.V. Weiss E.R. J. Neurochem. 2003; 84: 1040-1050Crossref PubMed Scopus (30) Google Scholar, 16Shi W. Sports C.D. Raman D. Shirakawa S. Osawa S. Weiss E.R. Biochemistry. 1998; 37: 4869-4874Crossref PubMed Scopus (41) Google Scholar, 17Raman D. Osawa S. Weiss E.R. Biochemistry. 1999; 38: 5117-5123Crossref PubMed Scopus (41) Google Scholar). In particular, a proline residue in the rhodopsin second intracellular loop distal to the ERY motif is involved (4Raman D. Osawa S. Gurevich V.V. Weiss E.R. J. Neurochem. 2003; 84: 1040-1050Crossref PubMed Scopus (30) Google Scholar, 17Raman D. Osawa S. Weiss E.R. Biochemistry. 1999; 38: 5117-5123Crossref PubMed Scopus (41) Google Scholar). In addition, computational modeling of molecular docking between rhodopsin and G proteins highlights that this proline and other residues of the second loop may directly engage transducin (18Filipek S. Krzysko K.A. Fotiadis D. Liang Y. Saperstein D.A. Engel A. Palczewski K. Photochem. Photobiol. Sci. 2004; 3: 628-638Crossref PubMed Scopus (153) Google Scholar). Although many of the biochemical behaviors observed for rhodopsin in the visual system generalize to the larger subfamily of class I rhodopsin-like GPCRs, significant differences remain. For example, regulatory behavior in nonvisual cell systems that does not normally apply to rhodopsin includes β-arrestin, clathrin-mediated receptor endocytosis (19Luttrell L.M. Lefkowitz R.J. J. Cell. Sci. 2002; 115: 455-465Crossref PubMed Google Scholar, 20Ferguson S.S. Pharmacol. Rev. 2001; 53: 1-24PubMed Google Scholar). Thus, in the absence of appropriate crystallographic data, the extent to which a rhodopsin paradigm applies to nonvisual GPCRs is unclear (21Demene H. Granier S. Muller D. Guillon G. Dufour M.N. Delsuc M.A. Hibert M. Pascal R. Mendre C. Biochemistry. 2003; 42: 8204-8213Crossref PubMed Scopus (24) Google Scholar, 22Chung D.A. Zuiderweg E.R. Fowler C.B. Soyer O.S. Mosberg H.I. Neubig R.R. Biochemistry. 2002; 41: 3596-3604Crossref PubMed Scopus (35) Google Scholar).In this study we used several GPCRs to investigate the ability of naturally occurring proline and alanine residues, which are present 6 amino acids downstream of the DRY motif, to modulate β-arrestin/receptor interactions. β-Arrestin translocation and receptor endocytosis were used as a direct read-out to assess the effects of mutation at this position on β-arrestin/receptor association for serotonin 2C, β2-adrenergic, α2a-adrenergic, and neuropeptide Y2 receptors. Our data combined with sequence analysis of over 175 human rhodopsin family GPCRs suggest that in these receptors a contiguous 10-amino acid region beginning with the DRY motif forms a phosphorylation-independent structural determinant for binding β-arrestin.EXPERIMENTAL PROCEDURESMaterials—[3H]Adenine for measurement of cAMP generation and 125I-cyanopindolol for receptor binding were purchased from PerkinElmer Life Sciences, and [3H]CGP-12177 was from Amersham Biosciences. Isoproterenol, propranolol, neuropeptide Y, and SB206553 were from Sigma. Norepinephrine was from Bioanalytical Systems (West Lafayette, IN). The anti-phospho-β2AR (Ser-355/Ser-356) was from Santa Cruz Biotechnology (Santa Cruz, CA).Plasmids—FLAG- and GFP-tagged 5-HT2c nonedited receptors were described previously (21Demene H. Granier S. Muller D. Guillon G. Dufour M.N. Delsuc M.A. Hibert M. Pascal R. Mendre C. Biochemistry. 2003; 42: 8204-8213Crossref PubMed Scopus (24) Google Scholar). NPY2R receptor was cloned by total RNA extraction from mouse brain using ProtoScript First Strand cDNA synthesis kit from New England Biolabs. β2AR, NPY2R, and α2aAR refer to receptors with a hemagglutinin tag at the N terminus. Receptor cDNA containing mutations for the β2AR-P138A, 5HT2cR-P159A, α2aAR-A138P, and NPY2R-H159P were generated by standard PCR methods using a proofreading polymerase (Pfu; Stratagene). β-Arrestin2-GFP was made as described (23Barak L.S. Ferguson S.S. Zhang J. Caron M.G. J. Biol. Chem. 1997; 272: 27497-27500Abstract Full Text Full Text PDF PubMed Scopus (392) Google Scholar).Antagonist Binding, Agonist Binding, cAMP Assays, and GTPγS Binding—These procedures in which receptor expression levels were closely matched between experimental groups have been described previously (5Oakley R.H. Laporte S.A. Holt J.A. Barak L.S. Caron M.G. J. Biol. Chem. 1999; 274: 32248-32257Abstract Full Text Full Text PDF PubMed Scopus (446) Google Scholar, 22Chung D.A. Zuiderweg E.R. Fowler C.B. Soyer O.S. Mosberg H.I. Neubig R.R. Biochemistry. 2002; 41: 3596-3604Crossref PubMed Scopus (35) Google Scholar).Receptor Phosphorylation and Sequestration Assay—Receptor phosphorylation and sequestration assay in HEK-293 cells have been done as described previously (24Barak 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). Measurement of receptor surface expression by ELISA was performed under nonpermeabilized conditions (25Marion S. Weiner D.M. Caron M.G. J. Biol. Chem. 2004; 279: 2945-2954Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar).Microscopy and β-Arrestin Translocation—Confocal microscopy of HEK-293 cells containing either β-arrestin2-GFP and one of the β2AR, α2AR, or NPY2R receptor variants or the 5HT2cR-GFP wild type or mutant was performed as described (23Barak L.S. Ferguson S.S. Zhang J. Caron M.G. J. Biol. Chem. 1997; 272: 27497-27500Abstract Full Text Full Text PDF PubMed Scopus (392) Google Scholar) using a Zeiss LSM-510.RESULTSOccurrence of Conserved Residues in the Second Intracellular Loop of Class I GPCRs—Examination of the human GRCR data base (www.g-pcr.org) demonstrated 360 class I rhodopsin-like entries of which 244 contained glutamic/aspartic residues followed by arginine residues (E/D) R at the DRY motif. 175 entries were randomly selected from a list of predominantly deorphanized class I GPCRs and were evaluated for the frequency of residue occurrence at positions 1-23 of the second loop, with the lead DRY motif residue defined as position 1. The second loop amino acids were then grouped according to their charge or hydrophobic potential.Fig. 1A shows these relative frequencies determined for receptors where the lead second loop residue is an aspartic acid. (Note, where the lead residue was glutamic acid, a similar frequency result was obtained (data not shown).) The Arg/Lys/His group is the most probable to occur in this loop region, particularly at positions 2 and 8.Fig. 1B presents a plot of the next most common group, the hydrophobic residues Iso/Leu/Val/Phe in red plotted among the basic group (blue). In Fig. 1B, the red and blue horizontal dashed lines show the frequency at which a member of the hydrophobic or basic group, respectively, would be expected to occur randomly. It is notable that the hydrophobic group members Iso/Leu/Val/Phe occur at position 6 98% of the time, a level comparable with the 99% occurrence of arginine at position 2. It is also apparent from Fig. 1A that at position 9 a proline (Pro-9) or alanine (Ala-9) occurs 90% of the time.Fig. 1C summarizes the position frequency analysis in terms of a composite of the most probable receptor sequence. Sub-sequences formed from the first 12 and 11 positions of the 23 positions match the mouse olfactory receptor Olfr843 and human prokineticin receptor 1 exactly, and the most probable match for the entire sequence occurs with the second loop of the human galanin receptor. In the composite second loop, the first 10 residues are highly conserved with positions 1-3 the DRY motif, positions 4-7 and 10 hydrophobic amino acids, position 8 a basic residue, and position 9 a proline/alanine combination. The second loops from position 11 onward show variability with a tendency toward basic residues.Mutagenesis in the Second Loop—Mutation of Arg-2 in the DRY motif has been shown to enhance β-arrestin binding, resulting in constitutively activated/desensitized receptors (24Barak 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, 27Wilbanks A.M. Laporte S.A. Bohn L.M. Barak L.S. Caron M.G. Biochemistry. 2002; 41: 11981-11989Crossref PubMed Scopus (72) Google Scholar). However, the functional dependence on β-arrestin binding from point substitutions beyond the DRY motif in residues 4-10 has received scant attention, although alanine substitution for Pro-9 in rhodopsin was demonstrated to decrease visual arrestin binding (4Raman D. Osawa S. Gurevich V.V. Weiss E.R. J. Neurochem. 2003; 84: 1040-1050Crossref PubMed Scopus (30) Google Scholar, 17Raman D. Osawa S. Weiss E.R. Biochemistry. 1999; 38: 5117-5123Crossref PubMed Scopus (41) Google Scholar). Because the crystal structure of rhodopsin in its inactive conformation is known, we computationally modeled through the ExPASy Proteomics Server software DeepView (28Guex N. Peitsch M. Schweve T. Diemand A. DeepView/Swiss-Pdb-Viewer. GlaxoSmithKline, Uxbridge, UK2001Google Scholar) if Ala-9 substitution could preserve a Pro-9 like conformation for the second loop. We used the recently published structure of rhodopsin (Protein Data Bank access code 1U19) (29Okada T. Sugihara M. Bondar A.N. Elstner M. Entel P. Buss V. J. Mol. Biol. 2004; 342: 571-583Crossref PubMed Scopus (930) Google Scholar), which has a completely resolved polypeptide chain and is also in agreement with the model of the rhodopsin oligomer (30Filipek S. J. Mol. Model. 2005; 11: 385-391Crossref PubMed Scopus (11) Google Scholar, 31Fotiadis D. Liang Y. Filipek S. Saperstein D.A. Engel A. Palczewski K. Nature. 2003; 421: 127-128Crossref PubMed Scopus (656) Google Scholar, 32Teller D.C. Okada T. Behnke C.A. Palczewski K. Stenkamp R.E. Biochemistry. 2001; 40: 7761-7772Crossref PubMed Scopus (626) Google Scholar).Secondary Structure Resulting from Substitution of Proline 9 with Alanine Based on Rhodopsin Crystal Data—To assess local conformational changes in the second loop resulting from an alanine for proline substitution (Fig. 2A), the differences in the planar (ω) and rotational (ϕ and ψ) bond angles (in degrees) about the C-α carbon were calculated (Fig. 2B). Not only is the alanine-computed conformation of the second loop practically identical to the native one, perturbations in the bond angles quickly dampen within 1-2 residues of the proline (Fig. 2B). Moreover, this region formed with the side chains of residues 3-10 centered about Pro-9 (Fig. 2C) occupies the center of an accessible region at the lower intracellular face of rhodopsin that has been demonstrated as a docking determinant for the G protein transducin (18Filipek S. Krzysko K.A. Fotiadis D. Liang Y. Saperstein D.A. Engel A. Palczewski K. Photochem. Photobiol. Sci. 2004; 3: 628-638Crossref PubMed Scopus (153) Google Scholar).FIGURE 2Geometry of the rhodopsin second intracellular loop. A, second intracellular loop of bovine rhodopsin shown with the alanine substitution at position 9. B, difference in degrees between the angles omega, psi, and phi of the C-α carbon from the 2.2-Å rhodopsin crystal structure and the resulting structure because of alanine substitution at position 9. C, putative arrestin-interacting face formed from residues 3, 4, and 7-10 and centered on proline 9 of the second intracellular loop.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Effects of Naturally Occurring Variations and Induced Proline Substitution in the Second Intracellular Loop of a Constitutively Active Serotonin Receptor—Serotonin 5HT2c receptors naturally exist in several distinct protein isoforms secondary to RNA editing (33Niswender C.M. Sanders-Bush E. Emeson R.B. Ann. N. Y. Acad. Sci. 1998; 861: 38-48Crossref PubMed Scopus (76) Google Scholar, 34Burns C.M. Chu H. Rueter S.M. Hutchinson L.K. Canton H. Sanders-Bush E. Emeson R.B. Nature. 1997; 387: 303-308Crossref PubMed Scopus (853) Google Scholar). Editing affects the second intracellular loop region of the 5-HT2cR starting two residues distal to the DRY motif, resulting in at least 14 distinct isoforms with different degrees of constitutive activity (34Burns C.M. Chu H. Rueter S.M. Hutchinson L.K. Canton H. Sanders-Bush E. Emeson R.B. Nature. 1997; 387: 303-308Crossref PubMed Scopus (853) Google Scholar, 35Liu Y. Emeson R.B. Samuel C.E. J. Biol. Chem. 1999; 274: 18351-18358Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). RNA editing changes amino acids 6, 8, and 10 (Ile, Asn, and Ile) that surround the second loop Pro-9 and impairs the ability of 5-HT2cR isoforms to constitutively internalize in a β-arrestin-dependent manner (25Marion S. Weiner D.M. Caron M.G. J. Biol. Chem. 2004; 279: 2945-2954Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). We therefore investigated whether Pro-9 in the 5HT2cR played an active role in this β-arrestin regulation by assessing the functional consequences of a Pro-9 to Ala-9 substitution. Additionally, introducing an Ala-9 was motivated by the fact that alanine is the most likely natural replacement residue to occur at this position in GPCRs (Fig. 1A).We first investigated whether substitution of Pro-9 in the most constitutively active 5HT2cR isoform (i.e. nonedited INI isoform) could affect the subcellular distribution of the receptor. Fig. 3A shows a typical intracellular vesicular pattern of the constitutively internalized GFP-tagged 5HT2cR in the absence of agonist, a pattern that is dependent on its interaction with β-arrestin (21Demene H. Granier S. Muller D. Guillon G. Dufour M.N. Delsuc M.A. Hibert M. Pascal R. Mendre C. Biochemistry. 2003; 42: 8204-8213Crossref PubMed Scopus (24) Google Scholar). By contrast, the Ala-9 5HT2cR has a much more pronounced plasma membrane presence. To quantify the extent to which the proline/alanine substitution modifies 5HT2cR distribution, we measured by ELISA the cell surface expression of the receptors in the presence of the inverse agonist SB206553 (Fig. 3B), which has been proven to disrupt β-arrestin/5HT2cR constitutive interaction (21Demene H. Granier S. Muller D. Guillon G. Dufour M.N. Delsuc M.A. Hibert M. Pascal R. Mendre C. Biochemistry. 2003; 42: 8204-8213Crossref PubMed Scopus (24) Google Scholar). 15 min of SB206553 treatment resulted in an absolute 26 ± 3% increase of Pro-9 5HT2cR expression at the cell surface. No significant variation of the Ala-9 5HT2cR was detected. After 30 min of inverse agonist treatment, 30 ± 3 and 8 ± 3% increases in cell surface expression were obtained for the Pro-9 and Ala-9 5HT2cRs, respectively, suggesting that in contrast to the 5HT2cR-INI a majority of the Ala-9 5HT2cRs already resides at the plasma membrane in the absence of agonist and therefore interacts much less well with β-arrestins.FIGURE 3Effects of proline substitution in the second intracellular loop of the constitutively active 5HT2cR. A, representative confocal images of HEK-293 cells transiently transfected with 4 μg of either 5HT2cR-GFP (top panel) or 5HT2cR-P159A-GFP (bottom panel) are shown. Pictures were taken in basal conditions. B, cell surface expression of FLAG-5HT2cR (▪) or FLAG-5HT2c-P159A (□) was measured by ELISA before and after 15 and 30 min of inverse agonist treatment (SB206553 2.5 μm) at 37 °C. Data represent the mean of three independent experiments done in triplicate. C and E, co-immunoprecipitation (IP) of β-arrestin2 (βarr2)-GFP or Gαq with FLAG-5HT2cR or FLAG-5HT2c-P159A in transiently transfected HEK-293 cells. IB, immunoblot. D and F, quantification of β-arrestin2-GFP or Gαq that was co-immunoprecipitated in three independent experiments each normalized by the amount of receptor expressed is presented. Bar, 10 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The basal intracellular pattern showing 5HT2cR in vesicles is a reflection of the ability of the receptor to interact constitutively with β-arrestin2 (25Marion S. Weiner D.M. Caron M.G. J. Biol. Chem. 2004; 279: 2945-2954Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). Therefore, by using a co-immunoprecipitation strategy, we tested whether the proline substitution affects this constitutive association (Fig. 3C). Under basal conditions an alanine substitution of Pro-9 led to a 4-fold decrease in the amount of β-arrestin2-GFP co-immunoprecipitated by the 5HT2cR (Fig. 3, D and C).It is well established that 5HT2c receptors interact with Gq to stimulate phospholipase C (36Chang M.S. Tam J.P. Sanders-Bush E. Sci. STKE 2000. 2000; : PL1Google Scholar, 37Price R.D. Weiner D.M. Chang M.S. Sanders-Bush E. J. Biol. Chem. 2001; 276: 44663-44668Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar) and that receptor RNA editing silences constitutive activity by affecting G protein coupling efficiency (37Price R.D. Weiner D.M. Chang M.S. Sanders-Bush E. J. Biol. Chem. 2001; 276: 44663-44668Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 38Niswender C.M. Copeland S.C. Herrick-Davis K. Emeson R.B. Sanders-Bush E. J. Biol. Chem. 1999; 274: 9472-9478Abstract Full Text Full Text PDF PubMed Scopus (296) Google Scholar). Consequently, we investigated by co-immunoprecipitation of the receptor with Gq (Fig. 3E) whether Pro-9 substitution would also affect Gq coupling to the constitutively active 5HT2cR. Under basal conditions and compared with the wild type, the Ala-9 5HT2cR produced a 4-fold decrease of co-immunoprecipitated Gq (Fig. 3F). Altogether these data provide strong evidence that the experimental substitution of Pro-9 as well as naturally occurring editing processes in positions 6, 8, and 10 regulate receptor coupling to the cognate G protein as well as β-arrestin interaction.Substitution of Proline 9 in β2-Adrenergic Receptor—We next evaluated whether the Pro-9 could modulate β-arrestin binding to a nonconstitutively active receptor. For this purpose we used the well characterized β2AR. We assessed ligand binding characteristics and G protein coupling for both wild type and mutant receptors (Table 1). The data presented in Table 1 are consistent with a relatively mild loss of G protein coupling for the Ala-9 β2AR. In order to assess in detail whether substitution of Pro-9 affects coupling between the receptor and its cognate G protein, the interaction between each receptor and the G protein was measured by receptor-stimulated binding of nonhydrolyzable GTP analogues (Fig. 4A). A 1 log decrease in affinity for the Ala-9 β2AR was observed (Table 1 and Fig. 4A).TABLE 1Binding characteristics of the Ala-9 and Pro-9 β2 ARsKD 125I-CypKhigh for isoR in high affinity statepmnm%Pro-9 β2AR61 ± 133.4 ± 1.539 ± 2Ala-9 β2AR26 ± 334 ± 1016 ± 1Maximum amount of GTPγS bindingGTPγS EC50Maximum amount of cAMPEC50 for cAMP productionnmnmPro-9 β2AR1.00 ± 0.057 ± 31.00 ± 0.054.9 ± 1Ala-9 β2AR0.83 ± 0.0481 ± 191.34 ± 0.074.4 ± 2 Open table in a new tab FIGURE 4Effects of proline substitution in the second intracellular loop of the β2AR. A and B, G protein coupling and cAMP signaling of the wild type and second loop substitution mutant β2ARs. β2AR (▪) and β2AR-AF (○) containing membranes prepared from HEK-293 cells transfected with cDNA for the bovine Gαs subunit were exposed to increasing concentrations of isoproterenol and evaluated for GTPγS binding. B, HEK-293 cells transiently transfected with β2AR (▪) and β2AR-AF (○) were exposed to increasing concentrations of isoproterenol for 10 min at 37 °C. A and B results are presented relative to the plateau response of the wild type receptor being defined as 1.00 and presented as mean ± S.D. Data are representative of four independent experiments. C and D, β-arrestin2 (β-arr2) translocation to the β2AR and Pro-9 mutant β2ARs. HEK-293 cells were transfected with 2.5 μg of cDNA for the β2AR (toppanels), or β2AR-AF (bottompanels) in addition to 1μg of cDNA for β-arrestin2-GFP. β-Arrestin2-GFP translocation was assessed at 37 °C before and after 20 μm isoproterenol. D, cytosolic β-arrestin fluorescence of cells expressing either the β2AR (▪) or β2AR-AF (○) was measured as a mean density per pixel. Data were collected in four independent experiments from thr
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