The Second Intracellular Loop of the Calcitonin Gene-related Peptide Receptor Provides Molecular Determinants for Signal Transduction and Cell Surface Expression
2005; Elsevier BV; Volume: 281; Issue: 3 Linguagem: Inglês
10.1074/jbc.m510064200
ISSN1083-351X
AutoresAlex C. Conner, John Simms, Stephen G. Howitt, Mark Wheatley, David R. Poyner,
Tópico(s)Neuroendocrine regulation and behavior
ResumoThe calcitonin gene-related peptide (CGRP) receptor is a heterodimer of a family B G-protein-coupled receptor, calcitonin receptor-like receptor (CLR), and the accessory protein receptor activity modifying protein 1. It couples to Gs, but it is not known which intracellular loops mediate this. We have identified the boundaries of this loop based on the relative position and length of the juxtamembrane transmembrane regions 3 and 4. The loop has been analyzed by systematic mutagenesis of all residues to alanine, measuring cAMP accumulation, CGRP affinity, and receptor expression. Unlike rhodopsin, ICL2 of the CGRP receptor plays a part in the conformational switch after agonist interaction. His-216 and Lys-227 were essential for a functional CGRP-induced cAMP response. The effect of (H216A)CLR is due to a disruption to the cell surface transport or surface stability of the mutant receptor. In contrast, (K227A)CLR had wild-type expression and agonist affinity, suggesting a direct disruption to the downstream signal transduction mechanism of the CGRP receptor. Modeling suggests that the loop undergoes a significant shift in position during receptor activation, exposing a potential G-protein binding pocket. Lys-227 changes position to point into the pocket, potentially allowing it to interact with bound G-proteins. His-216 occupies a position similar to that of Tyr-136 in bovine rhodopsin, part of the DRY motif of the latter receptor. This is the first comprehensive analysis of an entire intracellular loop within the calcitonin family of G-protein-coupled receptor. These data help to define the structural and functional characteristics of the CGRP-receptor and of family B G-protein-coupled receptors in general. The calcitonin gene-related peptide (CGRP) receptor is a heterodimer of a family B G-protein-coupled receptor, calcitonin receptor-like receptor (CLR), and the accessory protein receptor activity modifying protein 1. It couples to Gs, but it is not known which intracellular loops mediate this. We have identified the boundaries of this loop based on the relative position and length of the juxtamembrane transmembrane regions 3 and 4. The loop has been analyzed by systematic mutagenesis of all residues to alanine, measuring cAMP accumulation, CGRP affinity, and receptor expression. Unlike rhodopsin, ICL2 of the CGRP receptor plays a part in the conformational switch after agonist interaction. His-216 and Lys-227 were essential for a functional CGRP-induced cAMP response. The effect of (H216A)CLR is due to a disruption to the cell surface transport or surface stability of the mutant receptor. In contrast, (K227A)CLR had wild-type expression and agonist affinity, suggesting a direct disruption to the downstream signal transduction mechanism of the CGRP receptor. Modeling suggests that the loop undergoes a significant shift in position during receptor activation, exposing a potential G-protein binding pocket. Lys-227 changes position to point into the pocket, potentially allowing it to interact with bound G-proteins. His-216 occupies a position similar to that of Tyr-136 in bovine rhodopsin, part of the DRY motif of the latter receptor. This is the first comprehensive analysis of an entire intracellular loop within the calcitonin family of G-protein-coupled receptor. These data help to define the structural and functional characteristics of the CGRP-receptor and of family B G-protein-coupled receptors in general. G-Protein coupled receptors (GPCRs) 2The abbreviations used are: GPCRG-protein coupled receptorTMtransmembrane helixICLintracellular loopHAhemagglutininBSAbovine serum albuminTBSTris-buffered salineELISAenzyme-linked immunosorbant assayPBSphosphate-buffered salineMDmolecular dynamicsWTwild typeRAMPreceptor activity modifying proteinCLRcalcitonin receptor-like receptorCGRPcalcitonin gene-related peptide. 2The abbreviations used are: GPCRG-protein coupled receptorTMtransmembrane helixICLintracellular loopHAhemagglutininBSAbovine serum albuminTBSTris-buffered salineELISAenzyme-linked immunosorbant assayPBSphosphate-buffered salineMDmolecular dynamicsWTwild typeRAMPreceptor activity modifying proteinCLRcalcitonin receptor-like receptorCGRPcalcitonin gene-related peptide. comprise a large superfamily of membrane-spanning proteins encoded by 2-3% of the human genome. These receptors respond to an incredibly diverse array of stimuli from odorants, amines, peptides, and light through a series of broadly similar activation mechanisms and accessory proteins. The pharmaceutical implications of understanding these proteins cannot be underestimated, and the crystal structure of rhodopsin has presented many new avenues of study for this family (1Palczewski K. Kumasaka T. Hori T. Behnke C.A. Motoshima H. Fox B.A. Le Trong I. Teller D.C. Okada T. Stenkamp R.E. Yamamoto M. Miyano M. Science. 2000; 289: 739-745Crossref PubMed Scopus (5023) Google Scholar). G-protein coupled receptor transmembrane helix intracellular loop hemagglutinin bovine serum albumin Tris-buffered saline enzyme-linked immunosorbant assay phosphate-buffered saline molecular dynamics wild type receptor activity modifying protein calcitonin receptor-like receptor calcitonin gene-related peptide. G-protein coupled receptor transmembrane helix intracellular loop hemagglutinin bovine serum albumin Tris-buffered saline enzyme-linked immunosorbant assay phosphate-buffered saline molecular dynamics wild type receptor activity modifying protein calcitonin receptor-like receptor calcitonin gene-related peptide. Several structural and functional motifs are well characterized within the intracellular domains of the largest known family (A) of GPCRs. These include the conserved (D/E)R(Y/H) motif on the boundary between transmembrane helix 3 and the second intracellular loop (ICL2) and which is well documented to have a significant role in the activation mechanism (2Gershengorn M.C. Osman R. Endocrinology. 2001; 142: 2-10Crossref PubMed Scopus (115) Google Scholar) and the NPXXY motif of TM7, which can have diverse roles in the constitutive phosphorylation, internalization, and signaling of many family A GPCRs (3Kalatskaya I. Schussler S. Blaukat A. Muller-Esterl W. Jochum M. Proud D. Faussner A. J. Biol. Chem. 2004; 279: 31268-31276Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 4Urizar E. Claeysen S. Deupi X. Govaerts C. Costagliola S. Vassart G. Pardo L. J. Biol. Chem. 2005; 280: 17135-17141Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). In addition, residues within ICL2 juxtaposed to transmembrane helix 3 have been shown to be involved in receptor stability (5Erlenbach I. Kostenis E. Schmidt C. Serradeil-Le Gal C. Raufaste D. Dumont M.E. Pausch M.H. Wess J. J. Biol. Chem. 2001; 276: 29382-29392Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). In contrast, much less is known about the important amino acids of family B receptors, which include the calcitonin family of receptors and which tend to bind larger peptide agonists. There are no obvious common motifs between family A and family B receptors; nevertheless, a shared mechanism involving a ligand-induced conformational distortion between the distal regions of TMs 3 and 6 has been proposed for the parathyroid hormone receptor (6Sheikh S.P. Vilardarga J.P. Baranski T.J. Lichtarge O. Iiri T. Meng E.C. Nissenson R.A. Bourne H.R. J. Biol. Chem. 1999; 274: 17033-17041Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar) and the CGRP/adrenomedullin receptors (7Conner A.C. Hay D.L. Simms J. Howitt S.G. Schindler M. Smith D.M. Wheatley M. Poyner D.R. Mol. Pharmacol. 2005; 67: 20-31Crossref PubMed Scopus (53) Google Scholar). There are contrary reports arguing for the involvement of the second intracellular loop in receptor expression and activation within the family B GPCRs. A recent study on the VPAC1 receptor found that all residues of ICL2 could be mutated without alteration of receptor expression or adenylyl cyclase response to vasoactive intestinal peptide (8Couvineau A. Lacapere J.J. Tan Y.V. Rouyer-Fessard C. Nicole P. Laburthe M. J. Biol. Chem. 2003; 278: 24759-24766Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Conversely, a lysine residue in ICL2 of the parathyroid hormone (PTH) receptor was shown to be critical in activating the phospholipase C pathway while having no effect on the adenylyl cyclase response (9Iida-Klein A. Guo J. Takemura M. Drake M.T. Potts Jr., J.T. Abou-Samra A. Bringhurst F.R. Segre G.V. J. Biol. Chem. 1997; 272: 6882-6889Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). The family B receptor CLR is unusual among GPCRs in that it requires the co-expression of one of a family of accessory proteins called RAMPs (receptor activity modifying proteins) for cell trafficking and ligand interaction (10Aiyar N. Rand K. Elshourbagy N.A. Zeng Z. Adamou J.E. Bergsma D.J. Li Y. J. Biol. Chem. 1996; 271: 11325-11329Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar). A functional CGRP receptor is formed by a CLR/RAMP1 heterodimer (11Conner A.C. Simms J. Hay D.L. Mahmoud K. Howitt S.G. Wheatley M. Poyner D.R. Biochem. Soc. Trans. 2004; 32: 843-846Crossref PubMed Scopus (40) Google Scholar). In addition, an agonist-mediated signaling response requires the co-localization of a third membrane-associated accessory protein called the receptor component protein (12Evans B.N. Rosenblatt M.I. Mnayer L.O. Oliver K.R. Dickerson I.M. J. Biol. Chem. 2000; 275: 31438-31443Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar). Receptor component protein is thought to form an ionic interaction with the membrane adjacent to CLR (13Loiseau S.C. Dickerson I.M. Neuropeptides. 2004; 38 (abstr.): 6Google Scholar). There are no studies exploring the role of intracellular regions of CLR involved in CGRP binding, cAMP signaling, or protein expression; indeed there are very few mutational analyses of the second intracellular loop for any family B GPCRs. This report describes the systematic substitution of all residues of ICL2 of human CLR. Mutations were constructed either individually or in pairs replacing the side chains with a single methyl group (alanine). The naturally occurring alanine residues at position 221 and 224 were replaced with glycine. These mutants were transfected into mammalian cells in combination with a RAMP1 construct and analyzed using a tritiated cAMP radio receptor assay. The assay compared the CGRP-stimulated cAMP accumulation of mutant with wild-type CLR. Mutants with impaired function were further characterized for cellular expression, ligand affinity, and surface localization. We identify His-216 and Lys-227 as key residues for normal receptor function and utilize molecular modeling to reveal the likely spatial positioning of the second intracellular loop in the predicted active and inactive states of the receptor. Our findings are discussed in the context of previously proposed motifs within this region in other family B receptors. Materials—Human αCGRP was from Calbiochem. Peptides were dissolved in distilled water and stored as aliquots at -20 °C in non-stick microcentrifuge tubes (Thermo Life Sciences, Basingstoke, UK). Unless otherwise specified, chemicals were from Sigma or Fisher. Cell culture reagents were from Invitrogen or Sigma. [125I]Iodohistidyl8-human αCGRP (2000 Ci/mmol) was from Amersham Biosciences. Expression Constructs and Mutagenesis—Human CLR with an N-terminal hemagglutinin (HA) epitope tag (YPYDVPDYA) (14McLatchie L.M. Fraser N.J. Main M.J. Wise A. Brown J. Thompson N. Solari R. Lee M.G. Foord S.M. Nature. 1998; 393: 333-339Crossref PubMed Scopus (1852) Google Scholar) was provided by Dr. S. M. Foord (GlaxoSmithKline) and was subcloned into pcDNA3(-) (Invitrogen) before mutagenesis. Introduction of the epitope did not affect the pharmacology of the receptor (14McLatchie L.M. Fraser N.J. Main M.J. Wise A. Brown J. Thompson N. Solari R. Lee M.G. Foord S.M. Nature. 1998; 393: 333-339Crossref PubMed Scopus (1852) Google Scholar). Mutagenesis was carried out using the QuikChange site-directed mutagensis kit (Stratagene, Cambridge, UK), following the manufacturer's instructions. Forward and reverse oligonucleotide primers were designed with single base changes to incorporate amino acid point mutations alanine (or glycine) in the final CL protein and to engineer restriction sites to aid screening of mutants. The primers were synthesized by Invitrogen. The numbering of the residues accommodates a 22-amino acid signal protein before the start of the mature transcript (15Koller D. Born W. Leuthauser K. Fluhmann B. McKinney R.A. Fischer J.A. Muff R. FEBS Lett. 2002; 531: 464-468Crossref PubMed Scopus (29) Google Scholar). Plasmid DNA was extracted from the cultures using a Wizard-Prep DNA extraction kit according to the manufacturer's instructions (Promega, Southampton, UK). The plasmid DNA was eluted in 100 μl of sterile distilled water and stored at -20 °C. Sequences were confirmed by sequencing (Functional Genomics, Birmingham, UK). Cell Culture and Transfection—Cos-7 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) fetal bovine serum and 5% (v/v) penicillin/streptomycin in a humidified 95% air, 5% CO2 atmosphere. For transfection, the cells were plated onto either 12- and 48-well plates or 100-mm dishes. Cells were transfected using a mixture (per 1 μg of DNA) of 6 μl of 10 mm polyethyleneimine and 45 μl of 5% glucose solution incubated for 30 min at room temperature and added to an appropriate final volume of full media. 12- and 48-well plates were treated with 1 μg of DNA/well, and 100-mm dishes were treated with 10 μg of DNA/dish. Characterization of expressed receptors was performed 48-72 h after transfection. Membrane Preparation—The cells from each 100-mm plate were washed briefly with 1 ml of cold phosphate-buffered saline and scraped into a small volume of buffer (20 mm HEPES, 2 mm MgCl2, 1% (w/v) bovine serum albumin (BSA), pH 7.5). The cells were homogenized using an Ultra Turrax homogenizer (full speed for 20 s). The cells were then centrifuged at 20,000 × g for 30 min at 4 °C. The supernatant was removed, and the pellets were resuspended in 14 ml of buffer (as before) and used immediately for binding studies or stored at -70 °C. Radioligand Binding—Membranes were homogenized briefly before use, and 500 μl were incubated with 100 pm [125I]iodohistidyl8-human αCGRP and appropriate dilutions of human αCGRP for 60 min at room temperature. Nonspecific binding was measured in the presence of 1 μm CGRP. The samples were then centrifuged at 12,000 × g in a bench-top microcentrifuge for 5 min at room temperature. The pellets were washed twice with water, and the radioactivity was counted in a γ counter. Assay of cAMP Production—Growth medium was removed from the cells and replaced with Dulbecco's modified Eagle's medium containing 500 μm isobutylmethylxanthine for 30 min. αCGRP in the range 10 pm to 1 μm was added for a further 15 min. Ice-cold ethanol (95-100% v/v) was used to extract cAMP, which was subsequently measured by radio-receptor assay as previously described (16Poyner D.R. Andrew D.P. Brown D. Bose C. Hanley M.R. Br. J. Pharmacol. 1992; 105: 441-447Crossref PubMed Scopus (73) Google Scholar) Analysis of Cell-surface Expression of Mutants by Enzyme-linked Immunosorbant Assay (ELISA)—Cells in 12-well plates were transiently transfected with wild type (WT) or mutant HA epitope-tagged human CL and RAMP 1. The transfected cells were treated with 3.7% formaldehyde for 15 min after aspiration of growth medium. The cells were then washed 3 times with 0.5 ml of Tris-buffered saline (TBS). Nonspecific binding of the antibody was blocked with 1% BSA in TBS for 45 min. The cells were treated with 250 μl of primary antibody (mouse, anti-HA antibody 12CA5 (Sigma) diluted 1:1000 in TBS with 1% BSA) for 1 h, and the cells were washed again 3 times with 0.5 ml of TBS. A further block step was performed for 15 min before the cells were incubated with 250 μl of secondary antibody (anti-mouse, horseradish peroxidase-conjugated (Sigma) diluted 1:1000 in TBS) for 1 h. The cells were washed a further three times before development with O-phenylenediamine tablets (Bio-Rad) according to the manufacturer's instructions. Reactions were terminated with 100 μl/well of 1 m H2SO4. The absorbance measured by the ELISA showed a linear dependence on the DNA concentration used in the transfection. Immunohistocytochemistry—Cos-7 cells were seeded in 6-well plates containing nitric acid-washed glass coverslips (12 mm) and transfected using polyethyleneimine as described above. Cells were fixed and washed with phosphate-buffered saline (PBS) as described previously for the ELISA. Cells were blocked with 1% BSA in PBS for 45 min followed by incubation with an anti-HA primary antibody (diluted to 1:3000 in 3% (w/v) for 60 min. Cells were washed 3 times with PBS before reblocking with 1% BSA in PBS for 15 min at room temperature. Cells were labeled with a staining mixture including goat anti-mouse IgG conjugated to fluorescein isothiocyanate (Sigma) (diluted to 1:500 in 10% (v/v) goat serum in PBS), Alexa 546 phalloidin (Molecular Probes, Leiden, The Netherlands), and Hoescht 33342 (Sigma) for 60 min at room temperature in the dark. After a further three washes, coverslips were mounted on glass slides before confocal microscopy. Confocal Microscopy—Confocal microscopy was performed using a Nikon Optiphat II laser scanning microscope at 60× magnification under immersion oil. The HA-tagged receptors were visualized by exciting the secondary antibody, actin filaments were visualized by exciting the Alexa 546 phalloidin, and nuclei were visualized by exciting the Hoescht 33342. The appropriate wavelength was used according to manufacturer's instructions in each case. Images were captured at 10 random sites for each slide from three separate experiments. TM Prediction—Individual TM helix prediction of 58 diverse Family B GPCR sequences was performed using the web-based versions of TMHMM2 (17Krogh A. Larsson B. von Heijne G. Sonnhammer E.L. J. Mol. Biol. 2001; 305: 567-580Crossref PubMed Scopus (9080) Google Scholar) and HMMTOP2 (18Tusnady G.E. Simon I. Bioinformatics. 2001; 17: 849-850Crossref PubMed Scopus (1558) Google Scholar). A consensus prediction for the boundaries of TMIII and TMIV was generated by visual inspection, and from this initial survey, a ClustalW (19Wilbur W.J. Lipman D.J. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 726-730Crossref PubMed Scopus (1055) Google Scholar) profile alignment (using the Blosum matrix) was created against 58 peptide hormone family A GPCRs. The resulting alignment was used to generate an initial homology based model using the high resolution x-ray crystal structure of bovine rhodopsin as a template. Further refinement of the homology model was achieved through molecular dynamics (MD) simulations of the receptor embedded in a dipalmitoylphosphatidylcholine bilayer. A series (n = 5) of 5-ns MD simulations were carried out using the GRO-MOS96 force field parameter set, with minor modifications, as implemented in GROMACS (20Lindahl E. Hess B. Spoel D.V.D. J. Mol. Model. 2001; 7: 306-317Crossref Google Scholar). The resulting trajectories were concatenated and used to produce the final refined model of the CLR. The active state of the CLR was achieved through the use of a modified rhodopsin template, which was consistent with experimentally derived distance restraints obtained from the literature (21Dunham T.D. Farrens D.L. J. Biol. Chem. 1999; 274: 1683-1690Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 22Struthers M. Yu H. Kono M. Oprian D.D. Biochemistry. 1999; 38: 6597-6603Crossref PubMed Scopus (37) Google Scholar, 23Gouldson P.R. Kidley N.J. Bywater R.P. Psaroudakis G. Brooks H.D. Diaz C. Shire D. Reynolds C.A. Proteins. 2004; 56: 67-84Crossref PubMed Scopus (81) Google Scholar, 24Altenbach C. Cai K. Klein-Seetharaman J. Khorana H.G. Hubbell W.L. Biochemistry. 2001; 40: 15483-15492Crossref PubMed Scopus (95) Google Scholar, 25Altenbach C. Klein-Seetharaman J. Cai K. Khorana H.G. Hubbell W.L. Biochemistry. 2001; 40: 15493-15500Crossref PubMed Scopus (104) Google Scholar). Homology models of the active CLR were refined through the use of MD simulations as described above. Prediction of the ICL2 of Rhodopsin and the CLR—RAPPER was used to predict from first principles (ab initio) the conformations of ICL2 from rhodopsin and the CLR. Briefly, 1000 loop backbone conformations were generated using RAPPER assuming idealized stereochemistry for all heavy atoms (N, Cα, C, O). The side-chain orientations for the predicted backbone conformations were modeled using SCWRL (26Bower M.J. Cohen F.E. Dunbrack Jr., R.L. J. Mol. Biol. 1997; 267: 1268-1282Crossref PubMed Scopus (482) Google Scholar) within the environment of the remaining, non-modeled protein. Generated fragments were initially scored using an all-atom statistical potential (scop-e4-allatoms-x-ray-scores scoring set) as described by Samudrala and Moult (27Samudrala R. Moult J. J. Mol. Biol. 1998; 275: 895-916Crossref PubMed Scopus (379) Google Scholar). The ensemble of loop conformations was filtered on the basis of a RADPF score such that no more than the top 50 models were retained for energy minimization. Minimization was performed using a l-BFGS minimization method which utilized the AMBER all-atom force-field (parm99) together with the Still GB/SA solvation model, as implemented in the TINKER (28Pappu R.V. Marshall G.R. Ponder J.W. Nat. Struct. Biol. 1999; 6: 50-55Crossref PubMed Scopus (2) Google Scholar). Minimization was performed until either convergence or a 0.1-kcal mol-1 cutoff point was reached. Only atoms belonging to either the loop region or the N/C-terminal anchor residues were allowed to move during minimization. Minimized fragments were subsequently ranked according to the conformational free energy of the loop. Data Analysis—Curve fitting was done with PRISM Graphpad 4 (Graphpad Software Inc., San Diego, CA). For cAMP studies, the data from each concentration-response curve were fitted to a sigmoidal concentration-response curve to obtain the maximum response and -logEC50 (pEC50). For radioligand binding experiments curves were fitted to obtain maximum and minimum amounts of binding and -logIC50 (pIC50). Because the radioligand was present at concentrations well below its Kd, the IC50 values were effectively identical to the Ki values. To estimate Bmax values with 125I-labeled CGRP, the data were fitted to a sigmoidal curve, calculating the amount bound from the specific activity of the radioligand (this was progressively reduced by dilution with unlabeled CGRP). pEC50, pIC50, and Bmax values were compared by paired Student's t test. Comparisons were only made between wild-type (WT) and mutant data from concomitantly transfected cells. A control WT experiment was always performed alongside a mutant experiment. Identification of the Boundaries of ICL2—According to the Swiss-Prot data base entry Q16602 (www.ExPasy.org), ICL2 of the CLR would be predicted to encompass residues from (and including) Leu-215 to Met-230. An alignment of this region of 57 family B GPCRs in Table 1 shows the most conserved amino acids. The TM/loop boundaries were more precisely defined by using a consensus of previously validated TM prediction methods and remote sequence alignment to a known structure. Visual inspection of the TM prediction results revealed that, although the boundaries for TM4 were well defined, those for TM3 exhibited variability. Essentially, the predicted boundaries for TM3 were clustered into two populations. The first population, which included the majority of sequences studied (40 of 58), exhibited a TM3 boundary at the equivalent position to residues 214 and 243 (including the signal peptide) of the CLR. Significantly, this would position the conserved cysteine residue located at the extracellular end of TM3 in the same positions as that observed in family A receptors. The boundaries for TM3 of the second, smaller population was between the equivalent residues to 227 and 247 (CLR). We have, therefore, used the boundaries exhibited by the first, larger population for subsequent loop modeling studies. As previously mentioned, the predicted boundaries for TM4 were well defined and included residues 250-270 (CLR). Furthermore, sequence alignment of the predicted TM4 segment against the residues corresponding to the high resolution x-ray crystal structure of rhodopsin positioned a conserved tryptophan residue found in both family A and B GPCRs in the same position.TABLE 1Conservation of residues within ICL2 of CLR ICL2 boundaries are assumed from the model presented in this study. The table compares CLR with 57 diverse GPCR examples from family B taken from www.expasy.ch.CLR% IdentityCLR% IdentityCLR% IdentityTyr-214100Val-22115Gln-22818Leu-215100Ala-22227His-22919His-21656Val-22321Leu-23034Thr-21747Phe-22442Met-23126Leu-21871Ala-22529Trp-23219Ile-21948Glu-22650Tyr-23345Val-22036Lys-22728 Open table in a new tab Effect of ICL2 Substitution Mutants on cAMP Accumulation—To identify the contribution to CGRP-stimulated signaling provided by residues within this ICL2 and adjoining segments, residues between Tyr-214 and Tyr-233 inclusive (see Fig. 1) were mutated to alanine either individually or in pairs to generate the constructs (Y214A/L215A)CLR, (H216A)CLR, (T217A)CLR, (L218A/I219A)CLR, (V220A/V221A)-CLR, (A222G)CLR, (V223A/F224A)CLR, (A225G)CLR, (E226A)CLR, (K227A)CLR, (Q228A/H229A)CLR, (L230A/M231A)CLR, (W232A)-CLR, and (Y233A)CLR. Each mutant receptor construct was co-expressed with a RAMP1 vector in Cos-7 cells, and the CGRP-stimulated cAMP accumulation was compared with wild-type CLR. In each case during this study, the natural α-CGRP agonist was used to generate dose response curves. As shown in Fig. 2a and Table 2, the pEC50 for cAMP production in response to CGRP was not significantly different for the seven alanine substitution mutants (Y214A/L215A)CLR, (T217A)CLR, (L218A/I219A)CLR, (V220A/V221A)CLR, (Q228A/H229A)CLR, (L230A/M231A)CLR, and (Y233A)CLR or the two glycine substitution mutants (A222G)CLR and (A225G)CLR when compared with WT CLR co-transfected with RAMP1. Additionally, none of the point mutations in this study had basal levels of stimulation in excess of that seen for the wild-type receptor (data not shown). This suggests none of these substitution mutations forms a constitutively active receptor.TABLE 2Functional parameters of WT/RAMP1 and mutant receptors Values are the mean ± S.E; the number of determinations is shown in parentheses. The maximum responses (Emax) were not significantly different from the concomitantly expressed WT receptor for all of the mutants in this study (data not shown).MutantWT pEC50pEC50(Y214A/L215A)CLR9.02 ± 0.21 (3)8.91 ± 0.36 (3)(H216A)CLR9.41 ± 0.13 (3)8.37 ± 0.37(3)aSignificantly different from WT, p < 0.01, as assessed by paired Student's t test(T217A)CLR9.36 ± 0.21 (3)9.33 ± 0.35 (3)(L218A/I219A)CLR9.66 ± 1.01 (3)9.37 ± 1.04 (3)(V220/V221A)CLR9.75 ± 0.36 (3)9.64 ± 0.11 (3)(A222G)CLR9.54 ± 0.12 (3)9.54 ± 0.34 (3)(V223A/F224A)CLR9.62 ± 0.05 (3)9.02 ± 0.13(3)bSignificantly different from WT, p < 0.05, as assessed by paired Student's t-test(A225G)CLR9.43 ± 0.21 (3)9.34 ± 0.19 (3)(E226A)CLR10.07 ± 0.09 (3)9.87 ± 0.20 (3)(K227A)CLR9.97 ± 0.37 (3)8.91 ± 0.23(3)aSignificantly different from WT, p < 0.01, as assessed by paired Student's t test(Q228A/H229A)CLR9.60 ± 0.75 (3)9.55 ± 0.52 (3)(L230A/M231A)CLR9.44 ± 0.21 (3)9.65 ± 0.39 (3)(W232A)CLR9.27 ± 0.10 (3)8.85 ± 0.32(3)bSignificantly different from WT, p < 0.05, as assessed by paired Student's t-test(Y233A)CLR8.84 ± 0.06 (3)8.50 ± 0.10 (3)a Significantly different from WT, p < 0.01, as assessed by paired Student's t testb Significantly different from WT, p < 0.05, as assessed by paired Student's t-test Open table in a new tab In contrast to the mutants described above, two constructs including a double alanine substitution of the valine and phenylalanine residues at positions 223 and 224 (V223A/F224A)CLR as well as a single mutation of tryptophan to alanine at position 232 (W232A)CLR, both resulted in a small loss in the ability of CLR to elevate cAMP in response to CGRP (Table 2 and Fig. 2, b and c). The disruption in signaling resulted in a significant reduction in the pEC50 value of 4- and 2.6-fold for mutants (V223A/F224A)CLR and (W232A)CLR, respectively. However, when the His-216 or Lys-227 were mutated to alanine, the perturbation of cAMP was larger, with greater than an order of magnitude reduction in EC50 (Table 2 and Fig. 2, d and e). This reduction corresponds to an 11- and 11.5-fold decrease for (H216A)CLR and (K227A)CLR, respectively. It is not without precedent that the effects of removing a basic residue such as Lys-227 are due to the destabilization of a salt bridge formed by an adjacent cationic side chain left without an ionic partner (in this case Glu-226), disrupting stability. To investigate this possibility, we constructed and analyzed a double mutant, (E226A/K227A)CLR. The signaling profile of this mutant was not significantly different to the effects seen by the (K227A)CLR mutant alone (pEC50 values for wild-type and (E226A/K227A)CLR were 8.79 ± 0.08 and 8.28 ± 0.09, respectively, n = 3). These data suggest a direct role for the basic lysine side chain in the activation mechanism of the receptor. ELISA Analysis of Cell-surface Expression—The mutants with disrupted cAMP signaling were subsequently analyzed for their comparative ability to traffic to the cell surface using a whole-cell enzym
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