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Assembly and Signaling of CRLR and RAMP1 Complexes Assessed by BRET†

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Département de Biochimie, Institut de Recherche en Immunologie et Cancérologie and Groupe de Recherche Universitaire sur le Médicament, Université de Montréal, C.P. 6128, Succursale Centre-Ville, Montréal, QC, Canada H3C 3J7
Cite this: Biochemistry 2007, 46, 23, 7022–7033
Publication Date (Web):May 16, 2007
https://doi.org/10.1021/bi0622470
Copyright © 2007 American Chemical Society
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    Abstract

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    Biochemical and functional evidence suggest that the calcitonin receptor-like receptor (CRLR) interacts with receptor activity-modifying protein-1 (RAMP1) to generate a calcitonin gene-related peptide (CGRP) receptor. Using bioluminescence resonance energy transfer (BRET), we investigated the oligomeric assembly of the CRLR−RAMP1 signaling complex in living cells. As for their wild-type counterparts, fusion proteins linking CRLR and RAMP1 to the energy donor Renilla luciferase (Rluc) and energy acceptor green fluorescent protein (GFP) reach the cell surface only upon coexpression of CRLR and RAMP1. Radioligand binding and cAMP production assays also confirmed that the fusion proteins retained normal functional properties. BRET titration experiments revealed that CRLR and RAMP1 associate selectively to form heterodimers. This association was preserved for a mutated RAMP1 that cannot reach the cell surface, even in the presence of CRLR, indicating that the deficient targeting resulted from the altered conformation of the complex rather than a lack of heterodimerization. BRET analysis also showed that, in addition to associate with one another, both CRLR and RAMP1 can form homodimers. The homodimerization of the coreceptor was further confirmed by the ability of RAMP1 to prevent cell surface targeting of a truncated RAMP1 that normally exhibits receptor-independent plasma membrane delivery. Although the role of such dimerization remains unknown, BRET experiments clearly demonstrated that CRLR can engage signaling partners, such as G proteins and β-arrestin, following CGRP stimulation, only in the presence of RAMP1. In addition to shed new light on the CRLR−RAMP1 signaling complex, the BRET assays developed herein offer new biosensors for probing CGRP receptor activity.

    †

     This work was supported by a grant from the Canadian Institute for Health Research (M.B.). M. Héroux holds a studentship from the Canadian Institute for Health Research. M.B. holds a Canada Research Chair in Signal Transduction and Molecular Pharmacology.

    *

     To whom correspondence should be addressed:  Institut de Recherche en Immunologie et Cancérologie, Université de Montréal, C.P. 6128, Succursale Centre-Ville, Montréal, QC, Canada H3C 3J7. Telephone:  (514) 343-6319. Fax:  (514) 343-7780. E-mail:  [email protected].

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    Cell surface trafficking of myc-CRLR (Figure S1), BRET between myc-RAMP1-Rluc and myc-CRLR-GFP (Figure S2A), effect of CGRP on the BRET between myc-CRLR-Rluc and myc-RAMP1-GFP (Figure S2B), and BRET between LRP6-Rluc and LRP6-GFP and between Fz1-Rluc and Fz1-GFP (Figure S3). This material is available free of charge via the Internet at http://pubs.acs.org.

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