Functional Calcitonin Gene-related Peptide Receptors Are Formed by the Asymmetric Assembly of a Calcitonin Receptor-like Receptor Homo-oligomer and a Monomer of Receptor Activity-modifying Protein-1

Abstract

In addition to their interactions with hetero-trimeric G proteins, seven-transmembrane domain receptors are now known to form multimeric complexes that can include receptor homoor hetero-oligomers and/or accessory proteins that modulate their activity. The calcitonin gene-related peptide (CGRP) receptor requires the assembly of the seven-transmembrane domain calcitonin receptor-like receptor with the single-transmembrane domain receptor activity-modifying protein-1 to reach the cell surface and be active. However, the relative stoichiometric arrangement of these two proteins within a receptor complex remains unknown. Despite recent advances in the development of protein-protein interactions assays, determining the composition and stoichiometric arrangements of such signaling complexes in living cells remains a challenging task. In the present study, we combined bimolecular fluorescence complementation (BiFC) with bioluminescence resonance energy transfer (BRET) to probe the stoichiometric arrangement of the CGRP receptor complex. Together with BRET competition assays, co-immunoprecipitation experiments, and BiFC imaging, dual BRET/BiFC revealed that functional CGRP receptors result from the association of a homo-oligomer of the calcitonin receptor-like receptor with a monomer of the accessory protein receptor activity-modifying protein-1. In addition to revealing the existence of an unexpected asymmetric oligomeric organization for a G protein-coupled receptor, our study illustrates the usefulness of dual BRET/BiFC as a powerful tool for analyzing constitutive and dynamically regulated multiprotein complexes.Increasing evidence indicates that "signalosomes," regrouping different components of a signaling pathway into multiprotein complexes with well defined stoichiometries, play crucial roles in defining the specificity and efficacy of signal transduction (1, 2). For instance, the calcitonin gene-related peptide (CGRP) 3 receptor results from the association of the calcitonin receptor-like receptor (CRLR), a seven-transmembrane domain (7TM) receptor belonging to the superfamily of G protein-coupled receptors, with a one-transmembrane domain protein, the receptor activity-modifying protein-1 (RAMP1), which is part of a three member family (RAMP1, -2, and -3). When expressed individually, both CRLR and RAMP1 are retained intracellularly in the endoplasmic reticulum (ER), whereas their association permits their common plasma membrane targeting and determines the pharmacological properties of the receptor (3-5). From these data, it was proposed that the formation of a functional CGRP receptor requires a 1:1 stoichiometric association of CRLR and RAMP1. Consistent with this idea, cross-linking experiments, using bivalent reagents, captured complexes consisting of one CRLR and one RAMP1 molecule (6). However, the observation that both CRLR and RAMP1 can form homo-oligomers (often refer to as dimers) when expressed alone (3, 6 -8) adds a new level of complexity that reactualizes the question of the number of CRLR and R...

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Complex Formation and Interactions between Transcription Factors Essential for Human Prolactin Receptor Gene Transcription

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