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
Journal of Biological Chemistry
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
... he 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. 3 The abbreviations used are: CGRP, calcitonin gene-related peptide; 7TM, seven-transmembrane domain; BiFC, bimolecular fluorescence complementation; BRET, bioluminescence resonance energy transfer; COS7, simian kidney fibroblasts; CRLR, calcitonin receptor-like receptor; ER, endoplasmic reticulum; Fz1, Frizzled 1; Fz4m, Frizzled 4 mutant; HEK, human embryonic kidney; RAMP1, receptor activity-modifying protein-1; RET, resonance energy transfer; Rluc, Renilla luciferase; YFP, yellow fluorescent protein; YC, C-terminal fragment of YFP; YN, N-terminal fragment of YFP; PBS, phosphate-buffered saline; HA, hemagglutinin. Downloaded from FIGURE 8. Functionality of the asymmetrical CRLR⅐RAMP1 complex. Agonist-induced ␤-arrestin2-Rluc recruitment to CRLR-YN⅐CRLR-YC complexes was assessed by BRET in transfected HEK293T cells, in the absence or presence of RAMP1, after 10 min of stimulation with increasing concentrations of CGRP (n ϭ 3).