Assembly and signaling of CRLR and RAMP1 complexes assessed by BRET

Biochemistry. 2007 Jun 12;46(23):7022-33. doi: 10.1021/bi0622470. Epub 2007 May 16.

Abstract

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 beta-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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Calcitonin Gene-Related Peptide / chemistry
  • Calcitonin Gene-Related Peptide / metabolism
  • Calcitonin Receptor-Like Protein
  • Cell Line
  • Cell Membrane / metabolism
  • Humans
  • Kidney
  • Luciferases / metabolism
  • Mutagenesis, Site-Directed
  • Polymerase Chain Reaction
  • Radioligand Assay
  • Receptors, Calcitonin / chemistry
  • Receptors, Calcitonin / genetics
  • Receptors, Calcitonin / metabolism*
  • Receptors, Calcitonin Gene-Related Peptide / metabolism
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / metabolism
  • Renilla / enzymology
  • Sequence Deletion
  • Signal Transduction
  • Transfection

Substances

  • CALCRL protein, human
  • Calcitonin Receptor-Like Protein
  • Receptors, Calcitonin
  • Receptors, Calcitonin Gene-Related Peptide
  • Recombinant Fusion Proteins
  • Luciferases
  • Calcitonin Gene-Related Peptide