Identification and pharmacological characterization of domains involved in binding of CGRP receptor antagonists to the calcitonin-like receptor.
The calcitonin-like receptor (CLR) and the calcitonin receptor (CTR) interact with receptor activity-modifying protein 1 (RAMP1) at the cell surface to form heterodimeric receptor complexes. CLR and CTR are members of the class II (family B) G-protein-coupled receptors (GPCR) and bind calcitonin gene-related peptide (CGRP) with similar affinities when coexpressed with RAMP1. The observation that various nonpeptide CGRP receptor antagonists display a higher affinity for the CLR/RAMP1 complex than for CTR/RAMP1 provided an opportunity to investigate the molecular determinants of the differential receptor affinities of these antagonists. A chimeric receptor approach was utilized to identify key domains within CLR responsible for conferring high-affinity antagonist binding. Initial chimera experiments implicated distinct regions within CLR as responsible for the affinities of structurally diverse CGRP receptor antagonists. Dissection of these key regions implicated amino acids 37-63 located in the amino terminus of CLR as responsible for the high-affinity interaction of one structural class, while transmembrane domain (TM) 7 was responsible for the interaction of a second class of antagonist. A unique binding interaction in the amino terminus of CLR is consistent with the observation that these compounds also interact with the extracellular region of RAMP1 and could suggest the formation of a binding pocket between the two proteins. Conversely, a compound which interacted with TM7 did not display a similar RAMP1 dependence, suggesting an allosteric mechanism of antagonism. Collectively, these data provide insight into two alternative mechanisms of antagonism for this unique heterodimeric receptor complex.
Salvatore
Christopher A
CA
Pain Research Department, Merck Research Laboratories, West Point, Pennsylvania 19486, USA. christopher_salvatore@merck.com
Mallee
John J
JJ
Bell
Ian M
IM
Zartman
C Blair
CB
Williams
Theresa M
TM
Koblan
Kenneth S
KS
Kane
Stefanie A
SA
D001483:Base Sequence; D001667:Binding, Competitive; D000077221:Calcitonin Gene-Related Peptide Receptor Antagonists; D058286:Calcitonin Receptor-Like Protein; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008024:Ligands; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D058262:Receptor Activity-Modifying Protein 1; D058261:Receptor Activity-Modifying Proteins; D018003:Receptors, Calcitonin / Q000378:metabolism*; D018015:Receptors, Calcitonin Gene-Related Peptide / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism; D015398:Signal Transduction
C545578:CALCRL protein, human; D000077221:Calcitonin Gene-Related Peptide Receptor Antagonists; D058286:Calcitonin Receptor-Like Protein; D047908:Intracellular Signaling Peptides and Proteins; D008024:Ligands; D008565:Membrane Proteins; C545502:RAMP1 protein, human; D058262:Receptor Activity-Modifying Protein 1; D058261:Receptor Activity-Modifying Proteins; D018003:Receptors, Calcitonin; D018015:Receptors, Calcitonin Gene-Related Peptide; D043562:Receptors, G-Protein-Coupled