Identification and Pharmacological Characterization of Domains Involved in Binding of CGRP Receptor Antagonists to the Calcitonin-like Receptor

Abstract

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.

Determinants of 1-Piperidinecarboxamide,N-[2-[[5-Amino-l-[[4-(4-pyridinyl)-l-piperazinyl]carbonyl]pentyl]amino]-1-[(3,5-dibromo-4-hydroxyphenyl)methyl]-2-oxoethyl]-4-(1,4-dihydro-2-oxo-3(2H)-quinazolinyl) (BIBN4096BS) Affinity for Calcitonin Gene-Related Peptide and Amylin Receptors—The Role of Receptor Activity Modifying Protein 1

“…Therefore it seems that whereas RAMP1 is a critical determinant of BIBN4096BS affinity for CGRP 1 and AMY 1(a) receptors, CL and CT (a) are also important, the data supporting the interaction of BIBN4096BS at the interface between these proteins. Salvatore et al (2006) came to similar conclusions in their recent investigation of the CL receptor domains involved in the binding of the BIBN4096BS analog, compound 1, in which the region delimited by amino acids 37 to 63 was implicated.…”

Identification and Characterization of a Small Molecule Antagonist of Human VPAC2 Receptor

“…An ago-allosteric positive modulator has been reported for GLP 1 (Knudsen et al, 2007). For calcitonin receptor-like receptor, both competitive and allosteric small molecule antagonists have been found, and they interact with the ECD and the 7TM domain of the receptor, respectively (Salvatore et al, 2006). Thus, inhibiting class II GPCRs allosterically by interacting with the 7TM domain of the receptor might be quite common for small molecule modulators.…”

Pharmacological Properties of MK-3207, a Potent and Orally Active Calcitonin Gene-Related Peptide Receptor Antagonist

“…Human CLR, RAMP1, RAMP2, and RAMP3 expression vector constructs were described by Salvatore et al (2008). The expression vector construct for the insert-negative human CT receptor (CTR) was described by Salvatore et al (2006). Rat CLR and RAMP1 expression vector constructs were described by Mallee et al (2002).…”