Abstract
SummaryAssociation of receptor activity-modifying proteins (RAMP1-3) with the G protein-coupled receptor (GPCR) calcitonin receptor-like receptor (CLR) enables selective recognition of the peptides calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) that have diverse functions in the cardiovascular and lymphatic systems. How peptides selectively bind GPCR:RAMP complexes is unknown. We report crystal structures of CGRP analog-bound CLR:RAMP1 and AM-bound CLR:RAMP2 extracellular domain heterodimers at 2.5 and 1.8 Å resolutions, respectively. The peptides similarly occupy a shared binding site on CLR with conformations characterized by a β-turn structure near their C termini rather than the α-helical structure common to peptides that bind related GPCRs. The RAMPs augment the binding site with distinct contacts to the variable C-terminal peptide residues and elicit subtly different CLR conformations. The structures and accompanying pharmacology data reveal how a class of accessory membrane proteins modulate ligand binding of a GPCR and may inform drug development targeting CLR:RAMP complexes.
Highlights
G protein-coupled receptors (GPCRs) are a large family of cell surface receptors that regulate a multitude of biological processes in response to a diverse array of stimuli and they are important drug targets
maltose binding protein (MBP)-RAMP1 or MBP-RAMP2 extracellular domain (ECD)-calcitonin receptor-like receptor (CLR) ECD fusion proteins in which the two ECDs were covalently tethered with a flexible (Gly-Ser)5 linker were designed to ensure complex stability and enforce 1:1 CLR:receptor activity-modifying proteins (RAMPs) stoichiometry
The tethered RAMP1-CLR ECD fusion was a monomer, whereas the tethered RAMP2-CLR ECD fusion purified as a dimer, but the physiological relevance of oligomerization is unknown
Summary
G protein-coupled receptors (GPCRs) are a large family of cell surface receptors that regulate a multitude of biological processes in response to a diverse array of stimuli and they are important drug targets. The class B/Secretin family GPCRs in humans include 15 receptors that are activated by diverse neuropeptides, peptide paracrine factors, and peptide endocrine hormones (Hoare, 2005). These receptors are less well understood than the larger class A/Rhodopsin family, despite their physiological and clinical importance. Crystal structures are available for class B GPCR ECDs with bound peptides related to PTH, CRF, GIP, and GLP-1 (Pal et al, 2010; Parthier et al, 2007; Pioszak et al, 2008, 2009; Pioszak and Xu, 2008; Runge et al, 2008; Underwood et al, 2010) and a consensus has emerged from these studies. For PTH, GIP, and GLP-1 families, the peptides are closest to the N terminus; for the CRF-related peptides, they are displaced to be closer to the C terminus
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