Structural biology of heteromeric class B G protein‐coupled receptors (LB216)

Abstract

The calcitonin family peptides calcitonin (CT), amylin (AMY), calcitonin gene‐related peptide (CGRP), and adrenomedullin (AM) regulate diverse processes including calcium homeostasis, blood glucose levels, vasodilation, and vascular development. Their receptors are heteromeric cell surface protein complexes comprised of a class B GPCR subunit, either the calcitonin receptor (CTR) or the calcitonin‐like receptor (CLR), in association with a peptide selectivity‐determining receptor activity modifying protein (RAMP) subunit. CTR alone is the CT receptor and CTR complexes with any of three RAMPs are AMY receptors. The CLR‐RAMP1 complex is a CGRP receptor, whereas CLR complexes with either RAMP2 or ‐3 are AM receptors. How RAMPs determine calcitonin family peptide selectivity is unclear and the receptor stoichiometries are a topic of debate. Here, we report unique methods for bacterial production of recombinant CLR‐RAMP extracellular domain (ECD) complexes engineered for structure/function studies and use of the novel constructs for peptide binding studies and crystallization. In addition, we describe recombinant CTR ECD production in HEK293T cells, and production of the integral membrane portions of the receptors in the E. coli inner membrane for structure/function studies. Our results indicate that the CTR and CLR ECDs are sufficient to determine peptide binding selectivity, identify peptide residues critical for receptor binding, shed light on the receptor stoichiometries, and provide methods that will facilitate structural studies of these therapeutically important GPCRs. Grant Funding Source: Supported by NIH grant R01GM104251