G protein-coupled receptors (GPCRs) are critical transduction gatekeepers for extracellular signals, which are involved in numerous physiological processes and disease pathogenesis. Consequently, GPCRs are attractive drug targets and account for approximately 30-40% of all drugs currently in the market. Structural determination of GPCRs in complex with transducer proteins aids understanding of the molecular mechanism underlying GPCR agonist interaction and signaling and holds potential for future therapeutic drug discovery. Recently, cryo-electron microscopy (cryo-EM) has become a powerful approach to solve GPCR-transducer complex structures.
The first breakthrough by cryo-EM was achieved with the calcitonin receptor (CTR) in complex with the heterotrimeric Gs-protein. The CTR is activated by calcitonin, a peptide agonist, but the receptor can also associate with one of three receptor activity-modifying proteins (RAMP1-3) to generate three distinct amylin receptors (AMY1-3, respectively). RAMPs also partner with the calcitonin receptor-like receptor (CLR), giving rise to the CGRP receptor (RAMP1) and adrenomedullin 1 (RAMP2) and 2 (RAMP3) receptors, respectively. Recently, we determined the full-length structure of the human CGRP receptor bound to CGRP and the heterotrimeric Gs transducer using cryo-EM, offering insights into the structural and functional aspects of core receptor (CLR) interaction with RAMP1, as well as how the ligand (CGRP) engages with the core of the CLR bundle in this complex that is required for receptor activation and signaling. Structure characterization of CTR/RAMP complexes is crucial to understand the structural and molecular details of CTR: RAMP interactions that allow the generation of amylin receptors. To achieve this aim, here we present the purification of the CTR with three RAMPs in complex with heterotrimeric Gs-protein. In addition, we have solved the structure of the AMY1 receptor complex using single-particle cryo-EM that combined with previous structures of the CTR reveals novel insights into the modulation of receptor phenotype by RAMP1.