SUMMARY/ABSTRACT: Three receptor activity-modifying proteins (RAMP1-3) in humans heterodimerize with the class B G protein- coupled receptors (GPCRs) calcitonin receptor (CTR) and calcitonin-like receptor (CLR) and modulate their responses to calcitonin (CT), amylin, calcitonin gene-related peptide (CGRP), and two adrenomedullin (AM) peptide hormones. These have functions in bone remodeling (CT), regulation of food intake and blood glucose (amylin), and regulation of blood and lymphatic vasculature function (CGRP and AM) by activating one or more of seven receptors arising from CTR, CLR, and the RAMPs (6 heterodimers + CTR alone). The receptors are drug targets for osteoporosis (CTR), diabetes and obesity (amylin receptors), migraine headache (CGRP receptor), and cardiovascular and lymphatic system disorders (AM receptors). How RAMPs modulate CTR and CLR ligand binding and signaling is incompletely understood. This limits our understanding of the molecular basis for the diverse peptide actions in vivo and hinders drug development. Notably, there is growing evidence that RAMPs interact with numerous GPCRs so understanding this model system will have broad impact. In prior funding cycles we used biochemistry, pharmacology, and X-ray crystallography to reveal how CT, CGRP, and the AM peptides bind their receptor extracellular domains (ECDs) and how the RAMP1/2 ECDs alter CLR ECD peptide selectivity. Guided by these results we developed ultra high affinity CGRP and AM variants including picomolar affinity antagonists and sustained signaling agonists that exhibit persistent cAMP signaling. Herein we continue to define the mechanisms of RAMP modulation of CTR and CLR ligand binding and signaling and further characterize our novel peptide variants, which may have therapeutic potential. In Aim 1 we define the kinetics of peptide binding at CGRP and AM receptors (CLR:RAMPs) and its relation to cAMP signaling kinetics using biochemical and pharmacological assays (SPR and BRET kinetic methods). In Aim 2 we continue to define the structural bases for RAMP modulation of peptide binding to the CLR and CTR ECDs using advanced molecular dynamics simulations and X-ray crystallography. In Aim 3 we employ a novel biochemical assay to define how RAMPs modulate agonist and G protein coupling to CTR to enable amylin signaling. Completion of this project will yield the following outcomes: 1) an understanding of how peptide binding kinetics affects cAMP signaling kinetics for CGRP and AM receptors and the molecular basis for sustained signaling of ultra high affinity variants, 2) a near complete high-resolution structural understanding of calcitonin family peptide binding to their receptor ECDs including pathways of CGRP and AM peptide binding and unbinding, and 3) clarification of longstanding amylin receptor pharmacology ambiguities and delineation of how RAMPs modulate CTR. This project will advance our understanding of accessory membrane protein modulation of GPCRs ...