The human calcitonin (CTR) and calcitonin-like (CLR) receptors are cell-surface class B G protein-coupled receptors (GPCRs) that associate with receptor activity modifying protein (RAMP) co-receptors to mediate signaling by calcitonin family peptide hormones. The distinct biological actions of the peptides adrenomedullin (AM), calcitonin gene-related peptide (CGRP), and amylin (AMY) are elicited by their binding to specific molecular complexes comprised of CLR or CTR and one of three RAMPs. AM and CGRP regulate the cardiovascular system by signaling through CLR-RAMP2 or 3 and CLR-RAMP1 complexes, respectively. AMY regulates blood glucose levels by signaling through CTR associated with RAMP1, 2, or 3. CLR/CTR-RAMP complexes are important drug targets. Therapeutic agents targeting CLR-RAMP complexes have the potential to treat acute myocardial infarction, pulmonary hypertension, migraine headache, cancer, and several other disorders. CTR-RAMP complexes are targets of the diabetes drug SymlinTM. Despite the clinical value of targeting CLR/CTR-RAMP complexes, the molecular mechanisms of peptide hormone recognition and RAMP modulation of CLR/CTR hormone specificity are poorly understood, in large part because of a lack of structural information for the peptide-receptor complexes. Hormone binding affinity and specificity are largely determined by the extracellular domains (ECDs) of the CLR, CTR, and RAMP integral membrane proteins. The goals of this proposal are to determine crystal structures of AM, CGRP, and AMY bound to their respective CLR/CTR ECD-RAMP ECD complexes in order to define the precise molecular architectures that determine selective peptide binding. In addition, we will correlate structure and function through biochemical and cell-based pharmacological experiments. We propose the following three aims: (1) Determine the mechanism of AM recognition by the human CLR-RAMP2 ECD complex. (2) Determine how RAMP1 confers CGRP selectivity to the CLR-RAMP1 ECD complex. (3) Determine the mechanism of AMY recognition by the human CTR-RAMP1 ECD complex. Achieving these aims will define the molecular bases for RAMP-altered class B GPCR hormone recognition and provide structural templates to guide the rational design of therapeutic agents targeting the receptors.

Public Health Relevance

This project addresses how peptide hormones bind to receptors on the surface of human cells. The hormones we study are important regulators of the cardiovascular system and blood glucose levels. The receptors are members of a large family of receptors known as GPCRs that regulate virtually all aspects of human physiology. Discovering how these hormones bind to their receptors is important because it will aid the development of drugs that target the receptors for the treatment of cardiovascular disorders and diabetes. These studies are also important for the general knowledge we will gain about GPCRs, which will be broadly applicable to several areas of human health and disease.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Molecular and Integrative Signal Transduction Study Section (MIST)
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Dunsmore, Sarah
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University of Oklahoma Health Sciences Center
Schools of Medicine
Oklahoma City
United States
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Booe, Jason M; Warner, Margaret L; Roehrkasse, Amanda M et al. (2018) Probing the Mechanism of Receptor Activity-Modifying Protein Modulation of GPCR Ligand Selectivity through Rational Design of Potent Adrenomedullin and Calcitonin Gene-Related Peptide Antagonists. Mol Pharmacol 93:355-367
Roehrkasse, Amanda M; Booe, Jason M; Lee, Sang-Min et al. (2018) Structure-function analyses reveal a triple ?-turn receptor-bound conformation of adrenomedullin 2/intermedin and enable peptide antagonist design. J Biol Chem 293:15840-15854
Lee, Sang-Min; Booe, Jason M; Gingell, Joseph J et al. (2017) N-Glycosylation of Asparagine 130 in the Extracellular Domain of the Human Calcitonin Receptor Significantly Increases Peptide Hormone Affinity. Biochemistry 56:3380-3393
Hay, Debbie L; Pioszak, Augen A (2016) Receptor Activity-Modifying Proteins (RAMPs): New Insights and Roles. Annu Rev Pharmacol Toxicol 56:469-87
J Gingell, Joseph; Simms, John; Barwell, James et al. (2016) An allosteric role for receptor activity-modifying proteins in defining GPCR pharmacology. Cell Discov 2:16012
Watkins, Harriet A; Chakravarthy, Madhuri; Abhayawardana, Rekhati S et al. (2016) Receptor Activity-modifying Proteins 2 and 3 Generate Adrenomedullin Receptor Subtypes with Distinct Molecular Properties. J Biol Chem 291:11657-75
Lee, Sang-Min; Hay, Debbie L; Pioszak, Augen A (2016) Calcitonin and Amylin Receptor Peptide Interaction Mechanisms: INSIGHTS INTO PEPTIDE-BINDING MODES AND ALLOSTERIC MODULATION OF THE CALCITONIN RECEPTOR BY RECEPTOR ACTIVITY-MODIFYING PROTEINS. J Biol Chem 291:8686-700
Booe, Jason M; Walker, Christopher S; Barwell, James et al. (2015) Structural Basis for Receptor Activity-Modifying Protein-Dependent Selective Peptide Recognition by a G Protein-Coupled Receptor. Mol Cell 58:1040-52
Lee, Sang-Min; Booe, Jason M; Pioszak, Augen A (2015) Structural insights into ligand recognition and selectivity for classes A, B, and C GPCRs. Eur J Pharmacol 763:196-205
Warner, Margaret L; Bell, Tufica; Pioszak, Augen A (2015) Engineering high-potency R-spondin adult stem cell growth factors. Mol Pharmacol 87:410-20

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