Abstract

The pituitary adenylate cyclase-activating polypeptide type I receptor (PAC1R) belongs to the secretin receptor family and is widely distributed in the central neural system and peripheral organs. Abnormal activation of the receptor mediates trigeminovascular activation and sensitization, which is highly related to migraine, making PAC1R a potential therapeutic target. Elucidation of PAC1R activation mechanism would benefit discovery of therapeutic drugs for neuronal disorders. PAC1R activity is governed by pituitary adenylate cyclase-activating polypeptide (PACAP), known as a major vasodilator neuropeptide, and maxadilan, a native peptide from the sand fly, which is also capable of activating the receptor with similar potency. These peptide ligands have divergent sequences yet initiate convergent PAC1R activity. It is of interest to understand the mechanism of PAC1R ligand recognition and receptor activity regulation through structural biology. Here we report two near-atomic resolution cryo-EM structures of PAC1R activated by PACAP38 or maxadilan, providing structural insights into two distinct ligand binding modes. The structures illustrate flexibility of the extracellular domain (ECD) for ligands with distinct conformations, where ECD accommodates ligands in different orientations while extracellular loop 1 (ECL1) protrudes to further anchor the ligand bound in the orthosteric site. By structure-guided molecular modeling and mutagenesis, we tested residues in the ligand-binding pockets and identified clusters of residues that are critical for receptor activity. The structures reported here for the first time elucidate the mechanism of specificity and flexibility of ligand recognition and binding for PAC1R, and provide insights toward the design of therapeutic molecules targeting PAC1R.

Highlights

  • pituitary adenylate cyclase-activating polypeptide (PACAP) is a 38-amino acid C-terminally amidated polypeptide (PACAP38) that was discovered as a hypothalamic neuropeptide to potentially induce cAMP levels in anterior pituitary cells.[1]

  • Because of the 68% sequence identity between PACAP27 and vasoactive intestinal polypeptide (VIP), PACAP is identified as a member of the glucagon/gastric inhibitory polypeptide (GIP)/secretin/VIP family—a hormone family consisting of evolutionarily related peptides that regulate the activity of class B G-protein coupled receptor (GPCR) family, known as secretin receptor family.[2]

  • Intravenous infusion of PACAP38 but not VIP induces delayed migraine-like headaches, indicating that PAC1R is playing a major role in migraine[6] and suggesting

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Summary

Introduction

PACAP is a 38-amino acid C-terminally amidated polypeptide (PACAP38) that was discovered as a hypothalamic neuropeptide to potentially induce cAMP levels in anterior pituitary cells.[1] The N-. PACAP and its receptors are broadly expressed in the central nervous system (CNS) and in most peripheral organs, and have been found to exert a variety of functions including control of neurotransmitter release, vasodilation, bronchodilation, activation of intestinal motility, neuroprotection, immune modulation, and stimulation of cell proliferation and/or differentiation.[4]. Abnormal activation and sensitization of the central trigeminovascular pain pathway mediate migraine and the release of these peptides.[5] Intravenous infusion of PACAP38 but not VIP induces delayed migraine-like headaches, indicating that PAC1R is playing a major role in migraine[6] and suggesting

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