Crystal Structure of the PAC1R Extracellular Domain Unifies a Consensus Fold for Hormone Recognition by Class B G-Protein Coupled Receptors

Shiva Kumar,H Eric Xu,Kunchithapadam Swaminathan,Augen Pioszak,Chenghai Zhang,Karl-Wilhelm Koch

doi:10.1371/journal.pone.0019682

Abstract

Pituitary adenylate cyclase activating polypeptide (PACAP) is a member of the PACAP/glucagon family of peptide hormones, which controls many physiological functions in the immune, nervous, endocrine, and muscular systems. It activates adenylate cyclase by binding to its receptor, PAC1R, a member of class B G-protein coupled receptors (GPCR). Crystal structures of a number of Class B GPCR extracellular domains (ECD) bound to their respective peptide hormones have revealed a consensus mechanism of hormone binding. However, the mechanism of how PACAP binds to its receptor remains controversial as an NMR structure of the PAC1R ECD/PACAP complex reveals a different topology of the ECD and a distinct mode of ligand recognition. Here we report a 1.9 Å crystal structure of the PAC1R ECD, which adopts the same fold as commonly observed for other members of Class B GPCR. Binding studies and cell-based assays with alanine-scanned peptides and mutated receptor support a model that PAC1R uses the same conserved fold of Class B GPCR ECD for PACAP binding, thus unifying the consensus mechanism of hormone binding for this family of receptors.

Highlights

G-Protein Coupled Receptors (GPCRs) are important regulators of many physiological functions and as such have attracted a lot of pharmacological interest for their roles in numerous diseases
Pituitary adenylate cyclase 1 Receptor (PAC1R), which belongs to class B of the GPCR family, was identified in 1994 as the receptor for the peptide hormone Pituitary Adenylate Cyclase Activating Polypeptide (PACAP) [1]
Association of PACAP with PAC1R was analysed by Alphascreen assays, which is based on proximity transfer of excitation energy from the donor to the acceptor beads in a dose dependent manner

Summary

Introduction

G-Protein Coupled Receptors (GPCRs) are important regulators of many physiological functions and as such have attracted a lot of pharmacological interest for their roles in numerous diseases. Pituitary adenylate cyclase 1 Receptor (PAC1R), which belongs to class B of the GPCR family, was identified in 1994 as the receptor for the peptide hormone Pituitary Adenylate Cyclase Activating Polypeptide (PACAP) [1]. Due to the fact that PACAP is homologous to another peptide hormone, Vasoactive Intestinal Peptide (VIP), there is redundancy in their receptors, which are sub-classified based on the differences in their relative affinities for the two hormones. VPACR has further been subdivided based on its affinity for helodermin [6], which is a bioactive peptide that was first isolated from the poisonous salivary gland secretions of the gila monster (Heloderma suspectum) [7]. Helodermin, a member of the exendin family of peptides and sequentially related to the PACAP/glucagon family, is present exclusively in the gila monster [8]

Methods

Results

Conclusion