Abstract

The VPAC₁ receptor belongs to family B of G protein-coupled receptors (GPCR-B) and is activated upon binding of the vasoactive intestinal peptide (VIP). Despite the recent determination of the structure of the N terminus of several members of this receptor family, little is known about the structure of the transmembrane (TM) region and about the molecular mechanisms leading to activation. In the present study, we designed a new structural model of the TM domain and combined it with experimental mutagenesis experiments to investigate the interaction network that governs ligand binding and receptor activation. Our results suggest that this network involves the cluster of residues Arg¹⁸⁸ in TM2, Gln³⁸⁰ in TM7, and Asn²²⁹ in TM3. This cluster is expected to be altered upon VIP binding, because Arg¹⁸⁸ has been shown previously to interact with Asp³ of VIP. Several point mutations at positions 188, 229, and 380 were experimentally characterized and were shown to severely affect VIP binding and/or VIP-mediated cAMP production. Double mutants built from reciprocal residue exchanges exhibit strong cooperative or anticooperative effects, thereby indicating the spatial proximity of residues Arg¹⁸⁸, Gln³⁸⁰, and Asn²²⁹. Because these residues are highly conserved in the GPCR-B family, they can moreover be expected to have a general role in mediating function.