The human VPAC₁ receptor: Three-dimensional model and mutagenesis of the N-terminal domain

The human VPAC₁ receptor for vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase activating peptide belongs to the class II family of G-protein-coupled receptors with seven transmembrane segments. Like for all class II receptors, the extracellular N-terminal domain of the human VPAC₁ receptor plays a predominant role in peptide ligand recognition. To determine the three-dimensional structure of this N-terminal domain (residues 1-144), the Protein Data Bank (PDB) was screened for a homologous protein. A subdomain of yeast lipase B was found to have 27% sequence identity and 50% sequence homology with the N-terminal domain (8-117) of the VPAC₁ receptor together with a good alignment of the hydrophobic clusters. A model of the N-terminal domain of VPAC₁ receptor was thus constructed by homology. It indicated the presence of a putative signal sequence in the N-terminal extremity. Moreover, residues (Glu³⁶, Trp⁶⁷, Asp⁶⁸, Trp⁷³, and Gly¹⁰⁹) which were shown to be crucial for VIP binding are gathered around a groove that is essentially negatively charged. New putatively important residues for VIP binding were suggested from the model analysis. Site-directed mutagenesis and stable transfection of mutants in CHO cells indicated that Pro⁷⁴, Pro⁸⁷, Phe⁹⁰, and Trp¹¹⁰ are indeed important for VIP binding and activation of adenylyl cyclase activation. Combination of molecular modeling and directed mutagenesis provided the first partial three-dimensional structure of a VIP-binding domain, constituted of an electronegative groove with an outspanning tryptophan shell at one end, in the N-terminal extracellular region of the human VPAC₁ receptor.