The Human Vpac(1) Receptor - Three-Dimensional Model And Mutagenesis Of The N-Terminal Domain

The human VPAC(1) 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 VPAC(1) 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) of the VPAC(1) receptor together with a good
alignment of the hydrophobic clusters. A model of the N-terminal domain of
VPAC(1) receptor was thus constructed by homology. It indicated the presence of a
putative signal sequence in the N-terminal extremity. Moreover, residues
(Glu(36), Trp(67), Asp(68), Trp(73), and Gly(109)) 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 Pro(74), Pro(87), Phe(90), and Trp(110) 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 VPAC(1) receptor.