The human VPAC(1) receptor - Three-dimensional model and mutagenesis of the N-terminal domain

Citation
L. Lins et al., The human VPAC(1) receptor - Three-dimensional model and mutagenesis of the N-terminal domain, J BIOL CHEM, 276(13), 2001, pp. 10153-10160
Citations number
33
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Biochemistry & Biophysics
Journal title
JOURNAL OF BIOLOGICAL CHEMISTRY
ISSN journal
0021-9258 → ACNP
Volume
276
Issue
13
Year of publication
2001
Pages
10153 - 10160
Database
ISI
SICI code
0021-9258(20010330)276:13<10153:THVR-T>2.0.ZU;2-O
Abstract
The human VPAC(1), receptor for vasoactive intestinal peptide (VIP) and pit uitary 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 VP AC(1), receptor plays a predominant role in peptide ligand recognition. To determine the three-dimensional structure of this N-terninal domain (residu es 1-144), the Protein Data Bank (PDB) was screened for a homologous protei n. A subdomain of yeast lipase B was found to have 27% sequence identity an d 50% sequence homology with the N-terminal domain (8-117) of the VPAC(1), receptor together with a good alignment of the hydrophobic clusters. A mode l of the N-terminal domain of VPAC(1), receptor was thus constructed by hom ology, It indicated the presence of a putative signal sequence in the N-ter minal extremity. Moreover, residues (Glu(36) Trp(67) Asp(68), Trp(73), and Gly(109)) which were shown tb be crucial for VIP binding are gathered aroun d a groove that is essentially negatively charged. New putatively important residues for VIP binding were suggested from the model analysis. Site-dire cted 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 b inding and activation of adenylyl cyclase activation, Combination of molecu lar modeling and directed mutagenesis provided the first partial three-dime nsional structure of a VIP-binding domain, constituted of an electronegativ e groove with an outspanning tryptophan shell at one end, in the N-terminal extracellular region of the human VPAC(1), receptor.