title: Role of second extracellular loop in the function of human vasoactive intestinal polypeptide/pituitary adenylate cyclase activating polypeptide receptor 1 (hVPAC1R).
abstract: To elucidate the functional role of the second extracellular loop of human vasoactive intestinal polypeptide/pituitary adenylate cyclase-activating polypeptide (VIP/PACAP) receptor (hVPAC1R), surface expression, ligand binding, and receptor activation were analyzed. Amino acids in the entire second extracellular loop were individually substituted by alanine by site-directed mutagenesis. The mutant and wild-type receptors were transiently expressed in HEK293 cells and purified cell membranes were tested for the ability to bind VIP, while the receptor activity was measured as potency of cAMP production analysed on intact cells. Surface expression of the substituted conserved residues, W286A, I289A, W294A, and W295A, was evidently decreased to 20-30% compared to the wild-type expression. W286A also showed an significantly reduced potency of cAMP production. Substituted residues as F280A, E281A, and G284A showed a significant reduction in the potency of stimulated cAMP production amounting to 8-46-fold, compared to the wild-type with unaffected surface expression and VIP binding. These results indicate that some residues in the second extracellular loop of the human VPAC1R participate in the active mechanism of a ligand-mediated response without being directly involved in the binding of VIP.
authors.affiliation: Department of Clinical Biochemistry, Bispebjerg Hospital, University of Copenhagen, Denmark. smk@biobase.dk
authors.forename: S M
authors.initials: SM
authors.lastname: Knudsen
authors.forename: J W
authors.initials: JW
authors.lastname: Tams
authors.forename: J
authors.initials: J
authors.lastname: Fahrenkrug
mesh_terms: D001665:Binding Sites / Q000502:physiology; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism; D018076:DNA, Complementary; D006801:Humans; D007457:Iodine Radioisotopes; D007668:Kidney / Q000166:cytology; D008024:Ligands; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D051236:Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide; D011979:Receptors, Pituitary Hormone / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018005:Receptors, Vasoactive Intestinal Peptide / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D051238:Receptors, Vasoactive Intestinal Polypeptide, Type I; D017386:Sequence Homology, Amino Acid; D014162:Transfection; D014660:Vasoactive Intestinal Peptide / Q000378:metabolism / Q000494:pharmacology
chemical_list: D018076:DNA, Complementary; D007457:Iodine Radioisotopes; D008024:Ligands; D051236:Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide; D011979:Receptors, Pituitary Hormone; D018005:Receptors, Vasoactive Intestinal Peptide; D051238:Receptors, Vasoactive Intestinal Polypeptide, Type I; D014660:Vasoactive Intestinal Peptide; D000242:Cyclic AMP