title: Evidence for the formation of a functional complex between vasoactive intestinal peptide, its receptor, and Gs in lung membranes.
abstract: The molecular weight of the vasoactive intestinal peptide (VIP) receptor in rat lung and its interaction with the stimulatory guanine nucleotide-binding protein (Gs) were assessed by covalent cross-linking, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and immunological techniques. Studies with two cross-linking agents indicated that the VIP receptor in this tissue is a single polypeptide of Mr = 54,000. The VIP-occupied receptor could be cross-linked to neighboring proteins after detergent solubilization; higher molecular weight complexes of Mr = 114,000 and 184,000 were formed. Immunoblotting with antisera against G-protein subunits demonstrated that both complexes contained the alpha-subunit of Gs as well as the 125I-VIP cross-linked receptor whereas only the Mr = 184,000 complex contained the beta-subunit. Pretreatment with GTP reduced the prominence of these complexes, verifying the functional nature of this receptor-Gs association. Studies with a third cross-linking agent, ethylene glycol bis(succinimidyl succinate), provided direct evidence of physically associated, ternary VIP-receptor-Gs complexes actually in the membrane milieu. That these complexes were functionally associated with shown by their inhibition by anti-Gs alpha anti-serum. Since treatment of membranes with guanosine 5'-O-(3-thiotriphosphate) resulted in the separation of the VIP-cross-linked receptor from Gs such that no cross-linking could occur, we conclude that the binding of GTP analogs induces a conformational change in Gs in the membrane milieu.
authors.affiliation: Department of Pharmacology, Vermont Center for Vascular Research, College of Medicine, University of Vermont, Burlington 05405.
authors.forename: J C
authors.initials: JC
authors.lastname: Kermode
authors.forename: A W
authors.initials: AW
authors.lastname: DeLuca
authors.forename: A
authors.initials: A
authors.lastname: Zilberman
authors.forename: J
authors.initials: J
authors.lastname: Valliere
authors.forename: S M
authors.initials: SM
authors.lastname: Shreeve
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D003432:Cross-Linking Reagents; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D019204:GTP-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000494:pharmacology; D015151:Immunoblotting; D008168:Lung / Q000378:metabolism*; D046911:Macromolecular Substances; D008297:Male; D008970:Molecular Weight; D051381:Rats; D011919:Rats, Inbred Strains; D011964:Receptors, Gastrointestinal Hormone / Q000302:isolation & purification / Q000378:metabolism*; D018005:Receptors, Vasoactive Intestinal Peptide; D013388:Succinimides; D014660:Vasoactive Intestinal Peptide / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D003432:Cross-Linking Reagents; D003902:Detergents; D046911:Macromolecular Substances; D011964:Receptors, Gastrointestinal Hormone; D018005:Receptors, Vasoactive Intestinal Peptide; D013388:Succinimides; D014660:Vasoactive Intestinal Peptide; D006160:Guanosine Triphosphate; D019204:GTP-Binding Proteins; C019358:disuccinimidyl suberate