Evidence for the formation of a functional complex between vasoactive intestinal peptide, its receptor, and Gs in lung membranes.
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.
Kermode
J C
JC
Department of Pharmacology, Vermont Center for Vascular Research, College of Medicine, University of Vermont, Burlington 05405.
DeLuca
A W
AW
Zilberman
A
A
Valliere
J
J
Shreeve
S M
SM
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*
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