Direct evidence for functional coupling of the vasoactive intestinal peptide receptor to Gi3 in native lung membranes.
Although vasoactive intestinal peptide (VIP) exerts many of its effects through stimulation of adenylyl cyclase, there is increasing evidence that other signaling pathways may contribute to its action. The role of inhibitory G proteins (Gi) in VIP-mediated signaling in the lung was assessed by a combination of equilibrium-binding and covalent cross-linking studies. Pertussis toxin treatment of rat lung membranes reduced the high affinity binding of 125I-VIP, implicating a member of the Gi family in signaling from the VIP receptor. The particular G protein involved was identified as Gi3 through capture of a VIP/receptor/ Gi3 ternary complex by covalent cross-linking. There was a progressive rise with increasing VIP concentration in formation of the complex reported by the cross-linking strategy. Guanine nucleotides and an anti-G alpha i3 antiserum suppressed formation of the VIP/receptor/Gi3 ternary complex, demonstrating its functional nature in native lung membranes. Inhibition of high affinity 125I-VIP binding by the anti-G alpha i3 antiserum verified this functionality. Taken together, these data suggest that receptor/ Gi3 coupling makes a significant contribution to VIP-mediated signaling in the lung and illustrate the value of covalent cross-linking as a strategy to define receptor/G protein complexes that arise under conditions in which the stoichiometry and microdomains of the native cell membrane are preserved.
Diehl
N L
NL
Department of Pharmacology, College of Medicine, University of Vermont, Burlington 05405, USA.
Kermode
J C
JC
Shreeve
S M
SM
D037361:Adenylate Cyclase Toxin; D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D000918:Antibody Specificity; D001921:Brain / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D003432:Cross-Linking Reagents; D019204:GTP-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism / Q000502:physiology*; D006160:Guanosine Triphosphate / Q000494:pharmacology; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D015151:Immunoblotting; D007700:Kinetics; D008168:Lung / Q000378:metabolism; D037342:Pertussis Toxin; D051381:Rats; D018005:Receptors, Vasoactive Intestinal Peptide / Q000302:isolation & purification / Q000378:metabolism / Q000502:physiology*; D015398:Signal Transduction; D014660:Vasoactive Intestinal Peptide / Q000378:metabolism / Q000494:pharmacology*; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D037361:Adenylate Cyclase Toxin; D003432:Cross-Linking Reagents; D018005:Receptors, Vasoactive Intestinal Peptide; D010566:Virulence Factors, Bordetella; D006165:Guanylyl Imidodiphosphate; D014660:Vasoactive Intestinal Peptide; D006160:Guanosine Triphosphate; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases