Effects of adenyl nucleotides and carbachol on cooperative interactions among G proteins.
Muscarinic agonists and adenyl nucleotides are noncompetitive modulators of sites labeled by [35S]GTP gamma S in washed cardiac membranes from Syrian golden hamsters. Specific binding of the radioligand and its inhibition by either GTP gamma S or GDP reveals three states of affinity for guanyl nucleotides. In the absence of adenyl nucleotide, carbachol promotes an apparent interconversion of sites from higher to lower affinity for GDP; the effect recalls that of guanyl nucleotides on the binding of agonists to muscarinic receptors. In the presence of 0.1 mM ATP gamma S, the binding of [35S]GTP gamma S is increased at concentrations up to about 50 nM and decreased at higher concentrations. At a radioligand concentration of 160 pM, binding exhibits a bell-shaped dependence on the concentration of both ATP gamma S and AMP-PNP; with ADP and ATP, there is a second increase in bound [35S]GTP gamma S at the highest concentrations of adenyl nucleotide. ATP gamma S and AMP-PNP also modulate the effect of GDP, which itself emerges as a cooperative process: that is, binding of the radioligand in the presence of AMP-PNP exhibits a bell-shaped dependence on the concentration of GDP; moreover, the GDP-dependent increase in bound [35S]GTP gamma S is enhanced by carbachol. The interactions among GDP, GTP gamma S, and carbachol can be rationalized quantitatively in terms of a cooperative model involving two sites tentatively identified as G proteins. Both GTP gamma S and GDP exhibit negative homotropic cooperativity; carbachol enhances the homotropic cooperativity of GDP and induces or enhances positive heterotropic cooperativity between GDP and [35S]GTP gamma S. An analogous mechanism may underlie the guanyl nucleotide-dependent binding of agonists to muscarinic receptors. The data suggest that the binding properties of G proteins and their associated receptors reflect cooperative effects within heterooligomeric arrays; agonist-induced changes in cooperativity may facilitate the exchange of GTP for bound GDP and thereby constitute the mechanism of G protein activation in vivo.
Chidiac
P
P
Department of Pharmacology, University of Toronto, Ontario, Canada.
Wells
J W
JW
D000227:Adenine Nucleotides / Q000494:pharmacology*; D000244:Adenosine Diphosphate / Q000008:administration & dosage / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000008:administration & dosage / Q000031:analogs & derivatives / Q000494:pharmacology; D000266:Adenylyl Imidodiphosphate / Q000008:administration & dosage / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D002217:Carbachol / Q000494:pharmacology*; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D019204:GTP-Binding Proteins / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism / Q000494:pharmacology; D006153:Guanosine Diphosphate / Q000378:metabolism / Q000494:pharmacology; D008297:Male; D008647:Mesocricetus
D000227:Adenine Nucleotides; D006153:Guanosine Diphosphate; D000266:Adenylyl Imidodiphosphate; C022571:adenosine 5'-O-(3-thiotriphosphate); D016244:Guanosine 5'-O-(3-Thiotriphosphate); D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D002217:Carbachol; D019204:GTP-Binding Proteins