Effects of adenyl nucleotides and carbachol on cooperative interactions among G proteins

Biochemistry. 1992 Nov 10;31(44):10908-21. doi: 10.1021/bi00159a035.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenine Nucleotides / pharmacology*
  • Adenosine Diphosphate / administration & dosage
  • Adenosine Diphosphate / pharmacology
  • Adenosine Triphosphate / administration & dosage
  • Adenosine Triphosphate / analogs & derivatives
  • Adenosine Triphosphate / pharmacology
  • Adenylyl Imidodiphosphate / administration & dosage
  • Adenylyl Imidodiphosphate / pharmacology
  • Animals
  • Binding Sites / drug effects
  • Carbachol / pharmacology*
  • Cricetinae
  • Dose-Response Relationship, Drug
  • GTP-Binding Proteins / metabolism*
  • Guanosine 5'-O-(3-Thiotriphosphate) / metabolism
  • Guanosine 5'-O-(3-Thiotriphosphate) / pharmacology
  • Guanosine Diphosphate / metabolism
  • Guanosine Diphosphate / pharmacology
  • Male
  • Mesocricetus


  • Adenine Nucleotides
  • Guanosine Diphosphate
  • Adenylyl Imidodiphosphate
  • adenosine 5'-O-(3-thiotriphosphate)
  • Guanosine 5'-O-(3-Thiotriphosphate)
  • Adenosine Diphosphate
  • Adenosine Triphosphate
  • Carbachol
  • GTP-Binding Proteins