EFFECTS OF ADENYL-NUCLEOTIDES AND CARBACHOL ON COOPERATIVE INTERACTIONS AMONG G-PROTEINS

被引:43
作者
CHIDIAC, P
WELLS, JW
机构
[1] UNIV TORONTO, DEPT PHARMACOL, TORONTO M5S 1A1, ONTARIO, CANADA
[2] UNIV TORONTO, FAC PHARM, TORONTO M5S 1A1, ONTARIO, CANADA
关键词
D O I
10.1021/bi00159a035
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Muscarinic agonists and adenyl nucleotides are noncompetitive modulators of sites labeled by [S-35]GTPgammaS in washed cardiac membranes from Syrian golden hamsters. Specific binding of the radioligand and its inhibition by either GTPgammaS 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 ATPgammaS, the binding of [S-35]GTPgammaS 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 ATPgammaS and AMP-PNP; with ADP and ATP, there is a second increase in bound [S-35]GTPgammaS at the highest concentrations of adenyl nucleotide. ATPgammaS 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 [S-35]GTPgammaS is enhanced by carbachol. The interactions among GDP, GTPgammaS, and carbachol can be rationalized quantitatively in terms of a cooperative model involving two sites tentatively identified as G proteins. Both GTPgammaS and GDP exhibit negative homotropic cooperativity; carbachol enhances the homotropic cooperativity of GDP and induces or enhances positive heterotropic cooperativity between GDP and [S-35]GTPgammaS. 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.
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页码:10908 / 10921
页数:14
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