5'-p-Fluorosulfonylbenzoyl guanosine as a probe for the GTP-binding protein in alpha 2-adrenergic receptor-adenylate cyclase systems.

An analog of GTP, 5'-p-fluorosulfonylbenzoyl guanosine (5'-p-FSO2BzGuo), appears to interact irreversibly with guanine nucleotide-binding sites on human platelet membranes. This conclusion is based on the observation that incubation of human platelet membranes with 1.4 mM 5'-p-FSO2BzGuo followed by extensive membrane washing results in a reduction in the density of binding sites for [3H]guanylylimidodiphosphate (Gpp(NH)p), a hydrolysis-resistant analog of GTP. The alpha 2-adrenergic receptor of human platelets is felt to interact with a GTP-binding protein that modulates alpha 2-receptor-agonist interactions and mediates inhibition of adenylate cyclase. The present data suggest that 5'-p-FSO2BzGuo attains saturating, or near saturating, occupancy of this alpha 2-receptor-associated GTP-binding protein, since incubation of human platelet membranes with 5'-p-FSO2BzGuo mimics the effects of optimal concentrations of Gpp(NH)p (0.1 mM) in reducing alpha 2-receptor affinity for agonist agents: 5'-p-FSO2BzGuo increases the EC50 for epinephrine competition for [3H]yohimbine antagonist binding to alpha 2-receptors from 0.15 to 1.5 microM and promotes a time- and concentration-dependent decrease in high affinity [3H]epinephrine agonist binding. The persistent effects of 5'-p-FSO2BzGuo on alpha 2-receptor-agonist interactions following extensive washing of the platelet membranes suggest that 5'-p-FSO2BzGuo modification of the alpha 2-receptor-associated GTP-binding protein is irreversible. Taken together, the above findings suggest that 5'-p-FSO2BzGuo may be the appropriate reagent to prepare in a radiolabeled form to affinity label the GTP-binding proteins in human platelet membranes and compare the properties of alpha 2-adrenergic receptor-associated GTP-binding protein(s) with those of the presumably distinct GTP-binding protein that mediates stimulation of adenylate cyclase.