Epinephrine induces changes in the subcellular distribution of the inhibitory GTP-binding protein G(iα-2) and a 38-kDa phosphorylated protein in the human platelet

Abstract

By using antibodies specific for α subunits of inhibitor GTP-binding proteins (G(iα) polypeptides) to probe Western blots of whole platelet protein, we detected G(iα-2) as the predominant G(iα) species present in platelets. The subcellular compartmentalization of distinct G(iα-2)-immunoreactive polypeptides coupled to thrombin and α2-adrenergic receptors was examined in Triton X-100 platelet lysates prepared by high-speed centrifugation. This treatment permitted separation of the Triton-insoluble membrane skeleton from Triton-soluble cell components. In cells treated with either α-thrombin or epinephrine, we observed that a greater proportion of G(iα-2) was localized in the Triton-soluble fraction than in the Triton-insoluble fraction. Pertussis toxin was found to catalyze ADP-ribosylation of G(iα-2) in whole platelets. In thrombin-stimulated cells, this activity was confined to the Triton-soluble fraction and was markedly lower than that of unstimulated cells. Epinephrine, on the other hand, promoted translocation of a portion of the pertussis toxin-sensitive G(iα-2) from the Triton-soluble fraction to the Triton-insoluble fraction. In addition, epinephrine stimulated translocation of a phosphorylated protein of ~ 38 kDa that was not ADP-ribosylated by pertussis toxin. This protein expressed immunoreactivity with the general G(iα) antiserum AS/7 but not with the G(iα-2) antiserum LE/3. These findings suggest a role for specific localization of G(iα) proteins in epinephrine-induced platelet responses.