A role for Gi in control of thrombin receptor-phospholipase C coupling in human platelets.
Stimulation of washed human platelets with alpha-thrombin was accompanied by aggregation, formation of inositol phosphates and phosphatidic acid, liberation of arachidonic acid, mobilization of intracellular Ca2+ stores, and influx of Ca2+ from the extracellular medium. Each of these responses was potentiated by a short pretreatment with epinephrine, although alone this agent was ineffective. A prolonged (5 min) stimulation with alpha-thrombin desensitized both phospholipase C and Ca2+ mobilization to a further thrombin challenge. Epinephrine added following thrombin desensitization restored both the ability of thrombin to release Ca2+ stores and stimulate inositol phospholipid hydrolysis. Resensitization was mediated by alpha 2-adrenergic receptors and lasted about 3 min, after which the Ca2+ levels returned again to basal levels. Pretreatment of platelets with phorbol dibutyrate at concentrations which specifically activate protein kinase C increased the rate of desensitization of the thrombin-induced release of Ca2+ stores and abolished the ability of epinephrine to restore the thrombin response. The protein kinase C inhibitor, staurosporine, blocked the inhibitory effect of phorbol ester and also reduced the rate of desensitization of thrombin and subsequent epinephrine action. These results suggest that thrombin activation of protein kinase C phosphorylates and inactivates a signaling protein which is common to both thrombin and alpha 2-adrenergic receptors. This protein is involved in thrombin stimulation of phospholipase C but is not directly stimulatory since epinephrine alone does not activate this enzyme. We searched for a known second messenger protein common to both thrombin and alpha 2-adrenergic receptors which was phosphorylated in intact platelets by protein kinase C in parallel with thrombin-induced desensitization. The alpha subunit of the inhibitory GTP-binding protein, Gi, was the only candidate which fulfilled all of these criteria as shown by immunoprecipitation. Therefore, we suggest that alpha i maintains the thrombin receptor in a state which can couple to phospholipase C when activated with thrombin. This permissive state of alpha i is blocked by phosphorylation by thrombin-activated protein kinase C.
Crouch
M F
MF
Molecular Biology Department, Burroughs Wellcome Co., Research Triangle Park, North Carolina 27709.
Lapetina
E G
EG
D016718:Arachidonic Acid; D001095:Arachidonic Acids / Q000097:blood; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D002118:Calcium / Q000097:blood; D002352:Carrier Proteins / Q000097:blood; D004837:Epinephrine / Q000494:pharmacology; D011464:Epoprostenol / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000502:physiology*; D006801:Humans; D007163:Immunosorbent Techniques; D007295:Inositol Phosphates / Q000097:blood; D010750:Phosphoproteins / Q000097:blood; D010974:Platelet Aggregation / Q000187:drug effects; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000097:blood; D011956:Receptors, Cell Surface / Q000187:drug effects / Q000378:metabolism*; D018179:Receptors, Thrombin; D017923:Sodium-Hydrogen Exchangers; D013917:Thrombin / Q000494:pharmacology; D010738:Type C Phospholipases / Q000378:metabolism*
D001095:Arachidonic Acids; D002352:Carrier Proteins; D007295:Inositol Phosphates; D010750:Phosphoproteins; D011956:Receptors, Cell Surface; D018179:Receptors, Thrombin; D017923:Sodium-Hydrogen Exchangers; D016718:Arachidonic Acid; D011464:Epoprostenol; D011493:Protein Kinase C; D010738:Type C Phospholipases; D013917:Thrombin; D019204:GTP-Binding Proteins; D002118:Calcium; D004837:Epinephrine