Inositol 1,4,5-trisphosphate-mediated Ca2+ release from platelet internal membranes is regulated by differential phosphorylation.
Platelets are activated by an increase in cytosolic Ca2+, and a portion of this increase is derived from inositol 1,4,5-trisphosphate (InsP3)-mediated Ca2+ release from internal stores via the InsP3 receptor. Cytosolic cAMP inhibits platelet activation, and experiments were designed to determine if cAMP-dependent phosphorylation affects the rate of InsP3-mediated Ca2+ release. Western blotting of platelet internal membranes with anti-InsP3 receptor and anti-actin binding protein antibodies revealed that the platelet contains type 1 InsP3 receptor and that it is distinct from actin binding protein. The platelet InsP3 receptor was shown to be phosphorylated by endogenous, membrane-bound kinases as well as by exogenous protein kinase A. Prior phosphorylation of the insP3 receptor by endogenous kinases inhibited additional protein kinase A-dependent phosphorylation by 60%. Furthermore, endogenous phosphorylation resulted in a 2-fold increase in the InsP3-mediated Ca2+ release rate relative to dephosphorylated controls. Following endogenous phosphorylation, additional phosphorylation by protein kinase A returned the Ca2+ release rate to control values, while protein kinase A-dependent phosphorylation of dephosphorylated membranes did not affect the release rate. These results suggest that the InsP3 receptor within intact platelets is phosphorylated by endogenous kinases which results in a high InsP3-mediated Ca2+ release rate, and that increases in cAMP result in additional phosphorylation that inhibits Ca2+ release, thus contributing to inhibition of platelet activation.
Quinton
T M
TM
Department of Biochemistry, University of Louisville School of Medicine, Kentucky 40292, USA.
Brown
K D
KD
Dean
W L
WL
D000818:Animals; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D015153:Blotting, Western; D001921:Brain / Q000378:metabolism; D002118:Calcium / Q000097:blood*; D015220:Calcium Channels / Q000302:isolation & purification / Q000378:metabolism*; D000242:Cyclic AMP / Q000097:blood; D006801:Humans; D066298:In Vitro Techniques; D015544:Inositol 1,4,5-Trisphosphate / Q000494:pharmacology*; D053496:Inositol 1,4,5-Trisphosphate Receptors; D007425:Intracellular Membranes / Q000187:drug effects / Q000378:metabolism*; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism*; D008861:Microsomes / Q000378:metabolism*; D010766:Phosphorylation; D051381:Rats; D018160:Receptors, Cytoplasmic and Nuclear / Q000302:isolation & purification / Q000378:metabolism*
D015220:Calcium Channels; C506926:ITPR1 protein, human; D053496:Inositol 1,4,5-Trisphosphate Receptors; D008565:Membrane Proteins; D018160:Receptors, Cytoplasmic and Nuclear; D015544:Inositol 1,4,5-Trisphosphate; D000242:Cyclic AMP; D002118:Calcium