Tm. Quinton et al., INOSITOL 1,4,5-TRISPHOSPHATE-MEDIATED CA2+ RELEASE FROM PLATELET INTERNAL MEMBRANES IS REGULATED BY DIFFERENTIAL PHOSPHORYLATION, Biochemistry, 35(21), 1996, pp. 6865-6871
Platelets are activated by an increase in cytosolic Ca2+, and a portio
n of this increase is derived from inositol 1,4,5-trisphosphate (InsP(
3))-mediated Ca2+ release from internal stores via the InsP(3) recepto
r. Cytosolic cAMP inhibits platelet activation, and experiments were d
esigned to determine if cAMP-dependent phosphorylation affects the rat
e of InsP(3)-mediated Ca2+ release. Western blotting of platelet inter
nal membranes with anti-InsP(3) receptor and anti-actin binding protei
n antibodies revealed that the platelet contains type 1 InsP(3) recept
or and that it is distinct from actin binding protein. The platelet In
sP(3) receptor was shown to be phosphorylated by endogenous, membrane-
bound kinases as well as by exogenous protein kinase A. Prior phosphor
ylation of the InsP(3) receptor by endogenous kinases inhibited additi
onal protein kinase A-dependent phosphorylation by 60%. Furthermore, e
ndogenous phosphorylation resulted in a 2-fold increase in the InsP(3)
-mediated Ca2+ release rate relative to dephosphorylated controls. Fol
lowing endogenous phosphorylation, additional phosphorylation by prote
in kinase A returned the Ca2+ release rate to control values, while pr
otein kinase A-dependent phosphorylation of dephosphorylated membranes
did not affect the release rate. These results suggest that the InsP(
3) receptor within intact platelets is phosphorylated by endogenous ki
nases which results in a high InsP(3)-mediated Ca2+ release rate, and
that increases in cAMP result in additional phosphorylation that inhib
its Ca2+ release, thus contributing to inhibition of platelet activati
on.