PLATELET SARCO ENDOPLASMIC RETICULUM CA(2+)ATPASE ISOFORM 3B AND RAP 1B - INTERRELATION AND REGULATION IN PHYSIOPATHOLOGY/

Platelet Ca2+ signalling involves intracellular Ca2+ pools, whose cont ent is controlled by sarco/endoplasmic reticulum Ca(2+)ATPases (SERCAs ). Among these, a key role is played by the inositol trisphosphate-sen sitive Ca2+ pool, associated with the SERCA 3b isoform. We have invest igated the control of this Ca2+ pool through the cAMP-dependent phosph orylation of the GTP-binding protein, Rap (Ras-proximate) 1b. We first looked for this Ca2+ pool target of regulation by studying the expres sion of the different SERCA and Rap 1 proteins in human platelets and various cell lines, by Western blotting and reverse transcription-PCR. Since co-expression of Rap 1b and SERCA 3b was obtained, we looked fo r their protein-protein interaction as a function of the cAMP-dependen t phosphorylation of Rap 1b. Co-immunoprecipitations of SERCA 3b and R ap 1b proteins were found in the absence of phosphorylation, induced b y the catalytic subunit of the cAMP-dependent protein kinase (csPKA). In contrast, upon pre-treatment of platelet membranes with csPKA, the SERCA 3b dissociated from the Rap 1b protein, in agreement with a role of its phosphorylated state in their interaction. Finally, we looked for adaptation of this complex in a platelet pathological model of hyp ertension. We investigated the expression of both proteins, as well as the cAMP-dependent phosphorylation of Rap Ib and SERCA 3b activity in platelets from control normotensive Wistar-Kyoto rats and from sponta neously hypertensive rats (SHRs). A decrease in SERCA 3b activity was associated with a decrease in Rap 1b endogenous phosphorylation in SHR platelets, consistent with a functional role in the regulation of the SERCA 3b-associated Ca2+ pool.