Evidence for a role of rap1 protein in the regulation of human platelet Ca2+ fluxes

Abstract

The relationship between the 22-24 kDa cyclic AMP (cAMP)-dependent phosphoprotein previously described as being involved in the regulation of human platelet membrane Ca2+ transport and a GTP-binding protein of low molecular mass (ras-like protein) was investigated. After isolation of plasma membranes and intracellular membranes, it was found that guanosine 5'-[y-thio]triphosphate (GTP[S]) bound to plasma membrane proteins ranging in molecular mass from 22 to 29 kDa, but not to intracellular membranes. The major GTP-binding protein appeared as a 24 kDa protein under reduced conditions and a 22 kDa protein under non-reduced conditions. A similar membrane location and electrophoretic mobility were found for both the cAMP phosphoprotein and the protein recognized by a specific anti-rapI antibody. The identity between the cAMP phosphoprotein and the rapl GTP-binding protein was further examined by studying the functional effect of GTP on plasma membrane Ca2+ transport. A maximal GTP[S] concentration of 40 /,M was found to:(1) inhibit to the same degree (40%) both Ca2+-ATPase activity and the Ca2+ transport function mediated by the Ca2+-ATPase; (2) inhibit the phosphorylation of the 22-24 kDa protein by the catalytic subunit of the cAMP-dependent protein kinase (C.Sub.); and (3) abolish the stimulation of Ca2+ uptake induced by C.Sub. It is concluded that the platelet cAMP phosphoprotein is indeed the rapl GTP-binding protein, and that it regulates plasma membrane Ca2+ transport, thus providing evidence for a new role of a ras-related protein.

Cyclic AMP Raises Intracellular Ca 2+ in Human Megakaryocytes Independent of Protein Kinase A

“…These findings are in correspondence with the hypothesis that Rap1 is a molecular "switch" that regulates SERCA activity: the cAMP-induced Rap1 activation is correlated with the inhibition of SERCA and a rise in [Ca 2ϩ ] i (present data), and the PKA-mediated phosphorylation of Rap1 relieves the inhibition, resulting in activation of Ca 2ϩ -ATPases and a decrease in [Ca 2ϩ ] i . 10,11 The latter property is also observed in Ca 2ϩ regulation by phospholamban in cardiac and muscle cells. 23,24 Phosphorylation of this 24-kDa protein by PKA triggers stimulation of cardiac sarcoplasmic reticulum Ca 2ϩ -ATPases, leading to a fall in [Ca 2ϩ ] i .…”

“…Two types of cAMP GEF for Rap1 have been reported: cAMP-GEF-I, also called exchange protein, directly activated by cAMP (Epac), and cAMP-GEF-II. Interestingly, earlier work by Corvazier et al 11 and LacabaratzPorret et al 10 indicated that Rap1 might be involved in Ca 2ϩ regulation in platelets. First, proteins in crude platelet plasma membrane vesicles, ranging in molecular mass from 22 to 29 kDa, bound GTP␥S.…”

“…10,11 Because the guanine nucleotide exchange factor (GEF) for Rap1, Epac1/2, is a target for PKA-independent signaling by cAMP, Rap1 could be the link between cAMP and Ca 2ϩ . 12,13 Using a pulldown technique based on the specific binding of active, GTP-bound Rap1 to the Rap-binding domain of RalGDS, we investigated the effect of iloprost and forskolin on Rap1 activation.…”

Conventional protein kinase C isoforms differentially regulate ADP- and thrombin-evoked Ca2+ signalling in human platelets

“…However, Tao and colleagues [12] have previously suggested that PKC may affect SERCA indirectly via phosphorylation of Rap1b. Given that PKC has been shown to phosphorylate and activate Rap1b [48,49] and that phosphorylation of Rap1b by other kinases is thought to regulate SERCA activity in platelets [50][51][52], Rap1b may be involved in the regulation of SERCA activity by PKC. Further studies investigating the phosphorylation of SERCA and association of Rap1b with SERCA may help distinguish between these possibilities.…”

Immunolocalization of Rap1 in the Rat Parotid Gland: Detection on Secretory Granule Membranes

“…In neutrophils, in which rap1 is located primarily on the specific granules, studies suggest that it plays a crucial role in phagocytosis (Maridonneau-Parini and de Gunzburg 1992). In platelets it has been linked to cytoskeletal reorganization (Fischer et al 1990), Ca 2 ϩ regulation (Corvazier et al 1992), and to regulation of PLC ␥ activity (Torti and Lapetina 1992).…”