The Ras-like guanine-nucleotide-binding protein Rap1 controls integrin ␣ IIb  3 activity and platelet aggregation. Recently, we have found that Rap1 activation can be blocked by the nitric oxide/cyclic guanosine monophosphate (NO/cGMP) signaling pathway by type 1 cGMP-dependent protein kinase (cGKI). In search of possible targets of NO/cGMP/cGKI, we studied the expression of Rap1-specific GTPaseactivating proteins (GAPs) and guanine nucleotide exchange factors (GEFs) in platelets. We could detect mRNAs for a new protein most closely related to Rap1GAP and for postsynaptic density-95 discslarge and zona occludens protein 1 (PDZ)-GEF1 and CalDAG-GEFs I and III. Using 5-rapid amplification of cDNA ends (RACE), we isolated the complete cDNA of the new GAP encoding a 715-amino acid protein, which we have termed Rap1GAP2. Rap1GAP2 is expressed in at least 3 splice variants, 2 of which are detectable in platelets. Endogenous Rap1GAP2 protein partially colocalizes with Rap1 in human platelets. In transfected cells, we show that Rap1GAP2 exhibits strong GTPase-stimulating activity toward Rap1. Rap1GAP2 is highly phosphorylated, and we have identified cGKI as a Rap1GAP2 kinase. cGKI phosphorylates Rap1GAP2 exclusively on serine 7, a residue present only in the platelet splice variants of Rap1GAP2. Phosphorylation of Rap1GAP2 by cGKI might mediate inhibitory effects of NO/cGMP on Rap1.
IntroductionPlatelets are of great physiologic importance as regulators of clot formation and inflammation in the vasculature, and they have been established as major therapeutic targets in cardiovascular disease. 1 Platelets contain high levels of Rap1, a Ras-like guanine-nucleotidebinding protein. Recently, Rap1 was identified as a potent regulator of integrin function. [2][3][4][5] For example, the regulation of lymphocyte and macrophage adhesion by integrins ␣L 2 and ␣B M2 , respectively, is controlled by Rap1. 6,7 Rap1 also facilitates the activation of platelet integrin ␣ IIb  3 , which is required for fibrinogen binding and aggregation. 8,9 Recently, Rap1 was shown to control the aggregation of mouse platelets. 10 Many different platelet agonists, including thrombin, adenosine diphosphate (ADP), collagen, and thromboxane, induce Rap1 activation. 11,12 However, the exact pathways involved in Rap1 activation in platelets are unknown. Data from other cell types suggest that receptor-mediated formation of second messengers can lead to the activation of Rap1-specific guanine nucleotide exchange factors (GEFs). Calcium and diacylglycerol activate CalDAG-GEFI (also termed RasGRP2), a protein detected in mouse megakaryocytes and platelets, 10,13 and CalDAG-GEFIII (also termed RasGRP3), involved in neuronal differentiation and B-cell development. 14,15 Cyclic adenosine monophosphate (cAMP) activates cAMP-dependent Epacs, 16,17 and C3G is regulated by tyrosine phosphorylation 18 and by binding to adaptor proteins. 19,20 Postsynaptic density-95 discs-large and zona occludens protein 1 (PDZ)-GEF1 is a ubiquitously expressed GEF of Rap1 and co...