A low Mr GTP‐binding protein, Rapl, in human platelets: localization, translocation and phosphorylation by cyclic AMP‐dependent protein kinase

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Abstract
Subcellular fractions were prepared from human platelet membranes by sucrose density gradient centrifuga‐tlon and the localization of a low Mr GTP‐binding protein, rapl protein (Rapl) was analysed by immunoblotting using a specific antibody. Rapl, which has been purified from human platelets, was found to be located in plasma membrane and a‐granule fractions in resting platelets. Treatment of isolated a‐granules with pronase led to proteolysis of Rapl, indicating that this protein is exposed to the cytoplasmic face of the granules. Degranulation of a‐ granules consists of translocation and subsequent fusion of the granules with the open canalicular system. Activation of this process by thrombin induced the redistribution of Rapl on the a‐granules to plasma membranes. On the other hand, Rapl is known to be phosphorylated by cyclic AMP‐dependent protein kinase (A‐kinase) in vitro and in vivo. In intact human platelets, phosphorylation of Rapl by A‐kinase in response to prostaglandin Ex (PGEi) was observed only in Rapl localized in plasma membranes and not on a‐granules, although Rapl was phosphorylated in a cell‐free system when plasma membranes and a‐granule membranes were exposed to A‐kinase as substrates. These results strongly suggest that Rapl in plasma membranes and the protein on a‐granules are regulated by different mechanisms, and have different functions.

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