Ramaschi G, Balduini C, Torti M, Sinigaglia F
Department of Biochemistry, University of Pavia, Italy.
Biochim Biophys Acta. 1994 Jan 5;1199(1):20-6. doi: 10.1016/0304-4165(94)90091-4.
Following activation of human platelets changes in cytoskeletal organization occur: some proteins, which are present in the cytosol or membrane-associated in resting platelets, are recovered in the Triton-insoluble residue in activated cells. Assembly and disassembly of complex effector units on the membrane and inside cells is under the control of low molecular weight GTP-binding proteins, particularly those in the ras family. We investigated the interaction of small GTP-binding proteins with the platelet cytoskeleton and the effect of high cAMP levels on these interactions. At least two GTP-binding proteins of 24 and 28 kDa were detected in the Triton-insoluble residue of resting platelets. Stimulation of platelets with thrombin or concanavalin A (Con A), under non-aggregating conditions, resulted in increased 24 kDa protein-bound GTP, which also contained a significant amount of rap1B. High cAMP levels differently affected this interaction depending on the type of agonist used. cAMP increased association of G-proteins with the cytoskeleton following Con A-activation, while it decreased G-proteins interaction after thrombin stimulation. The activation did not influence the cAMP-dependent phosphorylation of rap1B. No phosphoprotein corresponding to rap1B could be detected in the Triton-insoluble residues, however. These findings could be related to the different mechanisms of cytoskeletal protein recruitment in platelets activated with either thrombin or Con A.
人血小板激活后，细胞骨架组织会发生变化：一些存在于静息血小板胞质溶胶或与膜相关的蛋白质，在活化细胞的Triton不溶性残渣中被回收。膜上和细胞内复杂效应器单元的组装和解聚受低分子量GTP结合蛋白的控制，特别是ras家族中的那些蛋白。我们研究了小GTP结合蛋白与血小板细胞骨架的相互作用以及高cAMP水平对这些相互作用的影响。在静息血小板的Triton不溶性残渣中检测到至少两种分子量分别为24 kDa和28 kDa的GTP结合蛋白。在非聚集条件下，用凝血酶或伴刀豆球蛋白A（Con A）刺激血小板，导致与24 kDa蛋白结合的GTP增加，其中还含有大量的rap1B。高cAMP水平根据所用激动剂的类型对这种相互作用产生不同的影响。cAMP在Con A激活后增加了G蛋白与细胞骨架的结合，而在凝血酶刺激后降低了G蛋白的相互作用。这种激活并不影响rap1B的cAMP依赖性磷酸化。然而，在Triton不溶性残渣中未检测到与rap1B相对应的磷蛋白。这些发现可能与用凝血酶或Con A激活的血小板中细胞骨架蛋白募集的不同机制有关。
