Abstract
To investigate the function of the human Ras-related CDC42 GTP-binding protein (CDC42Hs) we studied its subcellular redistribution in platelets stimulated by thrombin-receptor activating peptide (TRAP) or ADP. In resting platelets CDC42Hs was detected exclusively in the membrane skeleton (9.6 +/- 1.5% of total) and the detergent soluble fraction (90 +/- 4%). When platelets were aggregated with TRAP or ADP, CDC42Hs (10% of total) appeared in the cytoskeleton and decreased in the membrane skeleton, whereas RhoGDI (guanine-nucleotide dissociation inhibitor) and CDC42HsGAP (GTPase-activating protein) remained exclusively in the detergent-soluble fraction. Upon prolonged platelet stimulation CDC42Hs disappeared from the cytoskeleton and reappeared in the membrane skeleton. Rac translocated to the cytoskeleton with a similar time course as CDC42Hs. When platelets were stimulated under conditions that precluded the activation of the alpha IIb beta 3 integrin and platelet aggregation, cytoskeletal association of CDC42Hs was abolished. Translocation of CDC42Hs to the cytoskeleton but not aggregation was also prevented by cytochalasins B or D or the protein tyrosine kinase inhibitor genistein. Platelet secretion and thromboxane formation were not required but facilitated the cytoskeletal association of CDC42Hs. The results indicate that in platelets stimulated by TRAP or ADP, a fraction of CDC42Hs translocates from the membrane skeleton to the cytoskeleton. This process is reversible and is mediated by activation of the alpha IIb beta 3 integrin and subsequent actin polymerization and protein-tyrosine kinase stimulation. CDC42Hs might be a new component of a signaling complex containing specific cytoskeletal proteins and protein-tyrosine kinases that forms after activation of the alpha IIb beta 3 integrin in platelets.
Highlights
From the Institut fiir Prophylaxe und Epidemiologie der Kreislaufkrankheiten bei der Unioersittit Mtinchen, Pettenkoferstrasse 9, 80336 Miinchen, Germany
After three washes of the blot, antibody binding was detected using the enhanced chemiluminescence (ECL) system (Amersham Corp.), Bands corresponding to CDC42Hs and RhoGDI were measured by laser densitometry (Ultroscan XL, Pharmacia Biotech Inc.), To examine the proteolytic cleavage of actin-binding protein and talin, platelet proteins were separated on a 6% SDS-polyacrylamide gel electrophoresis slab gel, followed by staining of the gel with 0.2% Coomassie Brilliant Blue, destaining, and drying
The degree of cytoskeletal association ofCDC42Hs correlated with the extent of platelet aggregation (Fig. 1); at 50% aggregation the CDC42Hs protein band was barely detectable, and at 95% aggregation the association was maximal
Summary
Vol 270, No 29, Issue of July 21, pp. 17321-17326, 1995 Printed in U.S.A. Integrin aUbP3-mediated Translocation of CDC42Hs to the Cytoskeleton in Stimulated Human Platelets*. To investigate the function of the human Ras-related CDC42 GTP-binding protein (CDC42Hs) we studied its subcellular redistribution in platelets stimulated by thrombin-receptor activating peptide (TRAP) or ADP. When platelets were aggregated with TRAP or ADP, CDC42Hs (10% of total) appeared in the cytoskeleton and decreased in the membrane skeleton, whereas RhoGDI (guaninenucleotide dissociation inhibitor) and CDC42HsGAP (GTPase-activating protein) remained exclusively in the detergent-soluble fraction. Platelet secretion and thromboxane formation were not required but facilitated the cytoskeletal association of CDC42Hs. The results indicate that in platelets stimulated by TRAP or ADP, a fraction of CDC42Hs translocates from the membrane skeleton to the cytoskeleton. The results indicate that in platelets stimulated by TRAP or ADP, a fraction of CDC42Hs translocates from the membrane skeleton to the cytoskeleton This process is reversible and is mediated by activation of the lXubf integrin and subsequent actin polymerization and proteintyrosine kinase stimulation. The CDC42Hs translocation is mediated by activation of the QIIbf integrin and is dependent on actin polymerization and proteintyrosine kinase activation
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