|Budget Amount *help
¥7,300,000 (Direct Cost : ¥7,300,000)
Fiscal Year 1996 : ¥2,400,000 (Direct Cost : ¥2,400,000)
Fiscal Year 1995 : ¥4,900,000 (Direct Cost : ¥4,900,000)
1.Molecular Structure and Functions of Thromboxane A^2 (TXA_2) receptor Isoforms of Human Platelets. In human platelets, we demonstrated two isoforms of the human TXA_2 receptor (TXR) which had been cloned from placenta (TXRalpha) and from endothelium (TXRbeta). The two isoforms expressed in cultured cells showed similar ligand binding characteristics and phospholipase C (PLC) activation but oppositely regulated adenylyl cyclase (AC) activity ; TXRalpha activated AC,while TXRbeta inhibited it. The Arg^<60> to Leu mutant of TXRalpha、which we had reported to impair PLC activation, also impaired AC stimulation, while that of TXRbeta impaired PLC activation but retained its activity to inhibit AC.These findings suggest that carboxyl-terminal tails of TXRs might be involved in the pathway linked to regulate AC activity.
2.Collagen-stimulated Activation of Protein-tyrosine Kinases through Platelet Membrane Glycoprotein VI (GPVI). We found two platelet protein-tyrosine kinases, c-Src and Syk, became rapidly activated in response to anti-GPVI antibody insensitively to elevation of cAMP in a manner similar to collagen stimulation. Furthermore, either anti-GPVI antibody or collagen stimulated cAMP-insensitive tyrosine phosphorylation of phospholipase C-gamma_2. In GPVI-deficient platelets with normal amounts of integrin alpha_2beta_1, collagen induced alpha_2beta_1-dependent c-Src activation accompanied by tyrosine phosphorylation of several substrates including cortactin. In contrast, severe defects were observed in collagen-stimulated Syk activation and tyrosine phosphorylation of phospholipase C-gamma_2, Vav, and focal adhesion kinase, implicating a specific requirement of GPVI for recruiting these molecules to signaling cascades evoked by collagen-platelet interaction.