| Budget Amount *help |
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1989: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1988: ¥1,200,000 (Direct Cost: ¥1,200,000)
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| Research Abstract |
Recently, we found that there is a novel GTP-binding protein (G-protein) in calf thymocytes which is associated not only physically but also functionally with soluble phosphoinositide-specific phospholipase C (PLC). It is quite possible that this G-protein is involved in the receptor-coupled regulation of PLC activity. In the present study, we purified this G-protein and investigated its properties. Furthermore, we also examined the physical and functional interactions between mitogen receptors(s), G-proteins and PLC.
In addition to the ability to bind[^<35>S]GTPgammaS, the G-protein exhibited GTPase activity. The G-protein showed a molecular weight of 25,000 on gel filtration, which was different from those of well known G-proteins such as Gs, Gi, Go and Gt.
Since we found that the G-protein might exist in the membrane fraction in a larger amount than in cytosol, this G-protein was purified from the calf thymocyte membrane. The purified G-protein (G21K) showed a molecular weight of 21,0
… More
00 on SDS-PAGE. Comparison of the partial amino acid sequences of G21K with those of known lower molecular weight G- proteins suggests that, although structurally similar to rho gene products, this is a novel G-protein.
Then the mechanism underlying the receptor-coupled PLC activation was examined. We found that mitogen (concanavalin A) receptor(s) possibly interacts with at least two kinds of G-proteins (Gi and a low molecular weight G-protein). The low molecular weight G-protein might be the G-protein involved in receptor-coupled PLC activation and Gi might be responsible for the inhibitory regulation of the cellular cAMP level which inhibits PLC activity. Furthermore, we also found that a part of the G-protein with an approximate molecular weight of 25,000 might be translocated from the membrane to the cytosol and then enhance the cytosol PLC and/or membrane PLC exposed to the cytoplasmic side. At present, it is not evident whether G21K is the G-protein which is associated with mitogen receptor and translocates from membrane to cytosol by mitogen stimulation. However, it is quite possible because both G21K and the receptor associated G-protein were ADP-ribosylated with botulism toxin. Less
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