|Budget Amount *help
¥3,400,000 (Direct Cost : ¥3,400,000)
Fiscal Year 2000 : ¥500,000 (Direct Cost : ¥500,000)
Fiscal Year 1999 : ¥700,000 (Direct Cost : ¥700,000)
Fiscal Year 1998 : ¥500,000 (Direct Cost : ¥500,000)
Fiscal Year 1997 : ¥1,700,000 (Direct Cost : ¥1,700,000)
The assay method for agonist-induced stimulation of high-affinity GTPase activity was developed and applied to membranes prepared from rat brain, postmortem human brain, and human blood platelets to investigate the functional interaction between some receptors and their coupled G proteins. This method was shown to be suitable for detection of the functional activation of the G proteins that are negatively coupled to adenylyl cyclase, i.e.G_i family. Indeed, it has been shown that glutamate-stimulated high-affintiy GTPase activity in rat cerebral cortical, striatal, and hippocampal membranes is mediated through group II metabotropic glutamate receptors, which are known to inhibit adenylyl cyclase activity when stimulated. Furthermore, it has been shown that activation of G proteins coupled with some receptors negatively coupled to adenylyl cyclase, especially GABA_B receptors, can be detected in frontal cortical membranes prepared from postmortem human brains. This method was also utilized to investigate the functional activation of G proteins by some peptides, e.g.wasp venom mastoparan and neuropeptide Y, which is not mediated specific receptors but takes place directly or receptor-independently. We also tried to identify the G protein subtypes involved in receptor-mediated high-affinity GTPase activity, and found that G_<i2> was involved in the GABA_B receptor-mediated response in rat cortical membranes. The stimulatory effects of epinephrine and thrombin on high-affinity GTPase activity in human platelet membranes were not significantly different between affective disorder patients and control subjects. On the other hand, cyclic AMP response element binding protein (CREB) levels determined with western blotting in postmortem frontal cortex were upregulated in depressed suicide victims compared with control subjects. This assay provides useful information about mechanisms of functional activation of G proteins, in particular G_i, even in native membranes.