Budget Amount *help |
¥6,600,000 (Direct Cost: ¥6,600,000)
Fiscal Year 1995: ¥1,900,000 (Direct Cost: ¥1,900,000)
Fiscal Year 1994: ¥4,700,000 (Direct Cost: ¥4,700,000)
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Research Abstract |
In order to elucidate regulatory mechanisms of histamine H2 receptor function at the molecular level, we have done the following experiments : (1) We artificially mutated thr ee potential N-glycosylation ites in the canine histamine H2 receptor gene and examined the effects of N-glycosylation mutation on the appar ent molecular weight, ligand recognition, intracellular signal transduction and cellular localization of the receptors. From the analysis on the SDS-polyacrylaminde gel electr ophoresis combined with tunicamycin treatment, we found that two of the thr ee potential glycosylation sites are actually glycosylaed. Furthermore, recognition of the ligand, signaltransduction and cellular localization of non-glycosylated mutant receptors are essentially unaffected. Therefore, we conclude that N-glycosylation is not vital for histamine H2 receptor function. (2) Activation of histamine H2 receptor brings about at least two intracellular signaling mechanism, one thr-ough cAMP-proteinkinase A pathway and another thr ough phopholipase C and C-kinase pathway. We examined the role of C-kinase activation on the histamine H2 receptor function. We found that C-kinase activation sensitizes the histamine H2 receptor. This effect was abolished by staur osporine pretreatment without affectiong the desensitizing mechanism, indicating the inter action of two signaling mechanisms plays role in modulating the receptor function. (3) In our preliminary experiments, we found that histamine H2 receptors are phosphor ylated after histamine stimulation. Detailed analysis of the enzymes involved and the phosphor ylation sites are in progress. (4) We found that the receptor internalization after histamine stimulation was compromised in the C-terminally truncated mutant H2 receptors This mutation, however, did not affect the desensitization phenomenon. Thus, C-terminal portion of the receptor appears to be important for receptor cycling with in the cells, but not for desensitization.
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