MOLECULAR MECHANISM OF REGULATION OF HISTAMINE H2RECEPTOR FUNCTION

为了从分子水平阐明组胺H_2受体功能的调控机制，我们进行了以下实验：（1）对犬组胺H_2受体基因中三个潜在的N-糖基化位点进行了人工突变，研究了N-糖基化突变对受体表观分子量、配体识别、细胞内信号转导和细胞定位的影响。通过SDS-聚丙烯酰胺凝胶电泳结合衣霉素处理的分析，我们发现三个潜在的糖基化位点中有两个实际上被糖基化了。此外，非糖基化突变受体的配体识别、信号转导和细胞定位基本上不受影响。因此，我们得出结论，N-糖基化是不是至关重要的组胺H2受体功能。(2)组胺H_2受体的激活至少引起两种细胞内信号传导机制，一种通过cAMP-蛋白激酶A途径，另一种通过磷脂酶C和C-激酶途径。我们研究了C-激酶激活对组胺H2受体功能的作用。我们发现，C-激酶激活致敏组胺H2受体。星形孢菌素预处理可消除这种效应，但不影响脱敏机制，表明两种信号机制的相互作用在调节受体功能中起作用。(3)在我们的初步实验中，我们发现组胺H2受体磷酸化后组胺刺激。有关酶和磷酸化位点的详细分析正在进行中。(4)我们发现，组胺刺激后的受体内化在C-末端截短的突变H2受体中受到损害。然而，这种突变并不影响脱敏现象。因此，受体的C-末端部分似乎对于细胞中的受体循环是重要的，但对于脱敏不是重要的。

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.