MOLECULAR MECHANISM OF REGULATION OF HISTAMINE H2RECEPTOR FUNCTION

为了在分子水平上阐明组胺H2受体功能的调控机制，我们做了以下实验：(1)人工突变犬组胺H2受体基因中3个潜在的n -糖基化位点，研究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。从sds -聚丙烯酰胺凝胶电泳联合tunicamycin处理的分析中，我们发现3个潜在糖基化位点中有2个实际上是糖基化的。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 -糖基化对组胺H2受体的功能不是至关重要的。(2)组胺H2受体的激活至少带来两种细胞内信号通路，一种是通过cAMP-proteinkinase A途径，另一种是通过磷脂酶C和C激酶途径。我们研究了c激酶活化对组胺H2受体功能的作用。我们发现c激酶激活使组胺H2受体增敏。这一作用在不影响脱敏机制的情况下被奥孢素预处理消除，说明两种信号机制的相互作用在调节受体功能中起作用。(3)在前期实验中，我们发现组胺H2受体在组胺刺激后发生磷酸化。有关酶和磷酸化位点的详细分析正在进行中。(4)我们发现，在c端截断突变体H2受体中，组胺刺激后的受体内化受到损害，但这种突变不影响脱敏现象。因此，受体的c端部分似乎对细胞内的受体循环很重要，但对脱敏并不重要。

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.