GROWTH HORMONE RECEPTOR AND SERUM BINDING PROTEIN

结合生长激素（GH）的膜结合蛋白和可溶性蛋白， 高亲和力存在于许多物种中。 我们对肝脏生长激素的研究 小鼠血清GH受体（GHR）和GH结合蛋白（GHBP）的变化提示 这些蛋白质是由单个GHR/GHBP的选择性剪接产生的 基因以产生编码膜结合GHR或可溶性GHBP的mRNA， 但这一假设尚未得到证实。 这个的主要目标 项目的特点是GHR和GHBP的机制， 在鼠标中生成。 将分析基因组印迹，以确定是否 每种蛋白质都有一个GHR/GHBP基因或单独的基因。 基因结构将通过基因组克隆、限制性内切酶图谱分析 和序列分析。 该项目的第二个目标是开发定量分析， GHBP和小鼠GH（mGH），并确定GHBP和 母体血清中的mGH。 初步数据表明GHBP干扰 通过现有的放射免疫测定（RIA）测量mGH。 一个RIA， 准确测量总mGH将开发使用 测定前提取方法以去除GHBP或通过产生 结合游离mGH和与GHBP结合的mGH的抗肽抗血清。 将对母体血清进行分级分离，以将GHBP结合的mGH与游离mGH分离。 mGH，并测量两种组分的mGH浓度， 测定整个妊娠期间mGH与mGHBP结合的分数。 到 建立GHBP的放射免疫分析方法，并在中国人中表达重组GHBP 仓鼠卵巢细胞并纯化。 重组GHBP将用于 开发RIA。 已经克隆的GHR中没有一个被证明能引起 生物反应。 该项目的第三个目标是开发一个 用于检测mGH与GHR结合的生物学效应的系统。 mGH对葡萄糖摄取和氧化的影响将在 用GHR cDNA转染的3 T3-F442 A脂肪细胞。 由于这一分析可能 由于内源性GHR的复杂性，一种替代方案将是使用细胞 用由胞外结构域组成的嵌合受体转染 小鼠催乳素受体的跨膜和细胞内 小鼠GHR的结构域。 小鼠对葡萄糖的摄取和氧化 将检测催乳素与该嵌合受体的结合。 这些 系统可能被证明是非常有用的，为未来的研究信号转导 通过GHR。 生长激素的作用及对生长激素浓度的调节 妊娠期间的母体血清还不太清楚。 中的研究 该项目将提供有关膜结合的重要基础信息， GH受体，在细胞水平介导GH作用，和 可溶性生长激素结合蛋白，这可能起着重要作用， 测定母体血清中GH的有效浓度。

Membrane-bound and soluble proteins that bind growth hormone (GH) with high affinity are present in many species. Our studies on the hepatic GH receptor (GHR) and serum GH binding protein (GHBP) of the mouse suggest these proteins are generated by alternative splicing of a single GHR/GHBP gene to yield mRNAs encoding either membrane-bound GHR or soluble GHBP, but this hypothesis has not been proven. The primary goal of this project is to characterize the mechanism by which GHR and GHBP are generated in the mouse. Genomic blots will be analyzed to determine if there is a single GHR/GHBP gene or individual genes for each protein. Gene structure will be examined by genomic cloning, restriction mapping and sequence analysis. A second goal of this project is to develop quantitative assays for GHBP and mouse GH (mGH) and to determine gestational profiles of GHBP and mGH in maternal serum. Preliminary data indicate that GHBP interferes with measurement of mGH by existing radioimmunassays (RIAs). An RIA to accurately measure total mGH will be developed either by using a pre-assay extraction method to remove GHBP or by generating an anti-peptide antiserum that binds both free mGH and mGH bound to GHBP. Maternal serum will be fractionated to separate GHBP-bound mGH from free mGH, and the mGH concentration of both fractions will be measured to determine the fraction of mGH bound to mGHBP throughout pregnancy. To develop an RIA for GHBP, recombinant GHBP will be expressed in Chinese hamster ovary cells and purified. The recombinant GHBP will be used to develop an RIA. None of the GHRs that have been cloned has been shown to elicit a biological response. The third goal of this project is to develop a system for examining the biological effect of mGH binding to GHR. Effects of mGH on glucose uptake and oxidation will be examined in 3T3-F442A adipocytes transfected with GHR cDNA. Since this analysis may be complicated by endogenous GHR, an alternative will be to use cells transfected with a chimeric receptor composed of the extracellular domain of the mouse prolactin receptor and the transmembrane and intracellular domains of mouse GHR. Glucose uptake and oxidation in response to mouse prolactin binding to this chimeric receptor will be examined. These systems may prove very useful for future studies of signal transduction by GHR. The function of GH and the regulation of GH concentrations in maternal serum during pregnancy is not well understood. The studies in this project win provide important basic information about membrane-bound receptors for GH, which mediate GH action at the cellular level, and about soluble GH binding protein, which probably plays an important role in determining the effective concentration of GH in maternal serum.