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 许多物种都有很高的亲和力。我们对肝脏生长激素的研究 受体(GHR)和血清生长激素结合蛋白(GHBP)提示 这些蛋白质是由单个GHR/GHBP的选择性剪接产生的 基因产生编码膜结合的GHR或可溶性GHBP的mRNAs， 但这一假设尚未得到证实。这样做的主要目标是 该项目旨在描述GHR和GHBP 在鼠标中生成。基因组印迹将被分析以确定 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、限制性内切酶图谱进行检测 和序列分析。 该项目的第二个目标是开发定量分析方法 GHBP和小鼠生长激素(MGH)，并测定GHBP和MGH的妊娠分布 孕妇血清中的MGH。初步数据表明，GHBP会干扰 用现有的放射免疫分析法(RIA)测定MGH。一项RIA到 准确测量总MGH将通过使用 检测前提取方法以去除GHBP或通过生成 结合游离MGH和结合GHBP的MGH的抗肽抗血清。 孕妇血清将被分离，以分离GHBP结合的MGH和游离的MGH MGH，这两个组分的MGH浓度将被测量到 测定妊娠期间与mGHBP结合的mGH的比例。至 建立GHBP的RIA，重组GHBP将在中文中表达 仓鼠卵巢细胞和纯化。重组GHBP将用于 制定RIA。 已克隆的生长激素受体中没有一种被证明能引起 生物反应。这个项目的第三个目标是开发一种 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 3T3-F442A型GHR基因转染的脂肪细胞。因为该分析可能 内源性GHR使情况变得复杂，另一种选择是使用细胞 用由胞外区组成的嵌合受体转染 小鼠催乳素受体与跨膜和细胞内 小鼠生长激素受体的结构域。小鼠对葡萄糖的摄取和氧化反应 将检查催乳素与该嵌合受体的结合情况。这些 这些系统可能会对未来的信号转导研究非常有用。 由GHR提供。 生长激素的作用及对生长激素浓度的调节 孕妇在怀孕期间的血清并不是很清楚。中的研究 该项目为膜结合研究提供了重要的基础信息 生长激素受体，它在细胞水平上介导生长激素的作用，以及 关于可溶性生长激素结合蛋白，它可能起着重要作用 测定母体血清中生长激素的有效浓度。

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.