Human Choriogonadotropin Signaling in Cell Proliferation

描述（由申请人提供）：hCG在人类生殖中起着关键作用，在怀孕的前三个月产生。它也产生于多种肿瘤，包括绒毛膜癌、葡萄胎和睾丸胚胎癌。在结构上，hCG在某些方面与肿瘤生长因子(TGF)- β、神经生长因子（NGF）和血小板衍生生长因子(PDGF)- β相似。它们都含有胱氨酸结基序，并与多种肿瘤的发生发展有关。hCG刺激细胞生长和分化，因此，它的功能极有可能是由MAK激酶级联、丝裂原活化蛋白激酶-激酶-激酶(MAPKKK)- mapkk -细胞外信号调节激酶（ERK）三成员调控的。本研究的目的是探讨MAP激酶通路在hCG作用下细胞生长和分化中的作用。通路特异性DNA微阵列技术将用于比较hCG和表皮生长因子（EGF）信号在大鼠SIGC-hCG/中与各自受体相互作用时的基因表达谱；lutropin受体（LHR）和sigc表皮生长因子受体（EGFR）。这将为我们提供一些关于两种信号通路共同的基因表达的信息。我们将研究ERK下游和MAPKKK上游的信号蛋白。最后，寻找可以组织这些激酶的支架蛋白。该研究将在大鼠SIGC中进行。SIGC具有上皮形态，在没有黄体化的培养中生长。初步研究的短期具体目标将包括：1)分别转染hCG/LH和EGF受体cdna的大鼠SIG细胞，选择稳定的转染物SIGC-hCG/LHR和SIGC-EGFR；2) SIGC-hCG/LHR和SIGC-EGFR的初步表征，包括a)通过125IhCG和125IEGF结合测定hCG/LH和EGF受体的存在和浓度，b)研究hCG对腺苷酸环化酶和磷脂酶C激活的影响，以及EGF对受体酪氨酸激酶激活的影响；3)研究hCG、EGF对Raf、MEK、ERK活性的影响；4)应用DNA芯片比较SIGC-hCG/LHR和SIGC-EGFR中hCG和EGF信号通路基因表达谱；5)利用特异性多克隆ai抗体联合或免疫沉淀技术分别测定ERK和Raf激酶的下游和上游信号蛋白；6)寻找MAP激酶模块的支架蛋白。

DESCRIPTION (provided by applicant): hCG plays a key role in human reproduction and is produced during the first trimester of pregnancy. It is also produced by a variety of tumors including choriocarcinoma, hydatidiform mole, and embryonic carcinoma of the testis. Structurally, hCG is similar in some respects to tumor growth factor (TGF)-Beta, nerve growth factor (NGF) and platelet-derived growth factor (PDGF)-Beta. All of them contain cystine knot motif and have been implicated in the development of a variety of tumors. hCG stimulates cell growth and differentiation and, therefore, it is highly likely that its function is regulated by the three member MAK kinase cascade, mitogen-activated protein kinase-kinase-kinase (MAPKKK)--MAPKK-extracellular signal-regulated kinase (ERK). The objectives of the proposed studies are to investigate the involvement of the MAP kinase pathway in the cell growth and differentiation by hCG. Pathway-specific DNA microarray technology will be employed to compare the gene expression profiles of hCG and epidermal growth factor (EGF) signaling when they interact with their respective receptors in signaling in a rat SIGC-hCG/; lutropin receptor (LHR) and SIGC-epidermal growth factor receptor (EGFR). This should give us some information on the expression of genes that are common to both signaling pathways. The signaling proteins downstream of ERK and upstream of MAPKKK will be investigated. Lastly, search for a scaffold protein on which these kinases might be organized will be made. The studies will be carried out with the rat SIGC. SIGC has an epithelial morphology and grows in culture without luteinization. Specific short-term goals of the initial studies will include: 1) Transfection of rat SIG cells with hCG/LH and EGF receptor cDNAs separately and selection of stable transfectants, SIGC-hCG/LHR and SIGC-EGFR; 2) Preliminary characterization of SIGC-hCG/LHR and SIGC-EGFR, which will involve a) determination of the presence and concentration of hCG/LH and EGF receptors by binding assays using 125IhCG and 125IEGF and b) study of the effect of hCG on the activation of adenylyl cyclase and phospholipase C and of EGF on the activation of receptor tyrosine kinase; 3) Study the effect of hCG and EGF on the activities of Raf, MEK and ERK; 4) Comparison of the gene expression profiles of signaling by hCG and EGF in SIGC-hCG/LHR and SIGC-EGFR by DNA microarrays; 5) Determination of the downstream and upstream signaling proteins of ERK and Raf kinases respectively by association or immunoprecipitation techniques using specific polyclonaI antibodies; and 6) Search for a scaffold protein for MAP kinase module will be made.