STABLE TRANSFECTION OF GH(3) CELLS WITH RAT GONADOTROPIN-RELEASING-HORMONE RECEPTOR COMPLEMENTARY DEOXYRIBONUCLEIC-ACID RESULTS IN EXPRESSION OF A RECEPTOR-COUPLED TO CYCLIC ADENOSINE 3',5'-MONOPHOSPHATE-DEPENDENT PROLACTIN-RELEASE VIA A G-PROTEIN
STABLE TRANSFECTION OF GH(3) CELLS WITH RAT GONADOTROPIN-RELEASING-HORMONE RECEPTOR COMPLEMENTARY DEOXYRIBONUCLEIC-ACID RESULTS IN EXPRESSION OF A RECEPTOR-COUPLED TO CYCLIC ADENOSINE 3',5'-MONOPHOSPHATE-DEPENDENT PROLACTIN-RELEASE VIA A G-PROTEIN
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DOI：
10.1210/en.135.1.315
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发表时间：
1994-07-01
期刊：
影响因子：
4.8
通讯作者：
CONN, PM
中科院分区：
文献类型：
--
作者：
KUPHAL, D;JANOVICK, JA;CONN, PM
GH(3)细胞通常在TRH刺激下释放PRL，目前已稳定转染了大鼠GnRH受体互补DNA （GGH(3)-1’细胞）。与亲本系不同，GGH(3)-1’细胞表达GnRH受体，可以使用代谢稳定的GnRH类似物在放射配体测定中测量。受体数量（11,000 +/- 2,800个受体/细胞，n = 3）和Kd (4.1 +/- 1.0 × 10(-8) M)；n = 3)，使用放射性碘化GnRH激动剂测定，以及GnRH激动剂和拮抗剂以及不相关物质的结合抑制值表明，该受体与大鼠垂体细胞培养物中表达的受体相似，尽管前者的结合亲和力约低1 log。与gnrh刺激的促性腺激素释放不同，gnrh刺激的PRL释放来自转染细胞系，完全依赖于蛋白质合成，cAMP满足第二信使的要求。该受体似乎通过霍乱毒素敏感的g蛋白与腺苷酸环化酶介导的PRL释放偶联。这些研究提供的功能证据支持了克隆受体是释放激素的生理受体的观点，并且该受体可以根据g蛋白在靶细胞中的可用性和可及性与g蛋白进行不同的偶联。
GH(3) cells, which normally release PRL in response to stimulation by TRH, have been stably transfected with rat GnRH receptor complementary DNA (GGH(3)-1 ' cells). Unlike the parent line, GGH(3)-1 ' cells express GnRH receptor, which can be measured in a radioligand assay using a metabolically stable GnRH analog. The number of receptors (11,000 +/- 2,800 receptors/cell; n = 3) and Kd (4.1 +/- 1.0 X 10(-8) M; n = 3), determined using a radioiodinated GnRH agonist, as well as binding inhibition values for GnRH agonists and antagonists and for unrelated substances suggest that this receptor is similar to those expressed in cell cultures derived from rat pituitaries, although the binding affinity is about 1 log lower in the former. Unlike GnRH-stimulated release of gonadotropins from primary pituitary cultures, which does not require protein synthesis and is not coupled to cAMP production, GnRH-stimulated PRL release from the transfected cell line is absolutely dependent on protein synthesis, and cAMP fulfills the requirements of a second messenger. The receptor appears to be coupled to adenylate cyclase-mediated PRL release through a cholera toxin-sensitive G-protein. These studies provide functional evidence to support the view that the cloned receptor is the physiological receptor for the releasing hormone, and that this receptor can differentially couple to G-proteins depending on their availability and accessibility in the target cell.