Guo C H, Janovick J A, Kuphal D, Conn P M
Oregon Regional Primate Research Center, Beaverton 97006, USA.
Endocrinology. 1995 Jul;136(7):3031-6. doi: 10.1210/endo.136.7.7789329.
G proteins consist of heterotrimeric alpha-, beta-, and gamma-subunits. To assess the role of the beta gamma-subunit complex in GnRH receptor-mediated signal transduction, GGH3-1' cells were transfected with plasmids PRK5-beta ARK1 (495-689) containing complementary DNA (cDNA) of the carboxyl-terminal (Gly495-Leu689) of beta-adrenergic receptor kinase 1 (beta ARK1). GGH3-1' cells are GH3 cells that have been stably transfected with rat GnRH receptor cDNA. The carboxyl region of beta ARK1 (Gly495-Leu689) binds to the beta gamma complex and thereby inhibits its action. Twenty-four hours after stimulation, PRL release, cAMP release, and inositol phosphate (IP) production were measured in these cells and in control cells transfected with vector PRK5 cDNA alone. In cells expressing the beta ARK1-(495-689) sequence there was inhibition of basal PRL release (69.3%), cAMP release (61.2%), and IP production (75.5%) compared to cells containing vector only. When cells expressing the beta ARK1 fragment were stimulated with a GnRH analog (Buserelin; 10(-7) M), maximal responses were inhibited (66.1% for PRL release, 52.3% for cAMP release, and 79.1% for IP production). Scatchard analysis of GnRH analog binding was also performed in the two groups of transfected cells. No significant differences in Kd or receptor numbers were found between beta ARK1-(495-689)-transfected cells and control cells containing the vector alone. These data suggest a role for the beta gamma complex in mediation of cAMP release, IP production, and hormone release in response to agonist occupancy of the GnRH receptor.
G蛋白由异源三聚体的α、β和γ亚基组成。为了评估βγ亚基复合物在促性腺激素释放激素（GnRH）受体介导的信号转导中的作用，用含有β-肾上腺素能受体激酶1（βARK1）羧基末端（Gly495-Leu689）互补DNA（cDNA）的质粒PRK5-βARK1（495-689）转染GGH3-1'细胞。GGH3-1'细胞是已稳定转染大鼠GnRH受体cDNA的GH3细胞。βARK1的羧基区域（Gly495-Leu689）与βγ复合物结合，从而抑制其作用。刺激24小时后，在这些细胞和仅用载体PRK5 cDNA转染的对照细胞中测量催乳素（PRL）释放、环磷酸腺苷（cAMP）释放和肌醇磷酸（IP）生成。与仅含载体的细胞相比，在表达βARK1-（495-689）序列的细胞中，基础PRL释放（69.3%）、cAMP释放（61.2%）和IP生成（75.5%）受到抑制。当用GnRH类似物（布舍瑞林；10⁻⁷ M）刺激表达βARK1片段的细胞时，最大反应受到抑制（PRL释放为66.1%，cAMP释放为52.3%，IP生成为79.1%）。还对两组转染细胞进行了GnRH类似物结合的Scatchard分析。在转染βARK1-（495-689）的细胞和仅含载体的对照细胞之间，未发现解离常数（Kd）或受体数量有显著差异。这些数据表明βγ复合物在介导cAMP释放、IP生成以及响应GnRH受体激动剂占据时的激素释放中发挥作用。
