Miller T L, Godfrey P A, Dealmeida V I, Mayo K E
Department of Biochemistry, Northwestern University, Evanston, Illinois 60208, USA.
Endocrinology. 1999 Sep;140(9):4152-65. doi: 10.1210/endo.140.9.6977.
The interaction of GHRH with membrane-bound receptors on somatotroph cells of the anterior pituitary is an important step in the regulation of GH synthesis and secretion. The identification of a G protein-coupled receptor for GHRH has made it possible to investigate the pathway by which GHRH regulates pituitary somatotroph cell function. To initiate an analysis of the mechanisms regulating expression and function of the GHRH receptor, the structure of the gene and its promoter region were analyzed. The coding sequence of the rat GHRH receptor gene is contained within 14 exons spanning approximately 15 kb of genomic DNA. Four transcription start sites are located within 286 bp upstream of the initiation codon. The 5' flanking region of the GHRH receptor gene acts as a functional promoter in rat pituitary tumor GH3 cells, and basal promoter activity is enhanced in GH3 and COS7 cells by cotransfection of an expression construct encoding the pituitary-specific transcription factor Pit-1. The rat GHRH receptor gene is subject to at least 1 alternative RNA processing event that generates 2 receptor isoforms differing by 41 amino acids within the third intracellular loop (IL) of the protein. The short isoform of the GHRH receptor is predominant in pituitary cells. The MtT/S pituitary tumor cell line was found to express the GHRH receptor, and different populations of these cells produce predominantly the long or short isoforms of the receptor messenger RNA, suggesting that the alternative splicing can be regulated. Functional analysis of the two GHRH receptor isoforms demonstrates that both bind GHRH, but only the short isoform signals through a cAMP-mediated pathway. Neither receptor isoform is able to stimulate calcium mobilization from internal stores after GHRH treatment. Our findings indicate that the pituitary-specific transcription factor Pit-1 is involved in the somatotroph-specific expression of the GHRH receptor gene and that functionally distinct receptor proteins are generated by an alternative RNA processing mechanism.
生长激素释放激素（GHRH）与腺垂体生长激素细胞上的膜结合受体相互作用是调节生长激素（GH）合成与分泌的重要步骤。生长激素释放激素的G蛋白偶联受体的鉴定使得研究生长激素释放激素调节垂体生长激素细胞功能的途径成为可能。为了开始分析调节生长激素释放激素受体表达和功能的机制，对该基因及其启动子区域的结构进行了分析。大鼠生长激素释放激素受体基因的编码序列包含在14个外显子中，跨越约15kb的基因组DNA。四个转录起始位点位于起始密码子上游286bp内。生长激素释放激素受体基因的5'侧翼区域在大鼠垂体肿瘤GH3细胞中作为功能性启动子，通过共转染编码垂体特异性转录因子Pit-1的表达构建体，GH3和COS7细胞中的基础启动子活性增强。大鼠生长激素释放激素受体基因至少经历1次可变RNA加工事件，产生2种受体异构体，它们在蛋白质的第三个细胞内环（IL）内相差41个氨基酸。生长激素释放激素受体的短异构体在垂体细胞中占主导地位。发现MtT/S垂体肿瘤细胞系表达生长激素释放激素受体，并且这些细胞的不同群体主要产生受体信使RNA的长或短异构体，这表明可变剪接可以被调节。对两种生长激素释放激素受体异构体的功能分析表明，两者都结合生长激素释放激素，但只有短异构体通过cAMP介导的途径发出信号。生长激素释放激素处理后，两种受体异构体均不能刺激细胞内钙库释放钙。我们的研究结果表明，垂体特异性转录因子Pit-1参与生长激素释放激素受体基因的生长激素细胞特异性表达，并且通过可变RNA加工机制产生功能不同的受体蛋白。
