DeAlmeida V I, Mayo K E
Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, Illinois 60208, USA.
Mol Endocrinol. 1998 May;12(5):750-65. doi: 10.1210/mend.12.5.0102.
The hypothalamic peptide GH-releasing hormone (GHRH) stimulates the release of GH from the pituitary through binding and activation of the GHRH receptor, which belongs to the family of G protein-coupled receptors. The objective of this study was to identify regions of the receptor critical for interaction with the ligand by expressing and analyzing truncated and chimeric epitope-tagged GHRH receptors. Two truncated receptors, GHRHdeltaN, in which part of the N-terminal domain between the putative signal sequence and the first transmembrane domain was deleted, and GHRHdeltaC, which was truncated downstream of the first intracellular loop, were generated. Both the receptors were deficient in ligand binding, indicating that neither the N-terminal extracellular domain (N terminus) nor the membrane-spanning domains with the associated extracellular loops (C terminus) are alone sufficient for interaction with GHRH. In subsequent studies, chimeric proteins between the receptors for GHRH and vasoactive intestinal peptide (VIP) or secretin were generated, using the predicted start of the first transmembrane domain as the junction for the exchange of the N terminus between receptors. The chimeras having the N terminus of the GHRH receptor and the C terminus of either the VIP or secretin receptor (GNVC and GNSC) did not bind GHRH or activate adenylate cyclase after GHRH treatment. The reciprocal chimeras having the N terminus of either the VIP or secretin receptors and the C terminus of the GHRH receptor (VNGC and SNGC) bound GHRH and stimulated cAMP accumulation after GHRH treatment. These results suggest that although the N-terminal extracellular domain is essential for ligand binding, the transmembrane domains and associated extracellular loop regions of the GHRH receptor provide critical information necessary for specific interaction with GHRH.
下丘脑肽生长激素释放激素（GHRH）通过与生长激素释放激素受体结合并激活该受体，刺激垂体释放生长激素，生长激素释放激素受体属于G蛋白偶联受体家族。本研究的目的是通过表达和分析截短的和嵌合的表位标记生长激素释放激素受体，来确定该受体中与配体相互作用至关重要的区域。构建了两个截短受体，GHRHdeltaN（其中假定信号序列和第一个跨膜结构域之间的部分N端结构域被删除）和GHRHdeltaC（其在第一个细胞内环下游被截短）。这两种受体都缺乏配体结合能力，表明N端胞外结构域（N端）和带有相关胞外环的跨膜结构域（C端）单独都不足以与GHRH相互作用。在随后的研究中，以第一个跨膜结构域的预测起始位置作为受体之间N端交换的连接点，构建了生长激素释放激素受体与血管活性肠肽（VIP）或促胰液素受体之间的嵌合蛋白。具有生长激素释放激素受体N端和VIP或促胰液素受体C端的嵌合体（GNVC和GNSC）在生长激素释放激素处理后不结合生长激素释放激素或激活腺苷酸环化酶。具有VIP或促胰液素受体N端和生长激素释放激素受体C端的反向嵌合体（VNGC和SNGC）在生长激素释放激素处理后结合生长激素释放激素并刺激cAMP积累。这些结果表明，虽然N端胞外结构域对于配体结合至关重要，但生长激素释放激素受体的跨膜结构域和相关胞外环区域为与生长激素释放激素的特异性相互作用提供了关键信息。
