Buggy J J, Livingston J N, Rabin D U, Yoo-Warren H
Institute for Metabolic Disorders, Miles, Inc., West Haven, Connecticut 06516, USA.
J Biol Chem. 1995 Mar 31;270(13):7474-8. doi: 10.1074/jbc.270.13.7474.
The binding of glucagon to its hepatic receptor triggers a G-protein-mediated signal that ultimately leads to an increase in hepatic glucose production (gluconeogenesis) and glycogen breakdown (glycogenolysis). In order to elucidate the structural domain(s) of the human glucagon receptor (hGR) involved in the selective binding of glucagon, a series of chimeras was constructed in which various domains of the hGR were replaced by homologous regions from the receptor for the glucoincretin hormone, glucagon-like peptide I (GLP-IR). hGR and GLP-IR are quite similar (47% amino acid identify) yet have readily distinguishable ligand binding characteristics; glucagon binds to the recombinant hGR expressed in COS-7 cells with a Kd that is 1000-fold lower than the Kd for glucagon binding to GLP-IR. In the present study, chimeric receptors were transiently expressed in COS-7 cells and analyzed for glucagon binding. Expression of each receptor chimera was confirmed by immunofluorescence staining using a hGR-specific monoclonal antibody. This report identifies several non-contiguous domains of the hGR that are important for high affinity glucagon binding. Most notable are the membrane-proximal half of the amino-terminal extension, the first extracellular loop, and the third, fourth, and sixth transmembrane domains.
胰高血糖素与其肝脏受体的结合触发了一种由G蛋白介导的信号，最终导致肝脏葡萄糖生成（糖异生）和糖原分解（糖原olysis）增加。为了阐明人胰高血糖素受体（hGR）中参与胰高血糖素选择性结合的结构域，构建了一系列嵌合体，其中hGR的各个结构域被来自胰高血糖素样肽I（GLP-IR）的胰高血糖素激素受体的同源区域所取代。hGR和GLP-IR非常相似（47%氨基酸同一性），但具有易于区分的配体结合特性；胰高血糖素以比其与GLP-IR结合的Kd低1000倍的Kd与COS-7细胞中表达的重组hGR结合。在本研究中，嵌合受体在COS-7细胞中瞬时表达，并分析其对胰高血糖素的结合情况。使用hGR特异性单克隆抗体通过免疫荧光染色确认每个受体嵌合体的表达。本报告确定了hGR的几个非连续结构域，这些结构域对高亲和力胰高血糖素结合很重要。最值得注意的是氨基末端延伸的膜近端一半、第一个细胞外环以及第三、第四和第六个跨膜结构域。
