Runge Steffen, Gram Christian, Brauner-Osborne Hans, Madsen Kjeld, Knudsen Lotte B, Wulff Birgitte S
Molecular Pharmacology, Novo Nordisk, DK-2760 Maaloev, Denmark.
J Biol Chem. 2003 Jul 25;278(30):28005-10. doi: 10.1074/jbc.M301085200. Epub 2003 Apr 29.
The glucagon and glucagon-like peptide-1 (GLP-1) receptors are homologous family B seven-transmembrane (7TM) G protein-coupled receptors, and they selectively recognize the homologous peptide hormones glucagon (29 amino acids) and GLP-1 (30-31 amino acids), respectively. The amino-terminal extracellular domain of the glucagon and GLP-1 receptors (140-150 amino acids) determines specificity for the carboxyl terminus of glucagon and GLP-1, respectively. In addition, the glucagon receptor core domain (7TM helices and connecting loops) strongly determines specificity for the glucagon amino terminus. Only 4 of 15 residues are divergent in the glucagon and GLP-1 amino termini; Ser2, Gln3, Tyr10, and Lys12 in glucagon and the corresponding Ala8, Glu9, Val16, and Ser18 in GLP-1. In this study, individual substitution of these four residues of glucagon with the corresponding residues of GLP-1 decreased the affinity and potency at the glucagon receptor relative to glucagon. Substitution of distinct segments of the glucagon receptor core domain with the corresponding segments of the GLP-1 receptor rescued the affinity and potency of specific glucagon analogs. Site-directed mutagenesis identified the Asp385 --> Glu glucagon receptor mutant that specifically rescued Ala2-glucagon. The results show that three distinct epitopes of the glucagon receptor core domain determine specificity for the N terminus of glucagon. We suggest a glucagon receptor binding model in which the extracellular ends of TM2 and TM7 are close to and determine specificity for Gln3 and Ser2 of glucagon, respectively. Furthermore, the second extracellular loop and/or proximal segments of TM4 and/or TM5 are close to and determine specificity for Lys12 of glucagon.
胰高血糖素和胰高血糖素样肽-1（GLP-1）受体属于B族同源七跨膜（7TM）G蛋白偶联受体，它们分别选择性识别同源肽激素胰高血糖素（29个氨基酸）和GLP-1（30 - 31个氨基酸）。胰高血糖素和GLP-1受体的氨基末端胞外结构域（140 - 150个氨基酸）分别决定了对胰高血糖素和GLP-1羧基末端的特异性。此外，胰高血糖素受体核心结构域（7TM螺旋和连接环）强烈决定了对胰高血糖素氨基末端的特异性。在胰高血糖素和GLP-1的氨基末端，15个残基中只有4个不同；胰高血糖素中的Ser2、Gln3、Tyr10和Lys12，以及GLP-1中相应的Ala8、Glu9、Val16和Ser18。在本研究中，将胰高血糖素的这四个残基逐个替换为GLP-1的相应残基，相对于胰高血糖素，降低了其对胰高血糖素受体的亲和力和效力。用GLP-1受体的相应片段替换胰高血糖素受体核心结构域的不同片段，挽救了特定胰高血糖素类似物的亲和力和效力。定点诱变鉴定出Asp385→Glu的胰高血糖素受体突变体，该突变体特异性地挽救了Ala2 - 胰高血糖素。结果表明，胰高血糖素受体核心结构域的三个不同表位决定了对胰高血糖素N末端 的特异性。我们提出了一种胰高血糖素受体结合模型，其中TM2和TM7的胞外末端分别靠近胰高血糖素的Gln3和Ser，并决定其特异性。此外，第二个胞外环和/或TM4和/或TM5的近端片段靠近胰高血糖素的Lys12并决定其特异性。
