Takhar S, Gyomorey S, Su R C, Mathi S K, Li X, Wheeler M B
Department of Medicine, University of Toronto, ON, Canada.
Endocrinology. 1996 May;137(5):2175-8. doi: 10.1210/endo.137.5.8612565.
Truncated forms of glucagon-like peptide-1 (tGLP-1) are potent endogenous stimuli of insulin secretion from pancreatic beta cells and have powerful antidiabetogenic effects. In the present study we sought to determine the precise regions of the tGLP-1 receptor (R) that are required for its efficient coupling to the adenylyl cyclase (AC) system since it is well established that cAMP is the primary second messenger activated by tGLP-1. The predicted third intracellular loop (IC3) of the rat tGLP-1R was systemically scanned using a mutagenic based strategy. The resulting receptor mutants were expressed in COS-7 cells and examined for cAMP formation in response to tGLP-1 stimulation (10nM) and [125I] tGLP-1(7-36) amide binding. A single block deletion (IC3-1) within the N-terminal region of IC3 (K334-L335-K336) resulted in a dramatic reduction in the cAMP response to tGLP-1 (7.1 +/- 1.4% of the wild type (wt) tGLP-1R response, n = 3, p < or = 0.01), while displaying comparable levels of expression, (expressed as the %Bmax of the wt-tGLP-1R (101 +/- 13%, n = 3, p > or = 0.05). This receptor mutation was further analyzed by stable expression in CHO-K1 cells. In agreement with the COS model, IC3-1 displayed comparable levels of receptor expression (97 +/- 16% Bmax of wt tGLP-1R, n = 3, p > or = 0.05) and affinity for tGLP-1(Kd of 460 +/- 15pM vs. 450 +/- 12pM wt tGLP-1R, n = 3, p > or = 0.05), but was unable to effectively stimulate cAMP production (7.7 +/- 0.4% of wt tGLP-1R, n = 3, p < or = 0.01) in response to tGLP-1 (10nM), No other mutation examined within the IC3 domain displayed a lack of correlation between binding activity and cAMP accumulation. Further analysis of the K334-L335-K336 sequence by substitution analysis revealed that a K334 to A substitution was the only modification to result in a striking attenuation of the cAMP response (28 +/- 1.9% of wt tGLP-1, n = 3, p < or = 0.01). These results strongly suggest that within the IC3 domain the N-terminal KLK sequence or a portion thereof (specifically K-334) is required for the efficient coupling of the tGLP-1 receptor to the AC system.
胰高血糖素样肽-1（tGLP-1）的截短形式是胰腺β细胞胰岛素分泌的强效内源性刺激物，具有强大的抗糖尿病作用。在本研究中，我们试图确定tGLP-1受体（R）与腺苷酸环化酶（AC）系统有效偶联所需的精确区域，因为cAMP是tGLP-1激活的主要第二信使，这一点已得到充分证实。采用基于诱变的策略对大鼠tGLP-1R预测的第三个细胞内环（IC3）进行了系统扫描。将所得的受体突变体在COS-7细胞中表达，并检测其对tGLP-1刺激（10nM）的cAMP生成以及[125I]tGLP-1（7-36）酰胺结合情况。IC3（K334-L335-K336）N端区域内的单个片段缺失（IC3-1）导致对tGLP-1的cAMP反应显著降低（为野生型（wt）tGLP-1R反应的7.1±1.4%，n = 3，p≤0.01），而其表达水平相当（以wt-tGLP-1R的%Bmax表示为101±13%，n = 3，p≥0.05）。通过在CHO-K1细胞中的稳定表达对该受体突变体进行了进一步分析。与COS模型一致，IC3-1显示出相当的受体表达水平（为wt tGLP-1R的97±16%Bmax，n = 3，p≥0.05）以及对tGLP-1的亲和力（Kd为460±15pM，而wt tGLP-1R为450±12pM，n = 3，p≥0.05），但在响应tGLP-1（10nM）时无法有效刺激cAMP产生（为wt tGLP-1R的7.7±0.4%，n = 3，p≤0.01）。在IC3结构域内检测的其他突变均未显示结合活性与cAMP积累之间缺乏相关性。通过替换分析对K334-L335-K336序列进行的进一步分析表明，K334突变为A是导致cAMP反应显著减弱的唯一修饰（为wt tGLP-1的28±1.9%，n = 3，p≤0.01）。这些结果强烈表明，在IC3结构域内，N端的KLK序列或其一部分（特别是K-334）是tGLP-1受体与AC系统有效偶联所必需的。
