The third cytoplasmic domain of the GLP-1[7-36 amide] receptor is required for coupling to the adenylyl cyclase system

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 systematically 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 [I-125] 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 less than or equal to 0.01), while displaying comparable levels of expression, (expressed as the %Bmax of the wt-tGLP-1R (101+/-13%, n=3, p greater than or equal to 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 greater than or equal to 0.05) and affinity for tGLP-1(K-d of 460+/-15pM vs. 450+/-12pM wt tGLP-1R, n=3, p greater than or equal to 0.05), but was unable to effectively stimulate cAMP production (7.7+/-0.4% of wt tGLP-1R, n=3, p less than or equal to 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 less than or equal to 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.