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.