SCANNING OF THE GLUCAGON-LIKE PEPTIDE-1 RECEPTOR LOCALIZES G-PROTEIN-ACTIVATING DETERMINANTS PRIMARILY TO THE N-TERMINUS OF THE 3RD INTRACELLULAR LOOP

It is well known that glucagon-like peptide-1 (7-36 amide) (tGLP-1) is a potent insulinotropic hormone with powerful antidiabetogenic effect s. In the present study we sought to determine the precise regions of the intracellular domains of the tGLP-1 receptor that are required for its efficient coupling to adenylyl cyclase because cAMP is the primar y candidate second messenger coupling tGLP-1 to insulin secretion. Rec ently, we identified an amino acid within the third intracellular loop , K334, that was required for efficient coupling of tGLP-1 receptor to adenylyl cyclase. A similar mutagenesis-based strategy was employed h ere to examine the first and second intracellular loops and to further define sequences in the third loop required for the efficient couplin g of the receptor to its second messengers. Receptor mutants were expr essed in COS-7 cells and examined for tGLP-1 binding and cAMP stimulat ion. Three alanine substitution mutations, V327A, I328A, and V331A, re sulted in significantly lower tGLP-1-stimulated cAMP production withou t reductions in receptor expression. Analysis of the first and second intracellular loops revealed only one mutation contained within the fi rst loop, R176A, where a significant reduction in cAMP activation was observed with normal receptor expression. These studies suggest that s pecific determinants of coupling for tGLP-1 receptor are primarily loc alized to the predicted junction of the fifth transmembrane helix and the third intracellular loop. We predict that V327, I328, and V331 for m part of a hydrophobic face that directly contacts the G protein.