ANTIBODIES AGAINST SPECIFIC EXTRACELLULAR EPITOPES OF THE GLUCAGON RECEPTOR BLOCK GLUCAGON BINDING

Polyclonal antibodies were prepared against synthetic peptides corresp onding to four different extramembrane segments of the rat glucagon re ceptor. The antibodies bound specifically to native glucagon receptor as judged by immunofluorescence microscopy of cultured cells expressin g a synthetic gene for the receptor, Antibodies to peptides designated PR-15 and DK-12 were directed against amino acid residues 103-117 and 126-137, respectively, of the extracellular N-terminal tail. Antibody to peptide KD-14 was directed against residues 206-219 of the first e xtracellular loop, and antibody to peptide ST-18, against the intracel lular C-terminal tail, residues 468-485. The DK-12 and KD-14 antibodie s, but not the PR-15 and ST-18 antibodies, could effectively block bin ding of I-125-labeled glucagon to its receptor in liver membranes, Inc ubation of these antibodies with rat liver membranes resulted in both a decrease in the maximal hormonal binding capacity and an apparent de crease in glucagon affinity for its receptor, These effects were aboli shed in the presence of excess specific peptide antigen. In addition, DK-12 and KD-14 antibodies, but not PR-15 and ST-18 antibodies, interf ered with glucagon-induced adenylyl cyclase activation in rat liver me mbranes and behaved as functional glucagon antagonists, These results demonstrate that DK-12 and KD-14 antibodies are pharmacologically acti ve glucagon antagonists and strongly suggest that residues 125-137 of the N-terminal tail and residues 206-219 of the first extracellular lo op contain determinants of ligand binding and may comprise the primary ligand-binding site on the glucagon receptor.