CHARACTERIZATION OF DELETION AND TRUNCATION MUTANTS OF THE RAT GLUCAGON RECEPTOR - 7 TRANSMEMBRANE SEGMENTS ARE NECESSARY FOR RECEPTOR TRANSPORT TO THE PLASMA-MEMBRANE AND GLUCAGON BINDING

Glucagon receptor mutants were characterized with the aim of elucidati ng minimal structural requirements for proper biosynthesis, ligand bin ding, and adenylyl cyclase coupling. One N-terminal deletion mutant an d five truncation mutants with progressively shorter C termini were ex pressed in transiently transfected monkey kidney (COS-1) cells. Each t runcation mutant was designed so that the truncated C-terminal tail wo uld remain on the cytoplasmic surface of the receptor. In order to cha racterize the cellular location of the expressed receptor mutants, a h ighly specific, high affinity antipeptide antibody was prepared agains t the extracellular, N-terminal tail of the receptor, Immunoblot analy sis and immunofluorescence microscopy showed that the presence of all seven putative transmembrane segments, but not an intact N-terminal ta il, was required for cell surface expression of the receptor, Membrane s from cells expressing receptor mutants lacking a large portion of th e N-terminal tail or any of the seven putative transmembrane segments failed to bind glucagon. Membranes from cells expressing the C-termina l tail truncation mutants, which retained all seven transmembrane segm ents, bound glucagon with affinities similar to that of the native rec eptor and activated cellular adenylyl cyclase in response to glucagon. These results indicate that all seven helices are necessary for the p roper folding and processing of the glucagon receptor, Glycosylation i s not required for the receptor to reach the cell surface, and it may not be required for ligand binding, However, the N-terminal extracellu lar portion of the receptor is required for ligand binding, Most of th e distal C-terminal tail is not necessary for ligand binding, and the absence of the tail may increase slightly the receptor binding affinit y for glucagon. The C-terminal tail is also not necessary for adenylyl cyclase coupling and therefore does not play a direct role in G prote in (G(s)) activation by the glucagon receptor.