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

被引:93
作者
UNSON, CG
CYPESS, AM
KIM, HN
GOLDSMITH, PK
CARRUTHERS, CJL
MERRIFIELD, RB
SAKMAR, TP
机构
[1] ROCKEFELLER UNIV,NEW YORK,NY 10021
[2] HOWARD HUGHES MED INST,MOLEC BIOL & BIOCHEM LAB,NEW YORK,NY 10021
[3] NIDDKD,MOLEC PATHOPHYSIOL BRANCH,BETHESDA,MD 20892
关键词
D O I
10.1074/jbc.270.46.27720
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Glucagon receptor mutants were characterized with the aim of elucidating minimal structural requirements for proper biosynthesis, ligand binding, and adenylyl cyclase coupling. One N-terminal deletion mutant and five truncation mutants with progressively shorter C termini were expressed in transiently transfected monkey kidney (COS-1) cells. Each truncation mutant was designed so that the truncated C-terminal tail would remain on the cytoplasmic surface of the receptor. In order to characterize the cellular location of the expressed receptor mutants, a highly specific, high affinity antipeptide antibody was prepared against the extracellular, N-terminal tail of the receptor, Immunoblot analysis and immunofluorescence microscopy showed that the presence of all seven putative transmembrane segments, but not an intact N-terminal tail, was required for cell surface expression of the receptor, Membranes from cells expressing receptor mutants lacking a large portion of the N-terminal tail or any of the seven putative transmembrane segments failed to bind glucagon. Membranes from cells expressing the C-terminal tail truncation mutants, which retained all seven transmembrane segments, bound glucagon with affinities similar to that of the native receptor and activated cellular adenylyl cyclase in response to glucagon. These results indicate that all seven helices are necessary for the proper folding and processing of the glucagon receptor, Glycosylation is not required for the receptor to reach the cell surface, and it may not be required for ligand binding, However, the N-terminal extracellular portion of the receptor is required for ligand binding, Most of the distal C-terminal tail is not necessary for ligand binding, and the absence of the tail may increase slightly the receptor binding affinity for glucagon. The C-terminal tail is also not necessary for adenylyl cyclase coupling and therefore does not play a direct role in G protein (G(s)) activation by the glucagon receptor.
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页码:27720 / 27727
页数:8
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