Synthesis and expression of a gene for the rat glucagon receptor. Replacement of an aspartic acid in the extracellular domain prevents glucagon binding.
In order to facilitate structure-function studies of the glucagon receptor by site-directed mutagenesis, we have designed and synthesized a gene for the rat glucagon receptor. The gene codes for the native 485-amino-acid protein but contains 91 unique restriction sites. To characterize gene expression, a highly specific, high affinity antipeptide antibody was prepared against the receptor. The synthetic gene was expressed in transiently transfected monkey kidney (COS-1) cells. COS cells expressing the synthetic receptor gene bound glucagon with affinity and specificity similar to that of hepatocytes containing native receptor. The transfected COS cells also showed increased intracellular cAMP levels in response to glucagon. The functional role of an aspartic acid residue in the NH2-terminal tail of the receptor was tested by site-directed mutagenesis. This site in the related growth hormone releasing factor receptor was shown to be responsible for the little mouse (lit) genetic defect that results in mice of small size with hypoplastic pituitary glands. Mutant glucagon receptors with amino acid replacements of Asp64 were expressed at normal levels in COS cells but failed to bind glucagon. These results indicate that amino acid Asp64 may play a key role in glucagon binding to receptor.
Carruthers
C J
CJ
Howard Hughes Medical Institute, Laboratory of Molecular Biology and Biochemistry, New York, New York 10021.
Unson
C G
CG
Kim
H N
HN
Sakmar
T P
TP
D000262:Adenylyl Cyclases / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004789:Enzyme Activation; D005813:Genes, Synthetic; D005934:Glucagon / Q000378:metabolism*; D000882:Haplorhini; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D051381:Rats; D018027:Receptors, Glucagon / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014162:Transfection
D018027:Receptors, Glucagon; D001224:Aspartic Acid; D005934:Glucagon; D000262:Adenylyl Cyclases