A cDNA construct allowing the expression of rat hepatic glucagon receptors.
We constructed a full open reading frame (ORF) for the rat hepatic glucagon receptor using two non functional pCDM8 clones of a putative glucagon receptor. Clone I was complete but contained, in addition, two small introns interrupting the frame, while clone II lacked 104 bp at the 5' end. We isolated Hind III/Dra III and BamH I/Not I fragments from clone I and a Dra III/BamH I fragment from clone II then ligated the three fragments with pBluescript SK(+) digested with Hind III/Not I. Following this 4 fragment ligation procedure, we obtained one correct insert in a clone we subcloned in the Hind III/Not I sites of pCDM8. This plasmid was used to transiently transfect COSGs1 cells with the lipofectin method. In COSGs1 membranes transfected with this correct plasmid (but not with a truncated plasmid) [125I]iodoglucagon binding was selectively displaced with glucagon (IC50 = 4 nM) and adenylate cyclase was stimulated with glucagon (K(act) = 10 nM) while related peptides were inefficient at 1 microM. This demonstrated that the receptor previously described is indeed the rat hepatic glucagon receptor.
Department of Biochemistry and Nutrition, Medical School, Université Libre de Bruxelles, Belgium.
M
M
Svoboda
E
E
Ciccarelli
M
M
Tastenoy
P
P
Robberecht
J
J
Christophe
D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA; D005934:Glucagon / Q000378:metabolism*; D000882:Haplorhini; D008099:Liver / Q000378:metabolism*; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010957:Plasmids; D051381:Rats; D011964:Receptors, Gastrointestinal Hormone / Q000235:genetics*; D018027:Receptors, Glucagon; D014162:Transfection
D011964:Receptors, Gastrointestinal Hormone; D018027:Receptors, Glucagon; D004247:DNA; D005934:Glucagon