A cDNA construct allowing the expression of rat hepatic glucagon receptors

Biochem Biophys Res Commun. 1993 Apr 15;192(1):135-42. doi: 10.1006/bbrc.1993.1392.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Line
  • Cloning, Molecular
  • DNA
  • Glucagon / metabolism*
  • Haplorhini
  • Liver / metabolism*
  • Molecular Sequence Data
  • Open Reading Frames
  • Plasmids
  • Rats
  • Receptors, Gastrointestinal Hormone / genetics*
  • Receptors, Glucagon
  • Transfection

Substances

  • Receptors, Gastrointestinal Hormone
  • Receptors, Glucagon
  • DNA
  • Glucagon

Associated data

  • GENBANK/D25302
  • GENBANK/L04796
  • GENBANK/S64630
  • GENBANK/S64631
  • GENBANK/S64632
  • GENBANK/S64635
  • GENBANK/S64636
  • GENBANK/S64637
  • GENBANK/X68692
  • GENBANK/Z19151