THE GLUCAGON RECEPTOR - ITS STRUCTURE AND EXPRESSION IN RAT-TISSUES

A rat DNA fragment was PCR amplified using two degenerate primers corr esponding to consensus sequences in the TM2 and TM3 segments of member s of the secretin receptor family. An original synthetic oligodeoxynuc leotide, based on the 3' end of this fragment, was next used for scree ning a cDNA library from rat liver poly(A) (+)RNA. From pCDM8 clones a 1455 nucleotides ORF could be deduced that encoded a 485 amino acid s equence with 7 putative TM segments and 42% identity with the GLP-1 re ceptor. In addition four small (85-300 bp) introns were variously repr esented. A full ORF, free of introns, was then constructed with two no n functional pCDM8 clones and a pBluescript SK(+) vector, using a 4 fr agment ligation procedure. This insert was finally subcloned in pCDM8 for transfecting COSGs1 cells. Membranes from these cells showed selec tive glucagon binding and adenylyl cyclase stimulation by glucagon. Th e expression of the same glucagon receptor was detected by PCR in rat islets (beta cells ?), stomach, kidney and adipocytes. In rat genomic DNA, when taking into account the full size of all introns, the size o f the exons/introns coding area for the receptor was about 3.8 kb.