par Christophe, Jean
Référence Biochimica et biophysica acta, MR. Reviews on biomembranes, 1241, 1, page (45-57)
Publication Publié, 1995
Article révisé par les pairs
Résumé : The 1455 bp rat hepatic glucagon receptor ORF encodes 485 amino acids for a G-protein coupled protein with 7 transmembrane (TM) segments. The deduced amino acid sequence shows 42% identity with the rat GLP-1 receptor. Transfection of this receptor into COSGs1 cells allows selective glucagon binding and adenylyl cyclase stimulation. It now appears that the rat glucagon receptor gene contains 12 exons, 7 of which code for the TM domain. The gene is transcribed into several pre-mRNAs, variously shortened at the 5' end. One mature intronless mRNA, after the splicing out of the 11 introns, is translated into the functional glucagon receptor. We detected by PCR the apparent expression of the same glucagon receptor in rat liver, heart, islets (β cells?), stomach, kidney and adipocytes, suggesting that one gene allows the expression of only one type of glucagon receptor product, in terms of amino acid sequence. To further analyze the structure-activity relationship of this important yet strictly localized receptor four lines of research are now obvious: (1) To examine the bearing of posttranslational processing by glycosylation, phosphorylation and palmitoylation. (2) The DNA encoding the glucagon receptor being now stably transfected in CHO cells, this will hopefully allow to identify, at the atomic level, the interaction of glucagon with the receptor-effector complex. Such a transfected receptor, well expressed and coupled to adenylate cyclase, can indeed serve as reference when testing plasmids with partial deletions or point mutations (to alter charges), and chimeric constructions (where a fragment of the glucagon receptor is substituted by the corresponding fragment of a parent receptor, e.g., the tGLP-1 receptor). Mutagenesis of extracellular Asn and Cys residues will reveal the importance of glycosylation and disulfide bridges as prerequisites for receptor function. This evaluation will probably require the use of specific antibodies to see whether a given mutation is not responsible for a mere three-dimensional delocalization and general instability (inactivity) of the receptor synthesized by CHO cells. The binding and functional data collected will not only reveal specific roles for each extra- and intracellular domain of the receptor, they will also indicate how the side chains of residues His1, Gly4, Asp9, Lys12 and Ser16 in glucagon are sterically involved in effector coupling, giving clues in our search for pharmacologically valid analogs. (3) Within the first 104 bp of the 5'-flanking region [91], the TGAGCTCA sequence starting at position - 96 is similar to the consensus sequence TGACGTCA for CRE, and the ACCCAGGC sequence starting at position -50 could be related to the consensus sequence CCCCAGGC for factor AP-2 (that responds to both PKC and PKA). It is important to evaluate the regulation of receptor mRNA transcription with a full characterization (primary DNA sequence, placement, spacing, multiplicity) of regions of promoter sites that contain cis-acting enhancers, such as cAMP-responsive element CRE and tissue-specific elements. These elements could be regulated positively or negatively by trans-acting transcription factors and cofactors reacting to either cAMP (via protein-protein recognition with the C subunit of PKA), phosphorylation, hormones (corticosterone, insulin) or nutrients (glucose, polyunsaturated fatty acids). Expression assays and transgenic mouse technology could be used to identify these gene regulatory elements and the cell-specific transcription factors that control the limited tissue distribution of this receptor. (4) Appropriate primers will allow a quantitative PCR assay of mRNA levels for glucagon receptors, under various pathological conditions. For instance, in congenital obesity or hypertension in rodents, a change in receptor number in the tissues may reflect alterations in transcription rate and/or mRNA stability. Besides, a precise cellular localization of the receptor mRNA, by in situ hybridization procedures, could delineate whether β and δ cells are capable of expressing glucagon receptors and of modulating this synthesis, in response to glucagon secreted by α cells in the same islets.