The gene for cytosolic phosphoenolpyruvate carboxykinase (GTP) [GTP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.32] from the rat was isolated from a recombinant library containing the rat genome in phage A Charon 4A. The isolated clone, APCK1, contains the complete gene for phosphoenolpyruvate carboxykinase and -7 kilobases (kb) of flanking sequence at the 5' end and 1 kb at the 3' terminus. Restriction endonuclease mapping, R-loop mapping, and partial DNA sequence assay indicate that the gene is -6.0 kb in length (coding for a mRNA of 2.8 kb) and contains eight introns. Southern blotting of rat DNA digested with various restriction enzymes shows a pattern predicted from the restriction map of APCK1. A control region at the 5' end of the gene contained in a 1.2-kb restriction fragment was isolated and subcloned into pBR322. This segment of the gene contains the usual transcription start sequences and a 24-base sequence virtually identical to the sequence found in the 5'-flanking region of the human proopiomelonocortin gene, which is known to be regulated by glucocorticoids. The 1.2-kb fragment of the phosphoenolpyruvate carboxykinase gene can be transcribed into a unique RNA fragment of predicted size by an in vitro transcription assay.Phosphoenolpyruvate carboxykinase (GTP) [GTP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.32; P-enolpyruvate carboxykinase] is a key regulatory enzyme in hepatic gluconeogenesis, and its synthesis rate is controlled by a number of hormones including glucagon (acting through cAMP) (1, 2), cAMP (1), insulin (2, 3), epinephrine (4), glucocorticoids (5), and thyroid hormone (6). Of particular importance are: cAMP, which can induce the synthesis rate of P-enolpyruvate carboxykinase from rat liver cytosol 8-fold in 90 min (7); insulin, which causes an equally rapid deinduction of enzyme synthesis when administered to diabetic animals (2); and glucocorticoids, which also stimulate the synthesis of the enzyme (5). These rapid changes in the synthesis of hepatic P-enolpyruvate carboxykinase are accompanied by equally rapid changes in the sequence abundance of the mRNA coding for the enzyme (8,9
The mouse H19 gene was identified by virtue of its coordinate regulation with the mouse ac-fetoprotein gene. Both genes are expressed in the fetal liver, gut, and visceral endoderm of the yolk sac and are repressed shortly after birth in the liver and gut. They are both under the control of two trans-acting loci: raf, which affects the adult basal levels of the two mRNAs, and Rif, which affects their inducibility during liver regeneration. One crucial difference between the two genes is the activation of the H19 gene in mesoderm derivatives, skeletal and cardiac muscle. As a strategy for explaining both the similarities and differences in their modes of expression, the regulatory domains
Truncated forms of glucagon-like peptide-1 are the most potent endogenous stimuli of insulin secretion and have powerful antidiabetogenic effects. To determine the structure and coupling mechanisms of the human GLP-1 receptor we have isolated two pancreatic islet cDNAs, encoding the 463 amino acid receptor and differing mainly in their 3' untranslated regions. The deduced amino acid sequence is 90% homologous with the rat GLP-1 receptor. Northern blot analysis shows expression of a single 2.7 kb transcript in pancreatic tissue. When expressed in COS-7 cells the recombinant receptor conferred specific, high affinity GLP-1(7-37) binding. GLP-1(7-37) increased intracellular cAMP in a concentration dependent manner and caused an increase in the free cytosolic calcium ([Ca2+]i) from an intracellular pool, characteristic of phospholipase C (PLC) activation. Thus, like the structurally related glucagon and parathyroid hormone receptors, the human GLP-1 receptor can activate multiple intracellular signaling pathways including adenylyl cyclase and PLC. Knowledge of the GLP-1 receptor structure will facilitate the development of receptor agonists and elucidation of the important role of GLP-1 in normal physiology and disease states.
The binding of glucagon to its hepatic receptor triggers a G-protein-mediated signal that ultimately leads to an increase in hepatic glucose production (gluconeogenesis) and glycogen breakdown (glycogenolysis). In order to elucidate the structural domain(s) of the human glucagon receptor (hGR) involved in the selective binding of glucagon, a series of chimeras was constructed in which various domains of the hGR were replaced by homologous regions from the receptor for the glucoincretin hormone, glucagon-like peptide I (GLP-IR). hGR and GLP-IR are quite similar (47% amino acid identify) yet have readily distinguishable ligand binding characteristics; glucagon binds to the recombinant hGR expressed in COS-7 cells with a Kd that is 1000-fold lower than the Kd for glucagon binding to GLP-IR. In the present study, chimeric receptors were transiently expressed in COS-7 cells and analyzed for glucagon binding. Expression of each receptor chimera was confirmed by immunofluorescence staining using a hGR-specific monoclonal antibody. This report identifies several non-contiguous domains of the hGR that are important for high affinity glucagon binding. Most notable are the membrane-proximal half of the amino-terminal extension, the first extracellular loop, and the third, fourth, and sixth transmembrane domains.
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