Role of the glucagon receptor COOH-terminal domain in glucagon-mediated signaling and receptor internalization.

The binding of glucagon to its hepatic receptor is known to result in a number of effects, including the intracellular accumulation of cAMP, the mobilization of intracellular Ca2+, and the endocytosis of glucagon and its receptor into intracellular vesicles. In this study, we begin to define the functional role of the COOH-terminal tail of the human glucagon receptor in glucagon-stimulated signal transduction and receptor internalization. We have created and expressed in Chinese hamster ovary (CHO) cells five truncation mutants in which the COOH-terminal 24, 56, 62, 67, and 73 amino acids have been removed. Cells expressing relevant truncated receptors were assayed for cell surface expression by immunofluorescence, for ligand-binding properties, for cAMP and Ca2+-mediated signal transduction properties, and for receptor endocytosis. In addition, a mutant receptor containing seven serine-to-alanine mutations in the COOH-terminal tail was studied. Our results reveal the following: 1) a region of the COOH-terminal tail that is required for proper cell surface expression, 2) the COOH-terminal 62 amino acids, which comprise the majority of the tail, are not required for ligand binding, cAMP accumulation, or Ca2+ mobilization, and 3) phosphorylation of the COOH-terminal tail is crucial for glucagon-stimulated receptor endocytosis.

Human glucagon receptor monoclonal antibodies: antagonism of glucagon action and use in receptor characterization.

This paper describes the development and characterization of the first monoclonal antibody specific for the recently cloned human glucagon receptor (hGR), and its use in probing receptor structure and function. We demonstrate specificity of one of the antibodies, CIV395.7A, by immunofluorescence staining and immunoprecipitation analysis. In addition, CIV395.7A specifically competes with glucagon for the hormone binding site on the receptor, indicating that the antibody's specific recognition epitope overlaps with the receptor's hormone binding domain. As a consequence, the mAB antagonizes glucagon-stimulated signal transduction as assayed by in vitro cAMP accumulation. Binding inhibition studies further reveal that the antibody specifically recognizes the human and rat GR, but not mouse. Using hGR/glucagon-like peptide I receptor chimeras, we have localized the recognition epitope of the antibody to the membrane-proximal half of the amino-terminal extension of the receptor, thus defining a domain on the receptor which is involved in glucagon binding.

Glucagon.glucagon-like peptide I receptor chimeras reveal domains that determine specificity of glucagon binding.

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.

Isolation and structural analysis of the 5' flanking region of the gene encoding the human glucagon receptor.

The gene encoding the human glucagon receptor, including several kb of upstream sequence, was isolated from a bacteriophage lambda FIX II library constructed from human placental DNA. We report here the novel sequence of the 5' flanking region of the gene, the identification of a previously unreported intron of 5 kb, and the identification of the transcription start point of the glucagon receptor-specific transcript, which estimates the length of the first exon to be 300 bp.

Regulation of rat glucagon receptor expression.

This report describes the isolation of a cDNA for the rat glucagon receptor by using the glucagon-like peptide 1 receptor cDNA as a probe. Northern blot analysis using the cDNA clone showed that the message encoding the receptor is approximately 2.3 kb in size and is expressed only in liver and kidney among seven tissues tested. To study how glucagon receptor expression is regulated in vivo, the levels of hepatic glucagon receptor mRNA were measured in diabetic mouse model, db/db and control (db/+) mice. Interestingly, the receptor mRNA levels were similar between diabetic and control mice. In contrast, the number of hepatic glucagon receptors in diabetic mice measured by binding assays was significantly higher than that found in normal mice. These results suggest that the major regulation in hepatic glucagon receptor expression in vivo is at the posttranscriptional level.

Islet cell antigen 512 is a diabetes-specific islet autoantigen related to protein tyrosine phosphatases.

Islet cell Ag 512 (ICA512) is a recombinant human Ag that was isolated from an islet cDNA expression library by screening with human insulin-dependent diabetes mellitus sera. Specificity of reaction with diabetic sera was demonstrated initially by immunoprecipitation with a small number of diabetic and normal serum samples. To permit quantitative and rapid serum testing, ICA512 was purified and adapted to an ELISA format. In this way, a sensitivity of 48% with newly diagnosed diabetic sera has been measured with a panel of 80 sera. DNA sequencing of ICA512-3, a cDNA that contains a 1644 bp open reading frame, suggests that it codes for a transmembrane protein having a single membrane-spanning segment and a cytoplasmic domain that is closely related to the first intracellular (catalytic) domain of the T cell protein tyrosine phosphatase, CD45. Northern blot analysis of poly(A)+ RNAs from several human tissues indicates that ICA512 mRNA is expressed in brain and pancreas.

Cloning and functional expression of the human glucagon-like peptide-1 (GLP-1) receptor.

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.

Okadaic acid increases glucose uptake in 3T3-L1 adipocytes by stimulating glucose transporter 1 expression.

We present evidence that chronic 24 hour treatment of 3T3-L1 adipocytes with the phosphatase inhibitor okadaic acid increases deoxyglucose uptake 25 fold with a maximal effect at a concentration of 35nM. This pharmacological response is associated with a 21 fold increase in expression of glucose transporter 1 (glut 1) mRNA. These findings are discussed with respect to glucose transporter gene regulation and insulin signalling and are compared to previous observations describing the acute effects of okadaic acid on glucose transporter translocation.

Cloning and chromosomal mapping of mouse seminal vesicle protein F.

Seminal vesicle proteins (SVPs) are made by male rodents, and form the copulatory plug following mating. Here we report a partial nucleotide sequence of a mouse clone homologous to rat SVP F. Unexpectedly, we found that SVP F-related transcripts are expressed at high levels in mouse skeletal muscle. We mapped mouse SVP F to mouse chromosome 15 using somatic cell hybrid lines.

Two regulatory domains flank the mouse H19 gene.

The mouse H19 gene was identified by virtue of its coordinate regulation with the mouse alpha-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 responsible for the expression of the H19 gene in liver were identified by transiently introducing the gene into a human hepatoma cell line. Two regions necessary for high-level expression of the gene could be identified, a promoter-proximal domain immediately preceding the start of transcription and an enhancer domain which lies between 5 and 6.5 kilobases 3' of the polyadenylation site. The 3' domain consists of two separable enhancer elements, each of which exhibits the properties of tissue-specific enhancers. Nucleotide sequence comparisons between the two H19 and three alpha-fetoprotein enhancers revealed limited similarities which are candidates for binding of common regulatory factors. Sequences which lie 3' of the gene are also required for the expression of the H19 gene following differentiation of teratocarcinoma cells into visceral endoderm.

The gene encoding the cytosolic form of phosphoenolpyruvate carboxykinase (GTP) from the chicken.

The gene for cytosolic phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) from the chicken was isolated from a recombinant library containing the chicken genome in phage lambda Charon 4A. The isolated clone, lambda PCK1cc, contains the complete gene for the enzyme as well as both 5' and 3' flanking sequences. The gene is approximately 8 kilobases in length divided into 8 exons, as demonstrated by restriction endonuclease mapping and DNA-RNA heteroduplex analysis. Southern blotting of chicken chromosomal DNA digested with various restriction enzymes shows a pattern predicted from the restriction map of lambda PCK1cc. The phosphoenolpyruvate carboxykinase gene is present as a single copy in the haploid chicken genome. The 5' region of the gene was defined by S1 nuclease mapping and by sequencing. Two mRNA species with discrete 5' ends were observed using S1 nuclease mapping. The ratio between the amounts of these multiple forms of mRNA is the same in chicken kidney and liver and is not affected by induction of the enzyme mRNA by cAMP. Examination of sequence homologies with the gene for rat cytosolic phosphoenolpyruvate carboxykinase indicates a putative control region contained in flanking sequences at the 5' end of the gene.

Isolation and characterization of the gene coding for cytosolic phosphoenolpyruvate carboxykinase (GTP) from the rat.

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 lambda Charon 4A. The isolated clone, lambda PCK1, contains the complete gene for phosphoenolpyruvate carboxykinase and approximately equal to 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 approximately equal to 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 lambda PCK1. 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.

Rapid changes in the concentration of phosphoenolpyruvate carboxykinase mRNA in rat liver and kidney. Effects of insulin and cyclic AMP.

Starvation and diabetes both caused a marked increase in the concentration of hepatic phosphoenolpyruvate caroboxykinase mRNA while the administration of insulin to diabetic rats or refeeding glucose to starved animals caused a marked reduction in the levels of enzyme mRNA as measured by hybridization using a cDNA probe.l The Administration of dibutyryl cAMP to a starved-refed cat caused an 8-fold induction of phosphoenolpyruvate carboxykinase mRNA in 1 h. Triamcinolone plus acidosis induced the levels of enzyme mRNA in kidney 3-fold within 6 h, however, starvation for 24h had only marginal effects. In all of the above conditions, the levels of phosphoenolpyruvate carboxykinase mRNA measured by hybridization assay agreed well with the relative levels of translatable mRNA for the enzyme. The half-time of phosphoenolpyruvate carboxykinase mRNA, determined after the administration of either alpha-amanitin or cordycepin to starved animals, was approximately 40 min. However, cycloheximide either alone or together with cordycepin, not only prevented the decrease in phosphoenolpyruvate carboxykinase mRNA sequence abundance, but induced it 2-fold. Cycloheximide itself, when injected into 21-day fetal rats in utero caused an induction of enzyme mRNA equal to that noted when dibutyryl cAMP was administered. The mRNA for phosphoenolpyruvate carboxykinase is approximately 2.8 kb in length, but nuclei from the livers of diabetic rats contain a number of putative precursor RNA species for the enzyme, up to 6.5 kb in size, all containing a poly(A) tail. Two hours after refeedng glucose to a starved rat, these nuclear RNA species could no longer be detected by hybridization to our cDNA probe.