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J C Hutton

Publications and source records attributed to J C Hutton.

At least 19 recordsLinked to original sources
PubMed2006

A new model of insulin-deficient diabetes: male NOD mice with a single copy of Ins1 and no Ins2.

AIMS/HYPOTHESIS: We describe a novel model of insulin-deficient diabetes with a single copy of the gene encoding insulin 1 (Ins1) and no gene encoding insulin 2 (Ins2). MATERIALS AND METHODS: We constructed five lines of mice: mice with two copies of Ins1 (NOD( Ins1+/+,Ins2-/-)), mice with a single copy of Ins1 (NOD( Ins1+/-,Ins2-/-)), mice with two copies of Ins2 (NOD( Ins1-/-,Ins2+/+)), mice with a single copy of Ins2 (NOD( Ins1-/-,Ins2+/-)) and NOD( Ins1+/-,Ins2-/-) mice with a transgene encoding B16:Ala proinsulin. RESULTS: By 10 weeks of age, all male NOD( Ins1+/-,Ins2-/-) mice were diabetic, whereas all female NOD( Ins1+/-,Ins2-/-) were not diabetic (p < 0.0001). In contrast, neither male nor female NOD( Ins1-/-,Ins2+/-) with a single copy of Ins2 (rather than single copy of Ins1) developed early diabetes and no mice with two copies of either gene developed early diabetes. Islets of the diabetic male NOD( Ins1+/-,Ins2-/-) at this early age had no lymphocyte infiltration. Instead there was heterogeneous (between islet cells) weak staining for insulin. Although only male NOD( Ins1+/-,Ins2-/-) mice developed diabetes, both male and female NOD( Ins1+/-,Ins2-/-) mice had markedly decreased insulin content. In NOD( Ins1+/+,Ins2-/-), there was also a significant decrease in insulin content, whereas NOD( Ins1-/-,Ins2+/+) mice, and even NOD( Ins1-/-,Ins2+/-) mice, were normal. Male NOD( Ins1+/-,Ins2-/-) mice were completely rescued from diabetes by introduction of a transgene encoding proinsulin. On i.p. insulin tolerance testing, male mice had insulin resistance compared with female mice. CONCLUSIONS/INTERPRETATION: These results suggest that Ins1 is a 'defective gene' relative to Ins2, and that the mouse lines created provide a novel model of sex-dimorphic insulin-deficient diabetes.

Animals↗
PubMed2006

Alternative splicing of G6PC2, the gene coding for the islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), results in differential expression in human thymus and spleen compared with pancreas.

AIMS/HYPOTHESIS: Autoimmunity to insulin, glutamic acid decarboxylase and the tyrosine-phosphatase-like protein IA-2 is associated with type 1 diabetes. The production of self-molecules in thymus and secondary lymphoid tissues is critical for self-tolerance; reduced levels may impair tolerance and predispose to autoimmunity, as shown for insulin. Alternative splicing causes differential expression of IA-2 gene (PTPRN) transcripts and IA-2 protein in human thymus and spleen compared with pancreas. IA-2 sequences not present in lymphoid tissues become autoimmune targets in type 1 diabetes. The beta cell molecule islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) is an autoantigen in the non-obese diabetic (NOD) mouse, a model of type 1 diabetes. IGRP is a candidate autoantigen in the human disease, but robust assays for IGRP autoantibodies and/or autoreactive T cells are not available. Both full-length and IGRP splice variants encoded by the G6PC2 gene are expressed in the pancreas. In this study we tested the hypothesis that IGRP splice variants could be differentially expressed in thymus and spleen compared with the pancreas. METHODS: We evaluated the expression of G6PC2 transcripts in matched human thymus, spleen and pancreas specimens by RT-PCR. RESULTS: Alternative splicing results in differential expression of G6PC2 transcripts in thymus and spleen compared with pancreas. The full-length transcript is expressed in human pancreas but not in thymus or spleen. Five alternative spliced forms are always expressed in pancreas but those lacking exons 2, 3 and 4, alone or in combination, were rarely detected in thymus or spleen. CONCLUSIONS/INTERPRETATION: Differential tissue expression might favour autoimmune responses to IGRP in humans; target epitopes may be encoded by exons 3 and 4, or at the junctions of the conserved exons in the spliced transcripts. This information may aid in designing synthetic peptides for the identification of IGRP-specific autoreactive T cells in patients with type 1 diabetes.

Alternative Splicing↗
PubMed2004

Identification and characterization of a cDNA and the gene encoding the mouse ubiquitously expressed glucose-6-phosphatase catalytic subunit-related protein.

Glucose-6-phosphatase (G6Pase) catalyzes the final step in the gluconeogenic and glycogenolytic pathways, the hydrolysis of glucose-6-phosphate (G6P) to glucose and phosphate. This paper describes the identification and characterization of a cDNA and the gene encoding the mouse ubiquitously expressed G6Pase catalytic subunit-related protein (UGRP). The open reading frame of this UGRP cDNA encodes a protein (346 amino acids (aa); Mr 38,755) that shares 36% overall identity (56% similarity) with the mouse G6Pase catalytic subunit (357 aa; Mr 40,454). UGRP exhibits a similar predicted transmembrane topology and conservation of many of the catalytically important residues with the G6Pase catalytic subunit; however, unlike the G6Pase catalytic subunit, UGRP does not catalyze G6P hydrolysis and does not contain a carboxy-terminal di-lysine endoplasmic reticulum retention signal. UGRP mRNA was detected by RNA blot analysis in every mouse tissue examined with the highest expression in heart, brain, testis and kidney. Database analysis showed that the mouse UGRP gene is composed of six exons, spans approximately 4.2 kbp of genomic DNA and is located on chromosome 11 along with the G6Pase catalytic subunit gene. The UGRP gene transcription start sites were mapped by primer extension analysis, and the activity of the mouse UGRP gene promoter was analyzed using luciferase fusion gene constructs. In contrast to the G6Pase catalytic subunit gene promoter, the UGRP promoter was highly active in all cell lines examined.

Amino Acid Sequence↗
PubMed2002

Identification and characterization of a human cDNA and gene encoding a ubiquitously expressed glucose-6-phosphatase catalytic subunit-related protein.

Glucose-6-phosphatase (G6Pase) catalyzes the final step in the gluconeogenic and glycogenolytic pathways, the hydrolysis of glucose-6-phosphate (G6P) to glucose and phosphate. This paper describes the identification and characterization of a human cDNA and gene encoding a ubiquitously expressed G6Pase catalytic subunit-related protein (UGRP). The ORF of this UGRP cDNA encodes a protein (346 amino acids (aa); M(r) 38 709) which shares 36% overall identity to the human G6Pase catalytic subunit (357 aa; M(r) 40 487). UGRP exhibits a similar predicted transmembrane topology and conservation of many of the catalytically important residues with the G6Pase catalytic subunit; however, unlike the G6Pase catalytic subunit, UGRP does not catalyze G6P hydrolysis. UGRP mRNA was detected by RNA blot analysis in every tissue examined with the highest expression in muscle. Database analysis showed that the human UGRP gene is composed of six exons, spans approximately 5.4 kbp of genomic DNA and is located on chromosome 17q21 with the G6Pase catalytic subunit gene. The UGRP gene transcription start sites were mapped by primer extension analysis, and the activity of the UGRP gene promoter was analyzed using luciferase fusion gene constructs. In contrast to the G6Pase catalytic subunit gene promoter, the UGRP promoter was highly active in all cell lines examined.

Amino Acid Sequence↗
PubMed2002

Proinsulin processing in the diabetic Goto-Kakizaki rat.

The biosynthesis and processing of proinsulin was investigated in the diabetic Goto-Kakizaki (GK) rat. Immunofluorescence microscopy comparing GK and Wistar control rat pancreata revealed marked changes in the distribution of alpha-cells and pronounced beta-cell heterogeneity in the expression patterns of insulin, prohormone convertases PC1, PC2, carboxypeptidase E (CPE) and the PC-binding proteins 7B2 and ProSAAS. Western blot analyses of isolated islets revealed little difference in PC1 and CPE expression but PC2 immunoreactivity was markedly lower in the GK islets. The processing of the PC2-dependent substrate chromogranin A was reduced as evidenced by the appearance of intermediates. No differences were seen in the biosynthesis and post-translational modification of PC1, PC2 or CPE following incubation of islets in 16.7 mM glucose, but incubation in 3.3 mM glucose resulted in decreased PC2 biosynthesis in the GK islets. The rates of biosynthesis, processing and secretion of newly synthesized (pro)insulin were comparable. Circulating insulin immunoreactivity in both Wistar and GK rats was predominantly insulin 1 and 2 in the expected ratios with no (pro)insulin evident. Thus, the marked changes in islet morphology and PC2 expression did not impact the rate or extent of proinsulin processing either in vitro or in vivo in this experimental model.

Animals↗
PubMed2002

Targeting of a phogrin-green fluorescent protein chimaera to insulin secretory granules of pancreatic beta-cells in transgenic mice.

We have generated a transgenic mouse expressing a chimaeric phogrin-enhanced green fluorescent protein (EGFP) targeted to pancreatic beta-cells by the rat insulin II promoter. The transgenic animals appear healthy, have normal weight gain and normal glucose tolerance. Morphological analyses of adult transgenic mice revealed that the fluorescent reporter molecule was specifically expressed in beta-cells of the pancreatic islet and not in extra-pancreatic tissues. The distribution of phogrin-EGFP in beta-cells, however, was heterogeneous with three distinct populations distinguishable by FACS analysis and immunofluorescence microscopy. Superficially-localized islets in the whole pancreas were readily visualized in the animals as was the developing endocrine pancreas in undissected 15.5 day old mouse embryos. We envisage that the animal will be an important resource for future investigations of islet development, regeneration and the molecular cell biology of insulin secretion.

Animals↗
PubMed2001

Secretagogue-dependent phosphorylation of the insulin granule membrane protein phogrin is mediated by cAMP-dependent protein kinase.

Phogrin, a 60/64-kDa integral membrane protein of dense-core granules in neuroendocrine cells, is phosphorylated in a Ca(2+)-sensitive manner in response to secretagogue stimulation of pancreatic beta-cells. Phosphorylation of the phogrin cytosolic domain by beta-cell homogenates was Ca(2+)-independent but stimulated by cAMP. Recombinant protein kinase A (PKA) could phosphorylate phogrin directly. High performance liquid chromatography analysis of tryptic phosphopeptides, combined with site-directed mutagenesis of candidate sites, revealed the presence of two phosphorylation sites at Ser-680 and Thr-699, located in the juxtamembrane region between the transmembrane span and the protein-tyrosine phosphatase homology domain of phogrin. Full-length wild-type phogrin, as well as mutant versions where Ser-680 and Thr-699 had been replaced either by alanines or by aspartic acid residues, were targeted to secretory granules in transfected AtT20 neuroendocrine cells. Stimulation of these cells with a range of secretagogues, including K(+), BaCl(2), and forskolin, demonstrated that the in vivo phosphorylation sites are the same as those identified in vitro. In MIN6 beta-cells, the PKA inhibitor H-89 prevented Ca(2+)-dependent phogrin phosphorylation in response to glucose, suggesting that Ca(2+) exerts its effect on phogrin phosphorylation through regulating the activity of PKA.

Amino Acid Sequence↗
PubMed2001

Cloning and characterization of the human and rat islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) genes.

Islet-specific glucose-6-phosphatase (G6Pase) catalytic subunit-related protein (IGRP) is a homolog of the catalytic subunit of G6Pase, the enzyme that catalyzes the terminal step of the gluconeogenic pathway. Its catalytic activity, however, has not been defined. Since IGRP gene expression is restricted to islets, this suggests a possible role in the regulation of islet metabolism and, hence, insulin secretion induced by metabolites. We report here a comparative analysis of the human, mouse, and rat IGRP genes. These studies aimed to identify conserved sequences that may be critical for IGRP function and that specify its restricted tissue distribution. The single copy human IGRP gene has five exons of similar length and coding sequence to the mouse IGRP gene and is located on human chromosome 2q28-32 adjacent to the myosin heavy chain 1B gene. In contrast, the rat IGRP gene does not appear to encode a protein as a result of a series of deletions and insertions in the coding sequence. Moreover, rat IGRP mRNA, unlike mouse and human IGRP mRNA, is not expressed in islets or islet-derived cell lines, an observation that was traced by fusion gene analysis to a mutation of the TATA box motif in the mouse/human IGRP promoters to TGTA in the rat sequence. The results provide a framework for the further analysis of the molecular basis for the tissue-restricted expression of the IGRP gene and the identification of key amino acid sequences that determine its biological activity.

Amino Acid Sequence↗
PubMed2001

IDA-1, a Caenorhabditis elegans homolog of the diabetic autoantigens IA-2 and phogrin, is expressed in peptidergic neurons in the worm.

The closely related mammalian proteins IA-2 and phogrin are protein tyrosine phosphatase-like receptor proteins spanning the membrane of dense core vesicles of neuroendocrine tissues. They are of interest as molecular components of the secretory machinery and as major targets of autoimmunity in type I diabetes mellitus. The Caenorhabditis elegans genome has a single copy of an IA-2/phogrin homolog ida-1 III (islet cell diabetic autoantigen), which encodes the ida-1 (B0244.2) gene product as a series of 12 exons over a 10-kb region of chromosome III. The full-length sequence of the ida-1 cDNA encoded a 767-amino acid type 1 transmembrane protein of 87 kDa. The PTP catalytic site consensus sequence of IDA-1, like IA-2 and phogrin, diverged and would not be active. Expression of green fluorescent protein (GFP) under the ida-1 gene promoter showed activity in a subset of around 30 neurons with sensory functions and the uv1 cells of the vulva in hermaphrodites. Males showed additional expression in male-specific neurons. In situ experiments in rat brain showing the distribution of IA-2 and phogrin suggested a complimentary and overlapping pattern compared with the proprotein convertases PC1 and PC2. In C. elegans, IDA-1-expressing cells comprised a subset of those expressing the PC2 homolog KPC-2 (C51E3. 7), consistent with IDA-1 being a component of neuropeptide-containing dense core vesicles. The results support the hypothesis that C. elegans IDA-1 is the functional homolog of IA-2 and phogrin in mammals. Analysis of the function of IDA-1 should contribute to our understanding of the function of these proteins in signal transduction, vesicle locomotion, and exocytosis.

Amino Acid Sequence↗
PubMed2001

Cell-specific processing of chromogranin A in endocrine cells of the rat stomach.

The rat stomach is rich in endocrine cells. The acid-producing (oxyntic) mucosa contains ECL cells, A-like cells, and somatostatin (D) cells, and the antrum harbours gastrin (G) cells, enterochromaffin (EC) cells and D cells. Although chromogranin A (CgA) occurs in all these cells, its processing appears to differ from one cell type to another. Eleven antisera generated to different regions of rat CgA, two antisera generated to a human (h) CgA sequences, and one to a bovine (b) CgA sequence, respectively, were employed together with antisera directed towards cell-specific markers such as gastrin (G cells), serotonin (EC cells), histidine decarboxylase (ECL cells) and somatostatin (D cells) to characterize the expression of CgA and CgA-derived peptides in the various endocrine cell populations of the rat stomach. In the oxyntic mucosa, antisera raised against CgA(291-319) and CGA(316-321) immunostained D cells exclusively, whereas antisera raised against bCgA(82-91) and CgA(121-128) immunostained A-like cells and D cells. Antisera raised against CgA(318-349) and CgA(437-448) immunostained ECL cells and A-like cells, but not D cells. In the antrum, antisera against CgA(291-319) immunostained D cells, and antisera against CgA(351-356) immunostained G cells. Our observations suggest that each individual endocrine cell type in the rat stomach generates a unique mixture of CgA-derived peptides, probably reflecting cell-specific differences in the post-translational processing of CgA and its peptide products. A panel of antisera that recognize specific domains of CgA may help to identify individual endocrine cell populations.

Animals↗
PubMed2001

Characterization of the mouse islet-specific glucose-6-phosphatase catalytic subunit-related protein gene promoter by in situ footprinting: correlation with fusion gene expression in the islet-derived betaTC-3 and hamster insulinoma tumor cell lines.

Glucose-6-phosphatase (G6Pase) is a multicomponent system located in the endoplasmic reticulum comprising a catalytic subunit and transporters for glucose-6-phosphate, inorganic phosphate, and glucose. We have recently cloned a novel gene that encodes an islet-specific G6Pase catalytic subunit-related protein (IGRP) (Ebert et al., Diabetes 48:543-551, 1999). To begin to investigate the molecular basis for the islet-specific expression of the IGRP gene, a series of truncated IGRP-chloramphenicol acetyltransferase (CAT) fusion genes were transiently transfected into the islet-derived mouse betaTC-3 and hamster insulinoma tumor cell lines. In both cell lines, basal fusion gene expression decreased upon progressive deletion of the IGRP promoter sequence between -306 and -66, indicating that multiple promoter regions are required for maximal IGRP-CAT expression. The ligation-mediated polymerase chain reaction footprinting technique was then used to compare trans-acting factor binding to the IGRP promoter in situ in betaTC-3 cells, which express the endogenous IGRP gene, and adrenocortical Y1 cells, which do not. Multiple trans-acting factor binding sites were selectively identified in betaTC-3 cells that correlate with regions of the IGRP promoter identified as being required for basal IGRP-CAT fusion gene expression. The data suggest that hepatocyte nuclear factor 3 may be important for basal IGRP gene expression, as it is for glucagon, GLUT2, and Pdx-1 gene expression. In addition, binding sites for several trans-acting factors not previously associated with islet gene expression, as well as binding sites for potentially novel proteins, were identified.

Animals↗
PubMed2001

T-cell epitope analysis on the autoantigen phogrin (IA-2beta) in the nonobese diabetic mouse.

The protein tyrosine phosphatases (PTPs) IA-2 and phogrin (IA-2beta) are major autoantigens in type 1 diabetes that possess common serological epitopes in their COOH termini. The epitopes recognized by the T-cells that cause the disease, however, remain to be defined. Eight phogrin-specific T-cell clones were generated from NOD mice, and their epitopes were mapped. The mapping was performed initially with recombinant gluthathione S-transferase-phogrin COOH deletion constructs and ultimately with overlapping synthetic peptides. Two dominant epitopes were identified: one (aa 629-649) immediately adjacent to the transmembrane domain (aa 604-628) and the second (aa 755-777) lying in the NH(2)-terminal region of the conserved PTP domain. T-cells that are specific to either of these peptides and that could destroy islet tissue in vivo though spontaneous T-cell proliferative responses were observed in prediabetic female NOD splenocytes only to the aa 755-777 epitope. In NOD female mice immunized with the epitope peptide, intramolecular determinant spreading occurred from the aa 629-649 epitope to the aa 755-777 epitope but not in the opposite direction. We concluded that the initial T-cell response to phogrin is restricted to a small number of dominant peptides and that it subsequently spreads to other regions of the molecule, including those containing the major humoral epitopes that are highly conserved between IA-2 and phogrin.

Amino Acid Sequence↗
PubMed2000

Translational regulation of proinsulin biosynthesis and proinsulin conversion in the pancreatic beta-cell.

Insulin secretion from the pancreatic beta -cell can be initiated in minutes, vary as much as 50-100-fold, and be sustained for several hours without need for changes in insulin gene transcription. Remarkably, the cellular content of the hormone and its molecular composition do not vary appreciably in the face of changes of insulin granule exocytosis. Minimal morphological changes are apparent, further indicating that the movement of lipids and membrane proteins between the granule storage pool, the plasma membrane, and Golgi are likewise tightly controlled. Such homeostasis is achieved by an interplay of signaling pathways originating from the metabolism of glucose with downstream targets at the level of translation of dense-core granule proteins, granule biogenesis, and membrane trafficking. Our scant knowledge in this area is confined mostly to a descriptive account of the fate of the major secreted components, principally insulin and the enzymes PC1, PC2, and CPH involved in the proteolytic conversion of proinsulin to insulin. A common theme seems to be the role of intracellular energy homeostasis in integrating the stimulus-secretion and stimulus-biosynthetic responses of this cell.

Amino Acid Sequence↗
PubMed2000

Humoral autoreactivity to an alternatively spliced variant of ICA512/IA-2 in Type I diabetes.

AIMS/HYPOTHESIS: The receptor tyrosine phosphatase like-protein ICA512/IA-2 occurs as a proteolytically-processed 65,000 Mr type 1 transmembrane glycoprotein in beta cells and is a major autoantigen of Type I (insulin-dependent) diabetes mellitus. We investigated whether alternative splicing could affect humoral autoreactivity to the molecule. METHODS: Genomic and cDNA sequence analysis showed the presence of a ICA512 variant in islets and lymphoid tissues with an in-frame deletion of exon 13 which produces a secreted form lacking aa 557-629 including the transmembrane domain (aa 577 to 600). The alternatively spliced protein is detectable by western blotting in normal islets and translated into a protein that is processed to a series of soluble forms of 25,000-35,000 Mr Radioimmuno-precipitation assays for anti-ICA512 autoantibodies were developed with the widely used ICA512.bdc construct (which has exon 13 deleted) and a series of full-length and modified ICA512/IA-2 molecules. RESULTS: The assays showed that ICA512.bdc and ICA512604-979 gave the best discrimination between diabetic and control sera. With ICA512604-979 a somewhat greater proportion of patients expressing antibodies were detected than with ICA512.bdc in the groups studied (70.5 % vs 63.2 % of prediabetic/new-onset and 25.0 vs 13.9% in patients with diabetes > 20 years). Conversely, a small proportion (3 % recent-onset and 6% > 20 years) had antibodies to ICA512.bdc but not ICA512(604-979). CONCLUSION/INTERPRETATION: Important epitopes lie within the exon 13 region and others can be generated by the alternative splicing. As the deltaexon 13 variant is probably secreted by the beta cell, it could be recognized by the cellular and humoral arm of the immune system in the absence of cellular damage.

Adolescent↗
PubMed2000

Evidence for recognition of novel islet T cell antigens by granule-specific T cell lines from new onset type 1 diabetic patients.

Type 1 diabetes is a T cell-mediated autoimmune disease where a number of islet beta-cell target autoantigens have been characterized on the basis of reactivity with autoantibodies. Nevertheless, there remains uncertainty of the nature of another group of autoantigens associated with the secretory granule fraction of islet beta-cells that appear to be targeted predominantly by autoreactive T cells. We have previously characterized CD4+, HLA-DR-restricted T cell lines from new onset type 1 diabetic patients that are specific for the secretory granule fraction of rat tumour insulinoma, RIN. The T cell line from the first patient, HS, proliferates in response to crude microsomal membranes prepared from a recently established, pure human islet beta-cell line NES2Y. In addition, the HS line also responds to secretory granule fractions prepared from a murine tumour insulinoma grown in RIP-Tag mice, showing the recognition of species-conserved antigen(s) in beta-cells. Using partially matched antigen-presenting cells, the HS T cells and another line derived from a second patient, MR, were shown to be restricted by disease-associated DRB1*0101 and DRB1*0404 alleles, respectively. Neither the HS or MR T cell lines proliferate in response to a large panel of candidate islet cell antigens, including insulin, proinsulin, glutamic acid decarboxylase, the protein tyrosine phosphatase IA-2/phogrin, imogen-38, ICA69 or hsp60. Our data provide compelling evidence of the presence of a group of antigens associated with the secretory granule fraction of islet beta-cells recognized by the T cell lines, whose definition may contribute to our knowledge of disease induction as well as to diagnosis.

Animals↗
PubMed1999

Secretagogue-dependent phosphorylation of phogrin, an insulin granule membrane protein tyrosine phosphatase homologue.

Phogrin, a 60/64 kDa integral membrane protein localized to dense-core secretory granules of neuroendocrine cells, was found to be reversibly phosphorylated in intact pancreatic beta-cells. Phosphorylation occurred in response to a variety of secretory stimuli, including glucose and depolarizing concentrations of K(+). In MIN6 cells, the glucose dose-response and time course of phogrin phosphorylation paralleled that of insulin secretion. Like secretion, glucose- or K(+)-stimulated phosphorylation required the presence of Ca(2+). The calmodulin antagonist W-7 and the Ca(2+)/calmodulin-dependent kinase II inhibitor KN-93 dose-dependently inhibited both phosphorylation and secretion, while the 'inactive' analogue KN-92 was effective only at significantly higher concentrations. Phosphorylation of phogrin was also stimulated in cells exposed to forskolin, an effect presumably mediated by protein kinase A (cAMP-dependent protein kinase). Under these conditions, phogrin phosphorylation could be dissociated from the secretory response. In MIN6 cells, as in pancreatic islets, cAMP potentiates rather than initiates insulin release. Thus our observations are consistent with a role for phogrin phosphorylation in the signal-transduction pathway at a site proximal to the exocytic event itself, possibly regulating secretory-granule mobilization and recruitment to the exocytic site.

Animals↗
PubMed1999

Molecular cloning of a pancreatic islet-specific glucose-6-phosphatase catalytic subunit-related protein.

A pancreatic islet-specific glucose-6-phosphatase-related protein (IGRP) was cloned using a subtractive cDNA expression cloning procedure from mouse insulinoma tissue. Two alternatively spliced variants that differed by the presence or absence of a 118-bp exon (exon IV) were detected in normal balb/c mice, diabetic ob/ob mice, and insulinoma tissue. The longer, 1901-bp full-length cDNA encoded a 355-amino acid protein (molecular weight 40,684) structurally related (50% overall identity) to the liver glucose-6-phosphatase and exhibited similar predicted transmembrane topology, conservation of catalytically important residues, and the presence of an endoplasmic reticulum retention signal. The shorter transcript encoded two possible open reading frames (ORFs), neither of which possessed His174, a residue thought to be the phosphoryl acceptor (Pan CJ, Lei KJ, Annabi B, Hemrika W, Chou JY: Transmembrane topology of glucose-6-phosphatase. J Biol Chem 273:6144-6148, 1998). Northern blot and reverse transcription-polymerase chain reaction analysis showed that the mRNA was highly expressed in pancreatic islets and expressed more in beta-cell lines than in an alpha-cell line. It was notably absent in tissues and cell lines of non-islet neuroendocrine origin, and no other major tissue source of the mRNA was found. During development, it was expressed in parallel with insulin mRNA. The mRNA was efficiently translated and glycosylated in an in vitro translation/membrane translocation system and readily transcribed into COS 1, HIT, and CHO cells using cytomegalovirus or Rous sarcoma virus promoters. Whereas the liver glucose-6-phosphatase showed activity in these transfection systems, the IGRP failed to show glucose phosphotransferase or phosphatase activity with p-nitrophenol phosphate, inorganic pyrophosphate, or a range of sugar phosphates hydrolyzed by the liver enzyme. While the metabolic function of the enzyme is not resolved, its remarkable tissue-specific expression warrants further investigation, as does its transcriptional regulation in conditions where glucose responsiveness of the pancreatic islet is altered.

Alternative Splicing↗