[show abstract][hide abstract] ABSTRACT: 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.
Journal of Endocrinology 01/2003; 175(3):637-47. · 4.06 Impact Factor
[show abstract][hide abstract] ABSTRACT: We have identified an alternatively spliced 5-hydroxytryptamine 2A receptor (5-HT(2A)-R) transcript by PCR of human brain cDNA using degenerate oligonucleotide primers to transmembrane (TM) domains 3 and 7 of the 5-HT(2)-R subfamily. The variant contains a 118-bp insertion at the exon II/III boundary of the 5-HT(2A)-R, which produces a frame shift in the coding sequence and a premature stop codon. PCR analysis showed that the truncated receptor (5-HT(2A-tr)) and native 5-HT(2A)-R were co-expressed in most brain tissues, with the highest levels being found in hippocampus, corpus collosum, amygdala and caudate nucleus. Western blot analysis of HEK-293 cells transfected transiently with a 5-HT(2A-tr) construct showed that a 30-kDa protein was expressed on cell membranes. Co-transfection studies showed no effect of the 5-HT(2A-tr) variant on 3H-ketanserin binding to the native 5-HT(2A)-R or on functional coupling of the 5-HT(2A)-R to 5-HT-stimulated Ca(2+) mobilization. The functional significance of the 5-HT(2A-tr) variant and other truncated receptors remains to be established.
Brain Research 10/2000; 876(1-2):238-44. · 2.88 Impact Factor
[show abstract][hide abstract] ABSTRACT: A variant of the PC12 pheochromocytoma cell line (termed A35C) has been isolated that lacks regulated secretory organelles and several constituent proteins. Northern and Southern blot analyses suggested a block at the transcriptional level. The proprotein-converting enzyme carboxypeptidase H was synthesised in the A35C cell line but was secreted by the constitutive pathway. Transient transfection of A35C cells with cDNAs encoding the regulated secretory proteins dopamine beta-hydroxylase and synaptotagmin I resulted in distinct patterns of mistargeting of these proteins. It is surprising that hybrid cells created by fusing normal PC12 cells with A35C cells exhibited the variant phenotype, suggesting that A35C cells express an inhibitory factor that represses neuroendocrine-specific gene expression.
Journal of Neurochemistry 08/1999; 73(1):21-30. · 3.97 Impact Factor
[show abstract][hide abstract] ABSTRACT: The role of intracellular Ca2+ in the proteolytic processing and intracellular transport of secretory granule proproteins was investigated by pulse-chase radiolabelling of isolated rat islets of Langerhans. The conversion of proinsulin was inhibited by depletion of medium Ca2+ with EGTA and by blocking the transport of Ca2+ into cells with the Ca2+-channel antagonists verapamil, nifedipine and NiCl2. Proinsulin conversion was also reduced by the endoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin, indicating that the process requires transport of Ca2+ into the endoplasmic reticulum. This was supported by the finding that proinsulin processing was inhibited when Ca2+ was depleted before or during pulse-labelling, but not after transport of the protein to post-endoplasmic-reticulum compartments. Similarly, the inhibition of proinsulin processing was reversed by re-introduction of medium Ca2+ around the time of radiolabelling, but not after 15 min of chase incubation. Ca2+ depletion also decreased proteolytic maturation of the prohormone convertases PC1, PC2 and carboxypeptidase H. Secretion experiments suggested that the rate and extent of proinsulin transport into secretory granules were inhibited marginally by Ca2+ depletion, whereas those of the convertases were markedly impeded. Inhibition of proinsulin conversion by Ca2+ depletion was thus not simply related to the Ca2+-dependencies of mature PC1 and PC2, but also to a requirement for endoplasmic reticulum Ca2+ in proteolytic maturation of the convertases and in their transfer to secretory granules. The results also suggest that the Ca2+ required for prohormone processing in the granules enters the secretory pathway via the endoplasmic reticulum.
[show abstract][hide abstract] ABSTRACT: The post-translational processing and intracellular sorting of the proinsulin-converting enzyme carboxypeptidase H (CPH) was studied in isolated rat islets of Langerhans. Pulse-chase-radiolabelling experiments using sequence-specific antisera showed that CPH was synthesized initially as a 57-kDa glycoprotein which was processed to a 54-kDa mature form by proteolytic processing at the N-terminus. Processing of the CPH precursor occurred rapidly (t(1/2) = 30) after an initial delay of 15-30 min and the enzyme was secreted in parallel with the insulin-related peptides in response to glucose-stimulation within 1 h after radiolabelling. This indicated that the proteins were packaged into nascent secretory granules at approximately the same rate following synthesis. Conversion of proinsulin and the 57-kDa form was inhibited markedly by chase incubation of islets at 20 degrees C, indicating that maturation of both proteins occurs in a post-Golgi compartment. Affinity purification of the enzyme from insulinoma subcellular fractions showed that the 57-kDa form was associated with endoplasmic reticulum or Golgi elements, and the 54-kDa form was present in secretory granules. Structural analysis showed that the granule form of the enzyme had an N-terminal amino acid sequence beginning at residue 42 of rat CPH, thereby implicating cleavage of the precursor after the fourth Arg in a site containing five consecutive Arg residues. These findings indicate that post-translational processing of CPH is mediated by an endoprotease which cleaves at sites containing multiple basic amino acid residues upon segregation of the enzyme to the secretory granules.
Molecular and Cellular Endocrinology 09/1995; 113(1):99-108. · 4.04 Impact Factor
[show abstract][hide abstract] ABSTRACT: Human prohormone convertase PC2 was expressed in Xenopus oocytes and its properties were compared with those of the Type-2 endopeptidase of rat insulin secretory granules, previously identified as PC2 [Bennett, Bailyes, Nielson, Guest, Rutherford, Arden and Hutton (1992) J. Biol. Chem. 267, 15229-15236]. Recombinant PC2 had the same substrate specificity as the Type-2 endopeptidase, cleaving at the CA-junction (Lys64, Arg65) of human des-31,32-proinsulin to generate insulin; little activity was found toward human des-64,65-proinsulin or proinsulin itself. Recombinant PC2 was maximally active in 5-7 mM Ca2+ (K0.5 = 1.6 mM) whereas the Type-2 endopeptidase was maximally active in 0.5-1 mM Ca2+ (K0.5 = 40 microM). Both enzymes had a pH optimum of 5.0-5.5 but the Type-2 endopeptidase was active over a wider pH range. Two molecular forms of recombinant PC2 (71 kDa and 68 kDa) were found, both had an intact C-terminus but differed by the presence of the propeptide. The endogenous PC2 comprised several overlapping forms (size range 64-68 kDa), approximately two-thirds of which lacked C-terminal immunoreactivity. Part of the size difference between recombinant and endogenous PC2 was attributable to differences in N-glycosylation. The different post-translational proteolytic modifications of recombinant and endogenous PC2 did not account for the different pH and Ca2+ sensitivities shown by the enzymes. A modulating effect of carbohydrate on enzyme activity could not be excluded.
[show abstract][hide abstract] ABSTRACT: Sulphonylureas stimulate insulin secretion by binding to a receptor in the pancreatic beta-cell plasma membrane resulting in inhibition of ATP-sensitive K+ channels, membrane depolarization and thus influx of Ca2+ through voltage-dependent Ca2+ channels. Sulphonylureas can also induce hormone release at fixed membrane potentials without Ca2+ entry suggesting that these drugs may have other modes of action. We have determined whether different forms of sulphonylurea-binding proteins are present in insulin-secreting cells and their subcellular localization by density gradient centrifugation. Binding studies using [3H]-glibenclamide showed that islet and insulinoma membranes contained a single high affinity sulphonylurea binding site (Kd = 1 nmol/l). Photo-crosslinking of the drug to the membranes resulted in labelling of two proteins with apparent molecular weights of 170 and 140 kDa. The same analyses of insulinoma subcellular fractions showed that the majority (> 90%) of binding proteins were localized to intracellular membranes with only minor levels (< 10%) on plasma membranes. The 170 kDa sulphonylurea binding protein was present in both plasma and granule membrane fractions whereas the 140 kDa form was not present in the plasma membrane fraction. The differences in the molecular forms and subcellular distribution of the receptor are consistent with sulphonylureas having multiple sites of action in the pancreatic beta cell.
[show abstract][hide abstract] ABSTRACT: The post-translational processing of chromogranin A (CGA) and the nature of the enzyme(s) involved were investigated in rat pancreatic islet and insulinoma tissue. Pulse-chase radiolabelling experiments using sequence-specific antisera showed that the 98 kDa (determined by SDS/PAGE) precursor was processed to an N-terminal 21 kDa peptide, a C-terminal 14 kDa peptide and a 45 kDa centrally located peptide with a rapid time course (t1/2 approx. 30 min) after an initial delay of 30-60 min. The 45 kDa peptide was, in turn, converted partially into a 5 kDa peptide with pancreastatin immunoreactivity and a 3 kDa peptide with WE-14 immunoreactivity over a longer time period. Incubation of bovine CGA with rat insulinoma secretory-granule lysate produced peptides of 18, 16 and 40 kDa via intermediates of 65 and 55 kDa. N-terminal sequence analysis indicated that cleavage occurred at the conserved paired basic sites Lys114-Arg115 and Lys330-Arg331, suggesting that cleavage of the equivalent sites (Lys129-Arg130 and Lys357-Arg358) in the rat molecule produced the initial post-translational products observed in intact pancreatic beta-cells. The enzyme activity responsible for the cleavage of bovine CGA co-chromatographed on DEAE-cellulose with the type-2 proinsulin endopeptidase and with PC2 immunoreactivity. The type-1 enzyme (PC1/3) appeared inactive towards CGA. The requirement for Ca2+ ions and an acidic pH for conversion was consistent with the involvement of a member of the eukaryote subtilisin family, and the composition of the released peptides in pulse-chase and secretion studies suggested that conversion occurred in the secretory-granule compartment. The overall catalytic rate as well as the relative susceptibilities of the Lys114-Arg115 and Lys330-Arg331 sites to cleavage were affected by pH, suggesting that the ionic environment of the processing compartment may play a role in the differential processing of CGA which is evident in various neuroendocrine cells.
[show abstract][hide abstract] ABSTRACT: A search for novel pancreatic islet cadherins was undertaken using the polymerase chain reaction with mouse beta TC3 cell line cDNA and degenerate primers based on conserved C-terminal sequences in neural (N), epithelial, and placental cadherin (CAD). A hitherto uncharacterized rodent sequence was detected which was then cloned from a mouse insulinoma cDNA library and shown to be the mouse equivalent of chicken retina CAD (R-CAD). The similarity of the mouse and chicken sequences was remarkable (eight nonconservative changes in the 747 amino acids of the mature protein sequence; 95% overall identity), indicating strong conservation of function. Mouse R-CAD was also closely homologous to N-CAD (72% identity), including those regions of N-CAD implicated in the cadherin-cadherin interaction and Ca2+ binding. In vitro translation of the cDNA indicated that mouse R-CAD enters the secretory pathway and undergoes posttranslational glycosylation and proteolytic cleavage. R-CAD mRNA was distributed widely in mouse tissues with high levels present in brain, skeletal muscle, and thymus. In the pancreas, R-CAD and N-CAD showed endocrine cell specificity and a differential expression in beta- and non-beta-cells. Messenger RNA expression was evident during early pancreatic development at a time when the first pluripotent hormone-producing cells differentiate to attain their adult phenotype and become organized in islet-like clusters. The presence of R-CAD and N-CAD in islets is consistent with the neurone-like properties of this tissue. Differences in CAD expression might explain the segregation of exocrine and endocrine cells during development of the pancreas and the characteristic morphological distribution of the different endocrine cells within the islet.
[show abstract][hide abstract] ABSTRACT: Proinsulin conversion in the insulin secretory granule is mediated by two sequence-specific endoproteases related to the Kex2 homologues, PC2 and PC3 (Bennett, D. L., Bailyes, E. M., Nielsen, E., Guest, P. C., Rutherford, N. G., Arden, S. D., and Hutton, J. C. (1992) J. Biol. Chem. 267, 15229-15236; Bailyes, E. M., Bennett, D. L., and Hutton, J. C. (1992) Enzyme, in press). Radiolabeling studies using isolated rat islets showed that PC2 was synthesized initially as a 76-kDa glycoprotein which was converted by limited proteolysis to the mature 64-66-kDa form. Conversion was initiated approximately 1 h after synthesis and proceeded via intermediates of 71, 68, and 66 kDa with a t1/2 of 140 min. Release of only the 66- and 64-66-kDa radiolabeled forms of PC2 was induced by glucose and then only at times more than 2 h following synthesis. Proinsulin conversion, by contrast, was more rapid (delay = 30 min, t1/2 = 60 min), and release commenced as soon as 1 h after synthesis with the secreted material being comprised of the precursor, intermediate, and mature forms of insulin. Ultrastructural analysis of islet beta cells showed that PC2 was concentrated in secretory granules. Subcellular fractionation combined with immunoblot analysis showed that insulinoma secretory granules contained only the mature 64-66-kDa form of PC2, whereas fractions enriched in Golgi and endoplasmic reticulum contained a mixture of the 76- and 66-kDa forms of the enzyme. These results indicate that post-translational proteolysis of PC2 is initiated before sorting into the regulated pathway of secretion and that the relative proportions of proinsulin and PC2 packaged into secretory granules will change with physiological conditions.
Journal of Biological Chemistry 12/1992; 267(31):22401-6. · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: Enzymological studies have implicated two Ca(2+)-dependent endopeptidases in the conversion of proinsulin to insulin; a type 1 activity which cleaves on the C-terminal side of Arg31-Arg32 and a type 2 activity which cleaves C-terminally to Lys64-Arg65 in the proinsulin sequence. The possibility that these enzymes are related to the recently discovered family of mammalian subtilisin-like gene products (furin, PC2, and PC3) and the yeast propheromone-converting enzyme (KEX-2), was investigated. Degenerate oligonucleotide primers flanking the putative catalytic domain within this gene family were used in a polymerase chain reaction to amplify related sequences from rat insulinoma cDNA. One major product of 700 base pairs was obtained which was greater than 99% identical to the corresponding rat PC2 sequence. This cDNA was subcloned into the bacterial expression vector pGEX-3X to generate a recombinant protein for antibody production. Western blot analysis showed the immunoreactivity was prominent in neuroendocrine tissues as a 65-kDa protein. It was concentrated in secretory granule-enriched fractions of insulinoma tissue, where it was present as a readily solubilized monomeric protein. Deglycosylation studies using endoglycosidase H and N-glycanase showed that the 65-kDa protein was comprised of approximately 9% carbohydrate, consistent with the presence of three consensus sequences for N-linked glycosylation in rat PC2. The immunoreactivity co-eluted with the type 2 proinsulin endopeptidase on gel filtration and ion-exchange chromatography and the antisera specifically immunoprecipitated type 2 activity from insulin granule extracts. N-terminal sequence analysis of the immunoreactive protein gave two sequences which corresponded to residues 109-112 and 112-119 of rat PC2. This indicated that posttranslational processing of PC2 itself occurs C-terminally to basic amino acids to produce the mature enzyme. It is concluded that PC2 is the type 2 endopeptidase involved in proinsulin conversion. Localization of PC2 immunoreactivity to other tissues of the diffuse neuroendocrine system suggests that the type 2 endopeptidase also functions in the processing of precursor forms of other prohormones and polypeptide neurotransmitters.
Journal of Biological Chemistry 08/1992; 267(21):15229-36. · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: The intracellular distribution and molecular heterogeneity of carboxypeptidase H was studied in rat insulinoma tissue and isolated islets of Langerhans by a combination of immunohistochemical, ultrastructural, subcellular fractionation, and immunoblotting analyses. Immunofluorescence microscopy of islets demonstrated the presence of carboxypeptidase H in both insulin-containing B cells and glucagon-containing A cells. Quantitative ultrastructural analyses of islet B cells indicated that the enzyme was concentrated in mature insulin secretory granules, clathrin-coated condensing granules, and to a lesser extent the Golgi apparatus. Carboxypeptidase H activity was localized principally to secretory granule subfractions of insulinoma tissue, where it was present for the major part (70%) as a form which is readily solubilizable at pH values prevailing in the granule interior (5.5). This species migrated as a diffuse band of 53-57 kilodaltons (kDa) on immunoblot analysis using antisera raised against the purified native enzyme. In contrast, the insoluble form which was associated with the granule membrane at pH 5.5, migrated as a relatively compact band of 55-57 kDa. Carboxypeptidase H activity was also present in subcellular fractions which contained Golgi membranes together with elements of the endoplasmic reticulum, and in a low density secretory granule fraction which may represent immature granules. The enzyme in these compartments, like the granule membrane species, migrated as a compact 55-57 kDa band on immunoblots. Two-dimensional electrophoretic immunoblot analysis of secretory granules suggested that both membrane and soluble forms of the enzyme were glycoproteins and that the terminal glycosylation was similar in both instances. Antiserum raised against the deduced C-terminal 11 amino acids of the cloned carboxypeptidase H sequence recognized the 55-57 kDa membrane component in granules but did not react with the 53-57 kDa soluble species. A major difference between the soluble and membrane forms therefore appears to be a structural modification or proteolytic removal of the C-terminal domain in the trans-Golgi or early secretory granule compartment. The concept that proteolysis is involved is further supported by the observation that the relative proportion of the high and low mol wt forms of the enzyme in different subcellular fractions correlated with that of proinsulin and insulin, respectively. The membrane association of the 55-57 kDa form of carboxypeptidase H is disrupted at pH values of 9 and is dependent on ionic strength. This further suggests that the C-terminus of the protein may have an important role in the sorting or concentration of the enzyme in vesicular elements of the regulated pathway of secretion.
[show abstract][hide abstract] ABSTRACT: Two-dimensional gel-electrophoretic analysis combined with fluorography and densitometric quantification was used to examine the effects of glucose on the biosynthesis of rat pancreatic islet proteins. An increase in the medium glucose concentration from 2.8 to 16.7 mM produced a 10-20 fold stimulation in the synthesis of 10 out of 260 detected islet proteins, as judged by incorporation of [35S]methionine during a 20 min incubation. The synthetic rates of the majority of the remaining proteins were stimulated by 2-4-fold. Greater resolution achieved by pulse-chase labelling and subcellular fractionation showed that, of 32 major proteins localized to insulin secretory granules, the biosynthesis of 25 were stimulated 15-30-fold by glucose. By contrast, only eight of 160 proteins in the soluble fraction showed a response of similar magnitude. It is concluded that there is a major and co-ordinated activation of the biosyntheses of proteins destined for secretory granules, which most likely occurs at the level of translational initiation and signal-recognition-particle-mediated translocation into the endoplasmic reticulum lumen. However, it is clear that not all granule proteins, or the majority of proteins translocated across the endoplasmic reticulum membrane, are affected in an equivalent manner. In addition, the synthesis of a small number of cytosolic proteins may be increased markedly by insulinotropic stimuli.
[show abstract][hide abstract] ABSTRACT: The release of carboxypeptidase H activity from isolated rat islets was determined and compared to the secretion of immunoreactive insulin. Analysis of pancreatic islet cells sorted into beta and non-beta types indicated that approx. 80% of islet carboxypeptidase H activity is present in the beta cell. The release of both insulin and carboxypeptidase H was stimulated markedly by increasing the glucose concentration in the medium from 2.8 to 28 mM. The fractional release was in accordance with the observed cellular distribution of both proteins. The secretory response was biphasic with time, with an initial rapid transient phase of release within 5 min, followed by a more sustained response. The concentration-dependencies of glucose stimulation of release of insulin and carboxypeptidase H were similar, with a threshold for stimulation around 5.6 mM-glucose and maximal stimulatory response at 16.7-28 mM-glucose. The release of both proteins was inhibited by 20 mM-mannoheptulose, removal of Ca2+ from the medium and addition of 1 microM-noradrenaline. The combination of 10 mM-4-methyl-2-oxopentanoate and 10 mM-glutamine stimulated the release of carboxypeptidase H and insulin, as did 3-isobutyl-1-methylxanthine and 350 microM-tolbutamide in the presence of glucose. It is evident that carboxypeptidase H is released from the pancreatic beta-cell by an exocytotic process from the same intracellular compartment as insulin. The release of carboxypeptidase H by a constitutive process was at best equivalent to 0.4%/h, or less than 2% of the maximal rate of release via the regulated pathway. It is concluded that carboxypeptidase H can be used as a sensitive index of beta-cell secretion and an alternative marker to the insulin-related peptides.
[show abstract][hide abstract] ABSTRACT: A two-site immunoradiometric assay for insulin is described which is both rapid (processing time 60 min) and highly sensitive (lower detection limit 2 pM). Insulin is bound by a 125I-labelled mouse monoclonal antibody raised against human proinsulin and binding assessed by immunoprecipitation with an immunoadsorbent prepared from guinea pig polyclonal antisera raised against bovine insulin. Human, rat, bovine and porcine insulins (10-600 pM) showed similar reactivities in the assay. The human insulin-like peptides, proinsulin, des-31,32-proinsulin and des-64,65-proinsulin (25 pM) had reactivities which were 44.7%, 63.2% and 73.4% of that of insulin, respectively. The assay was highly reproducible with a coefficient of variation of 2.3% for the highest human insulin standard (1000 pM) and 5.5% for the lowest (2 pM). The assay was suitable for determining the concentration of insulin in plasma of fasting human subjects, in normal and tumour-bearing rats and for in vitro studies of insulin secretion from rat pancreatic islets.
Molecular and Cellular Endocrinology 01/1990; 67(2-3):173-8. · 4.04 Impact Factor
[show abstract][hide abstract] ABSTRACT: The regulation of the biosynthesis of the insulin-secretory-granule matrix proteins insulin II, chromogranin A and carboxypeptidase H was studied in isolated rat islets of Langerhans. Islets were labelled with [35S]-methionine, and incorporation into total protein was determined by trichloroacetic acid precipitation and that into specific proteins by immunoprecipitation followed by polyacrylamide-gel electrophoresis and fluorography. Islets incubated in the presence of 16.7 mM-glucose incorporated 3 times as much [35S]-methionine into total protein as did islets incubated with 2.8 mM-glucose. The same conditions produced more than a 20-fold increase in incorporation into both proinsulin and chromogranin A, with no observable effect on carboxypeptidase H. The concentration-dependencies of the glucose-stimulated synthesis of chromogranin A and proinsulin were parallel, and in both cases the response to 16.7 mM-glucose was typified by an initial lag of 20 min, followed by a rapid activation to a new steady state over the ensuing 40 min. Synthesis of total protein, although activated to a lesser extent, responded with similar kinetics. Extracellular Ca2+ depletion did not affect the basal or glucose-stimulated biosynthesis of any of the proteins under investigation. Mannoheptulose (20 mM) abolished glucose-stimulated synthesis of insulin, chromogranin A and total protein, but had no effect on the synthesis of carboxypeptidase H. It is concluded that the biosynthesis of insulin and chromogranin A is regulated principally at the translational level by the same intracellular signal generated from the metabolism of glucose. Such regulation is not common to all insulin-secretory-granule proteins, since the synthesis of carboxypeptidase H was unaffected by the same stimulus.