Biomedical subjects

W B Huttner

Publications and source records attributed to W B Huttner.

At least 91 records · Page 5Linked to original sources

PubMed1991

Trimeric G-proteins of the trans-Golgi network are involved in the formation of constitutive secretory vesicles and immature secretory granules.

Non-hydrolysable analogues of GTP, such as GTP gamma S and GMP-PNP, have previously been shown to inhibit the formation of constitutive secretory vesicles (CSVs) and immature secretory granules (ISGs) from the trans-Golgi network (TGN). Using a cell-free system, we show here that the formation of these vesicles is also inhibited by [A1F4]-, a compound known to act on trimeric G-proteins. Addition of highly purified G-protein beta gamma subunits stimulated, in a differential manner, the cell-free formation of both CSVs and ISGs. ADP-ribosylation experiments revealed the presence of a pertussis toxin-sensitive G-protein alpha subunit in the TGN. We conclude that trimeric G-proteins regulate the formation of secretory vesicles from the TGN.

Adenosine Diphosphate Ribose↗

PubMed1991

Sulfation of Tyr1680 of human blood coagulation factor VIII is essential for the interaction of factor VIII with von Willebrand factor.

The acidic region of the Factor VIII light chain was studied with regard to structural requirements for the formation of a functional von Willebrand factor (vWF)-binding site. Factor VIII mutants lacking the B domain, with additional deletions and an amino acid replacement within the sequence 1649-1689 were constructed using site-directed mutagenesis and expressed in Cos-1 cells. These mutants, which were recovered as single-chain molecules with similar specific activities, were compared in their binding to immobilized vWF. Deletion of amino acids 741-1648 or 741-1668 did not affect the binding of Factor VIII to vWF. However, a mutant with a deletion of residues 741-1689 was no longer capable of interacting with vWF. This indicates a role for residues within the sequence 1669-1689 in the formation of a vWF-binding site. When recombinant Factor VIII was expressed in the presence of chlorate, an inhibitor of protein sulfation, the resulting Factor VIII displayed strongly reduced binding to vWF. vWF binding was completely abolished when within the sequence 1669-1689 the tyrosine residue Tyr1680, which is part of a consensus tyrosine sulfation sequence, was replaced by phenylalanine. The Factor VIII sequence 1673-1689 was identified as a high affinity substrate for tyrosylprotein sulfotransferase (Km = 57 microM) in cell-free sulfation studies. It is concluded that sulfation of Tyr1680 is required for the interaction of Factor VIII with vWF. Two synthetic peptides that represent the sequence 1673-1689, but differ with respect to sulfation of Tyr1680 are shown to have vWF binding affinity that is considerably lower than the Factor VIII protein. Several models to accommodate our findings are discussed.

Binding, Competitive↗

PubMed1991

Newly synthesized synaptophysin is transported to synaptic-like microvesicles via constitutive secretory vesicles and the plasma membrane.

The biogenesis of synaptic-like microvesicles (SLMVs) in neuroendocrine cells was investigated by studying the traffic of newly synthesized synaptophysin to SLMVs in PC12 cells. Synaptophysin was found to be sulfated, which facilitated the determination of its exit route from the trans-Golgi network (TGN). Virtually all [35S]sulfate-labeled synaptophysin was found to leave the TGN in vesicles which were indistinguishable from constitutive secretory vesicles but distinct from immature secretory granules and SLMVs. [35S]sulfate-labeled synaptophysin was rapidly transported from the TGN to the cell surface, with a t1/2 of approximately 10 min in resting cells. After arrival at the cell surface, [35S]sulfate-labeled synaptophysin cycled for at least 1 h between the plasma membrane and an intracellular compartment likely to be the early endosome. Up to approximately 40% of the [35S]sulfate-labeled synaptophysin eventually (after 3 h and later) reached SLMVs, which could be distinguished from the other post-TGN compartments by their lower buoyant density in a sucrose gradient and their selective inclusion upon permeation chromatography using a controlled-pore glass column. Our results suggest that newly synthesized membrane proteins of SLMVs in neuroendocrine cells, and possibly of small synaptic vesicles in neurons, reach these organelles via the TGN----plasma membrane----early endosome.

Animals↗

PubMed1991

Expression of chromogranin A mRNA in small cell carcinoma of the lung.

Chromogranin A (CgA) is the most specific neuroendocrine marker but in small cell carcinoma of the lung (SCCL) CgA immunoreactivity cannot always be detected, possibly owing to poor granularity. The localization of CgA mRNA could be a more useful indicator of CgA synthesis in SCCL. We have therefore studied the expression of CgA mRNA in SCCL, using in situ hybridization and Northern blot analysis, comparing it with CgA immunoreactivity. The expression of CgA mRNA and immunoreactivity in other types of lung tumours was also investigated. Freshly fixed, surgically resected SCCL (n = 5), adenocarcinoma (n = 10), squamous cell carcinoma (n = 10), large cell carcinoma (n = 5), and primary lymphoma (n = 2) were examined. Weak immunoreactivity to CgA was seen in one SCCL but all five strongly expressed CgA mRNA. The specificity of the in situ hybridization signal and the probe was confirmed by the use of a sense probe and Northern blot analysis. Non-SCCLs were negative for both CgA mRNA and immunoreactivity. This study provides evidence for CgA synthesis in SCCL and confirms the specificity of CgA for neuroendocrine differentiation. In situ hybridization appears to be a very useful tool for investigating the nature of tumour cells, especially in the absence of the end product of the gene transcript.

Blotting, Northern↗

PubMed1991

Secretogranin I (chromogranin B) mRNA accumulation is hormonally regulated in GH3B6 rat pituitary tumor cells.

Secretogranin I (SgI; chromogranin B) belongs to a class of acidic tyrosine-sulfated secretory proteins believed to play a role in the secretory process of endocrine cells. Our aim here was to compare the levels of SgI mRNA to that of prolactin (PRL) and growth hormone (GH), using rat pituitary cell lines. As far as the constitutive expression is concerned, we found a positive correlation between SgI mRNA and PRL mRNA levels. However, the neuropeptide TRH (50 nM) inhibited the accumulation of SgI mRNA in GH3B6 cells whereas, as expected, it induced a rapid and sustained increase in PRL mRNA accumulation. By contrast, 17 beta-estradiol (1 nM) stimulated the accumulation of both SgI and PRL mRNAs, with the same EC50 (18-59 pM). Reciprocally, treatment with dexamethasone (100 nM) reduced the level of SgI and PRL mRNAs to 23% and 29% of control, respectively, but led to a 2.1-fold increase in the GH mRNA level. Altogether, the present work shows that SgI gene expression is subject to multiple hormonal regulations and occasionally parallels the regulation of the PRL gene but never that of the GH gene, under the conditions tested.

Animals↗

PubMed1991

Exocytotic and endocytotic membrane traffic in neurons.

Because neurons are highly polarized and capable of various modes of neurosecretion the exocytotic and endocytotic membrane traffic in these cells is more complex than in other eukaryotic cells. Progress in our understanding of neuronal membrane traffic and organelle biogenesis has come from recently discovered analogies to epithelial and endocrine cells.

Animals↗

PubMed1991

The granin (chromogranin/secretogranin) family.

The chromogranins/secretogranins, referred to in abbreviated form as granins, are a family of acidic secretory proteins that are found in the secretory granules of a wide variety of endocrine cells and neurons, being stored together with many different peptide hormones and neuropeptides. The recent elucidation of their primary structure has provided insights into possible functions of these proteins. Moreover, the granins have been successfully used as markers for normal and neoplastic endocrine and neuronal cells, as well as model proteins to understand the sorting mechanism involved in the formation of secretory granules.

Amino Acid Sequence↗

PubMed1991

Characterization of the immature secretory granule, an intermediate in granule biogenesis.

The events in the biogenesis of secretory granules after the budding of a dense-cored vesicle from the trans-Golgi network (TGN) were investigated in the neuroendocrine cell line PC12, using sulfate-labeled secretogranin II as a marker. The TGN-derived dense-cored vesicles, which we refer to as immature secretory granules, were found to be obligatory organellar intermediates in the biogenesis of the mature secretory granules which accumulate in the cell. Immature secretory granules were converted to mature secretory granules with a half-time of approximately 45 min. This conversion entailed an increase in their size, implying that the maturation of secretory granules includes a fusion event involving immature secretory granules. Pulse-chase labelling of PC12 cells followed by stimulation with high K+, which causes the release of secretogranin II, showed that not only mature, but also immature secretory granules were capable of undergoing regulated exocytosis. The kinetics of secretion of secretogranin II, as well as those of a constitutively secreted heparan sulfate proteoglycan, were reduced by treatment of PC12 cells with nocodazole, suggesting that both secretory granules and constitutive secretory vesicles are transported to the plasma membrane along microtubules. Our results imply that certain membrane proteins, e.g., those involved in the fusion of post-TGN vesicles with the plasma membrane, are sorted upon exit from the TGN, whereas other membrane proteins, e.g., those involved in the interaction of post-TGN vesicles with the cytoskeleton, may not be sorted.

Animals↗

PubMed1991

Milieu-induced, selective aggregation of regulated secretory proteins in the trans-Golgi network.

Regulated secretory proteins are thought to be sorted in the trans-Golgi network (TGN) via selective aggregation. The factors responsible for this aggregation are unknown. We show here that two widespread regulated secretory proteins, chromogranin B and secretogranin II (granins), remain in an aggregated state when TGN vesicles from neuroendocrine cells (PC12) are permeabilized at pH 6.4 in 1-10 mM calcium, conditions believed to exist in this compartment. Permeabilization of immature secretory granules under these conditions allowed the recovery of electron dense cores. The granin aggregates in the TGN largely excluded glycosaminoglycan chains which served as constitutively secreted bulk flow markers. The low pH, high calcium milieu was sufficient to induce granin aggregation in the RER. In the TGN of pituitary GH4C1 cells, the proportion of granins conserved as aggregates was higher upon hormonal treatment known to increase secretory granule formation. Our data suggest that a decrease in pH and an increase in calcium are sufficient to trigger the selective aggregation of the granins in the TGN, segregating them from constitutive secretory proteins.

Animals↗

PubMed1991

Argyrophilic carcinoma of the male breast. A neuroendocrine tumor containing predominantly chromogranin B (secretogranin I).

Argyrophilic tumors were diagnosed in 28 of 134 (20.8%) consecutive male patients who had a carcinoma of the breast removed between 1961 and 1990. Histologically, most argyrophilic tumors showed uniform cellularity and prevalent expansive growth. Ultrastructural observation disclosed the presence of electron-dense cored granules in the cytoplasm of the tumor cells. By immunocytochemistry, 17 of 28 argyrophilic tumors (60.7%) contained chromogranin B (secretogranin I)-immunoreactive cells, whereas chromogranin A was present in four of these 17 tumors only (14.2%). Immunoblotting studies showed chromogranin B immunoreactivity similar to that found in normal neuroendocrine cells. Despite these findings, which would argue for a distinct morphologic and immunochemical entity, no statistically significant differences between argyrophilic and common male breast carcinomas were found when a number of clinicopathologic features and relapse-free survival were considered.

Adult↗

PubMed1991

Subcellular localization of secretogranin II and synaptophysin by immunoelectron microscopy in differentiated hypothalamic neurons in culture.

Secretogranin II (SgII), a tyrosine-sulfated secretory protein, is a widespread component of endocrine and neuronal cells. In the present study we used mouse hypothalamic neurons differentiated in culture and studied the subcellular localization of SgII by two methods, i.e., by the use of immunoperoxidase or immunogold electron microscopy. By immunoperoxidase labeling, SgII was mainly detected in the matrix of large dense-core vesicles (LDCVs). In addition, usually in nerve terminals containing LDCVs, peroxidase reaction product was also found in association with the membrane of small synaptic vesicles (SSVs). By immunogold labeling, SgII was detected only in the matrix of LDCVs. We also compared the localization of SgII and synaptophysin (SY), an integral membrane protein of SSVs, by double labeling, using a combination of pre-embedding immunogold and -peroxidase techniques for SgII and SY, respectively. In perikarya, SgII-positive LDCVs were observed in the vicinity of the Golgi complex and scattered in the cytoplasm. In contrast, SY labeling was restricted to electron-translucent vesicles and tubular membranes in the Golgi area. Moreover, membrane structures positive for both SgII and SY were not found either in the Golgi zone or in other regions of the cytoplasm. In synaptic boutons, immunolabeling of LDCVs and SSVs with anti-SgII and anti-SY, respectively, was mutually exclusive. In summary, within the limitation of the methods used, our data are consistent with the notion that SgII and SY are segregated from each other on exit from the trans-Golgi network, than follow two distinct membrane traffic pathways, and that the presence of SgII on the membrane of some SSVs is due to endocytosis.

Animals↗

PubMed1990

Requirement for GTP hydrolysis in the formation of secretory vesicles.

The specificity of vesicular transport in a cell is determined by the formation of vesicles with specific contents from a donor compartment and their selective fusion with the appropriate acceptor compartment. Several of the latter fusion steps have been investigated in detail using cell-free systems, and work with these systems as well as genetic evidence has revealed a role for GTP-binding proteins in membrane fusion processes. We have reconstituted the formation of constitutive secretory vesicles and immature secretory granules from the trans Golgi network in a cell-free system. We show here that the budding of both types of post-Golgi vesicles is inhibited by non-hydrolysable analogues of GTP, which suggests a more widespread role for GTP-binding proteins in membrane traffic than previously assumed.

Animals↗

PubMed1990

Conversion of recombinant hirudin to the natural form by in vitro tyrosine sulfation. Differential substrate specificities of leech and bovine tyrosylprotein sulfotransferases.

Hirudin, a tyrosine-sulfated protein secreted by the leech Hirudo medicinalis, is one of the most potent anticoagulants known. The hirudin cDNA has previously been cloned and has been expressed in yeast, but the resulting recombinant protein was found to be produced in the unsulfated form, which is known to have an at least 10 times lower affinity for thrombin than the naturally occurring tyrosine-sulfated hirudin. Here we describe the in vitro tyrosine sulfation of recombinant hirudin by leech and bovine tyrosylprotein sulfotransferase (TPST). With both enzymes, in vitro sulfation of recombinant hirudin occurred at the physiological site (Tyr-63) and rendered the protein biochemically and biologically indistinguishable from natural hirudin. However, leech TPST had an over 20-fold lower apparent Km value for recombinant hirudin than bovine TPST. Further differences in the catalytic properties of leech and bovine TPSTs were observed when synthetic peptides were tested as substrates. Moreover, a synthetic peptide corresponding to the 9 carboxyl-terminal residues of hirudin (which include Tyr-63) was sulfated by leech TPST with a similar apparent Km value as full length hirudin, indicating that structural determinants residing in the immediate vicinity of Tyr-63 are sufficient for sulfation to occur.

Adrenal Medulla↗

PubMed1990

Analysis of the substrate specificity of tyrosylprotein sulfotransferase using synthetic peptides.

Tyrosylprotein sulfotransferase (TPST) catalyzes the sulfation of proteins at tyrosine residues. We have analyzed the substrate specificity of TPST from bovine adrenal medulla with a novel assay, using synthetic peptides as substrates. The peptides were modeled after the known, or putative, tyrosine sulfation sites of the cholecystokinin precursor, chromogranin B (secretogranin I) and vitronectin, as well as the tyrosine phosphorylation sites of alpha-tubulin and pp60src. Varying the sequence of these peptides, we found that (i) the apparent Km of peptides with multiple tyrosine sulfation sites decreased exponentially with the number of sites; (ii) acidic amino acids were the major determinant for tyrosine sulfation, acidic amino acids adjacent to the tyrosine being more important than distant ones; (iii) a carboxyl terminally located tyrosine residue may be sulfated. Moreover, TPST catalyzed the sulfation of a peptide corresponding to the tyrosine autophosphorylation site of pp60v-src (Tyr-416) but not of a peptide corresponding to the non-autophosphorylation site of pp60c-src (Tyr-527). These results experimentally define structural determinants for the substrate specificity of TPST and show that this enzyme and certain autophosphorylating tyrosine kinases have overlapping substrate specificities in vitro.

Adrenal Medulla↗

PubMed1990

Occurrence of tyrosine sulfate in proteins--a balance sheet. 1. Secretory and lysosomal proteins.

1. The abundance of tyrosine sulfate in secretory proteins and in various classes of cellular proteins has been quantified and compared to protein-bound carbohydrate sulfate. 2. HepG2 cells and fibroblasts, two cell types showing only the constitutive pathway of secretion, and PC12 cells, which show both the constitutive and the regulated pathway of secretion, were subjected to pulse-chase and/or long-term labelling with [35S]sulfate and [3H]tyrosine, followed by analysis of proteins in the cells and medium. Under both conditions of labelling, 65-92% of the protein-bound tyrosine sulfate and 44-84% of the protein-bound carbohydrate sulfate were found to be secretory. In HepG2 cells, the frequency of sulfation of tyrosine residues, which can be determined independently from protein abundance and the rate of protein synthesis, was 8-22 times higher in proteins secreted into the medium than in cellular proteins. 3. All cell lines studied contained significant amounts, not only of carbohydrate sulfate, but also of tyrosine sulfate in specific cellular proteins. As shown for fibroblasts, these tyrosine-sulfated proteins were retained within the cells for at least 100 min of chase following a pulse with [35S]sulfate and were almost completely recovered in a light membrane fraction after subcellular fractionation. 4. Lysosomes were found to contain small, but significant, amounts of protein-bound tyrosine sulfate in addition to protein-bound carbohydrate sulfate. Protein-bound tyrosine sulfate in lysosomes reached a peak at 20 min of chase and rapidly disappeared thereafter, whereas protein-bound carbohydrate sulfate accumulated after 20 min of chase. Examination of the known sequences of eleven lysosomal enzymes revealed the presence of potential tyrosine sulfation sites in five of them. 5. Our results show that secretory proteins are the most abundant, but not exclusive, in vivo substrates for tyrosine sulfation and suggest the presence of soluble tyrosine-sulfated proteins in lysosomes and other, as yet unidentified, organelles of the secretory pathway. In the following paper in this journal we describe the abundance of tyrosine sulfate in integral membrane proteins.

Amino Acid Sequence↗

PubMed1990

Occurrence of tyrosine sulfate in proteins--a balance sheet. 2. Membrane proteins.

1. The abundance of tyrosine sulfate in membrane proteins was quantified in four different cell lines and compared to that in soluble cellular and secreted proteins. 2. Upon metabolic labelling of HepG2, Ltk-, AtT20 and PC12 cells with [35S]sulfate or [3H]tyrosine, a fraction enriched in integral membrane proteins was found to contain small, but significant, amounts of protein-bound tyrosine sulfate (up to 2.5% of the total cellular plus secreted protein-bound tyrosine sulfate). On the other hand, the frequency of sulfation of tyrosine residues of membrane proteins was within the same order of magnitude as that of secreted proteins, indicating that the low abundance of tyrosine sulfate in membrane proteins was largely a reflection of the low abundance of these proteins themselves. Consistent with this conclusion were the results of an analysis showing that 14 out of 32 selected membrane-spanning proteins contain potential tyrosine sulfation sites. 3. In HepG2 cells, three tyrosine-sulfated integral membrane glycoproteins of molecular mass 100, 125 and 150 kDa were identified. Characterization of the 150-kDa tyrosine-sulfated membrane protein revealed that it was protected from proteolysis in intact cells, suggesting a localization in an intracellular organelle. 4. Together with the results reported in the preceding paper in this journal, our data suggest that tyrosine sulfation occurs in various classes of trans-Golgi-derived proteins, soluble as well as membrane, and extracellularly exposed as well as intracellularly retained, proteins. This suggests that tyrosine sulfation may have a variety of physiological functions, depending on the individual tyrosine-sulfated protein or protein class.

Amino Acid Sequence↗

PubMed1990

The organisation of the mouse chromogranin B (secretogranin I) gene.

A cosmid clone containing the gene for mouse chromogranin B (secretogranin I), a secretory protein found in secretory granules of most endocrine cells and neurons, was isolated and sequenced. The chromogranin B protein was found to be encoded by 5 exons which correspond to the cleaved signal peptide, the short N-terminal sequence preceding the disulfide-bonded loop structure, the disulfide-bonded loop structure itself, the large, variable region comprising approximately 90% of the protein, and the conserved C-terminal sequence. The promoter region of the chromogranin B gene is very GC-rich and contains a CATAA motif, a cAMP-responsive element and an Sp1 binding site.

Amino Acid Sequence↗

PubMed1990

Cell-free protein sorting to the regulated and constitutive secretory pathways.

To elucidate the mechanism of secretory granule formation, we here identify the first intermediate in this process, the immature secretory granule, in the neuroendocrine cell line PC12 and demonstrate the packaging of a regulated secretory protein, secretogranin II, to immature secretory granules in a cell-free system. The formation of immature secretory granules was as fast (t1/2 approximately 5 min) as that of constitutive secretory vesicles identified by the presence of a rapidly secreted heparan sulfate proteoglycan. Using the cell-free system, the formation of post-Golgi secretory vesicles was found to be dependent upon ATP. Two distinct populations of vesicles were formed: immature secretory granules containing secretogranin II and constitutive secretory vesicles containing the heparan sulfate proteoglycan. These results show that in a cell-free system, a constitutive and a regulated secretory protein are sorted upon exit from the trans-Golgi network.

Adenosine Triphosphate↗