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R B Kelly

Publications and source records attributed to R B Kelly.

At least 127 records · Page 7Linked to original sources
PubMed1985

The exocrine protein trypsinogen is targeted into the secretory granules of an endocrine cell line: studies by gene transfer.

The exocrine protein rat anionic trypsinogen has been expressed and is secreted from the murine anterior pituitary tumor cell line AtT-20. We examined which secretory pathway trypsinogen takes to the surface of this endocrine-derived cell line. The "constitutive" pathway externalizes proteins rapidly and in the absence of an external stimulus. In the alternate, "regulated" pathway, proteins are stored in secretory granules until the cells are stimulated to secrete with 8-Br-cAMP. On the basis of indirect immunofluorescence localization, stimulation of release, and subcellular fractionation, we find that trypsinogen is targeted into the regulated secretory pathway in AtT-20 cells. In contrast, laminin, an endogenous secretory glycoprotein, is shown to be secreted constitutively. Thus it appears that the transport apparatus for the regulated secretory pathway in endocrine cells can recognize not only endocrine prohormones, but also the exocrine protein trypsinogen, which suggests that a similar sorting mechanism is used by endocrine and exocrine cells.

Adrenocorticotropic Hormone↗
PubMed1985

Selective packaging of human growth hormone into synaptic vesicles in a rat neuronal (PC12) cell line.

We have introduced the gene for human growth hormone (hGH) into PC12 cells, a rat pheochromocytoma-derived cell line with neuronal characteristics, and have isolated stable cell lines that express this protein. hGH is stored within the cells in membrane-bounded vesicles that are indistinguishable from the endogenous catecholaminergic synaptic vesicles. When the transfected cells are stimulated by carbachol or direct depolarization, they release norepinephrine and hGH with parallel kinetics. Treatment of the transfected cells with nerve growth factor results in a twofold increase in the amounts of hGH stored in and secreted from the cells. Not all proteins are packaged into the synaptic vesicles, since the rate of release of laminin, a soluble secreted protein endogenous to PC12 cells, is not stimulated by carbachol. This neuronal cell line therefore possesses at least two distinct pathways for secretion and can selectively package a foreign endocrine hormone into the regulated pathway.

Animals↗
PubMed1985

Secretory protein targeting in a pituitary cell line: differential transport of foreign secretory proteins to distinct secretory pathways.

The mouse pituitary cell line, AtT-20, packages the adrenocorticotropic hormone (ACTH) in secretory vesicles and releases it when the cell is stimulated with secretagogues. These cells have the capacity, after transfection with the appropriate DNA, to package heterologous peptide hormones into the regulated secretory vesicles (Moore, H. P. H., M. D. Walker, F. Lee, and R. B. Kelly, 1983, Cell, 35:531-538). To test if other secreted proteins prefer a different route to the surface, we have transfected AtT-20 cells with DNAs coding for a fragment of a membrane protein, the vesicular stomatitis virus G protein from which the membrane spanning domain has been deleted (Rose, J. K., and J. E. Bergmann, 1982, Cell, 17:813-819). We found that the secreted vesicular stomatitis virus G proteins were not transported to the regulated secretory vesicles. Instead they preferentially exited the cell by the constitutive pathway previously found in these cells (Gumbiner, B., and R. B. Kelly, 1982, Cell, 28:51-59). In contrast, human growth hormone transfected into the cells by the same procedure was transported to the regulated pathway with a similar efficiency as the endogenous hormone ACTH. Transport of the secreted G protein to the regulated pathway, if it occurs at all, is at least 30-fold less efficient than peptide hormones. We conclude that the transport machinery in AtT-20 cells must selectively recognize different secreted proteins and sort them into distinct secretory pathways.

Animals↗
PubMed1984

Calmodulin is tightly associated with synaptic vesicles independent of calcium.

A protein in highly purified synaptic vesicles from elasmobranch electric organ is recognized by two specific antisera that recognize different determinants of calmodulin. The protein is indistinguishable from authentic calmodulin by migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence or absence of calcium. It is tightly associated with the intact synaptic vesicle membrane even in the absence of calcium. It is on vesicles rather than membrane contaminants and cytoplasmically oriented since a calmodulin antibody (sheep anti-calmodulin antibody) immunoprecipitates at least 86% of intact synaptic vesicles. Surprisingly, another calmodulin antiserum (rabbit anti-calmodulin serum) specifically precipitates less than 20% of the intact vesicles. This antiserum (rabbit anti-calmodulin serum) also detects 4-15 times less calmodulin immunoreactivity than sheep anti-calmodulin antibody by radioimmunoassay of vesicles solubilized with nondenaturing detergents. The difference essentially disappears if the vesicle calmodulin is solubilized in sodium dodecyl sulfate. We suggest that the antigenic determinant recognized by rabbit anti-calmodulin serum is concealed in vesicle-associated calmodulin and may be involved in binding calmodulin to the vesicle.

Animals↗
PubMed1984

Sorting and secretion of adrenocorticotropin in a pituitary tumor cell line after perturbation of the level of a secretory granule-specific proteoglycan.

A mouse anterior pituitary tumor cell line (AtT-20) that secretes adrenocorticotropin and beta endorphin sorts the proteins it transports to the surface into two exocytotic pathways. AtT-20 cells also synthesize a secretory granule-specific sulfated molecule and secrete it on stimulation (Moore, H.-P., B. Gumbiner, and R. B. Kelly, 1983, J. Cell Biol., 97:810-817). We show here that this molecule is sensitive to proteolysis and that the residual sulfated material co-migrates with a chondroitin sulfate standard on thin-layer electrophoresis. Furthermore, this sulfated molecule is completely sensitive to chondroitinase ABC digestion. Thus the secretory granule-specific sulfated molecule is a proteoglycan with chondroitin sulfate side chains. We examined the role of proteoglycans in the sorting and secretion of adrenocorticotropin in AtT-20 cells by severely decreasing the amount of this vesicle-specific proteoglycan in two ways. First, a xyloside was used to inhibit proteoglycan biosynthesis; second, a variant of the AtT-20 cell line was isolated that synthesized little of the sulfated proteoglycan. In neither case was the sorting or secretion of adrenocorticotropin detectably altered, suggesting that the proteoglycan is not required for these processes.

Adrenocorticotropic Hormone↗
PubMed1983

A highly antigenic proteoglycan-like component of cholinergic synaptic vesicles.

A monoclonal antibody, tor70, recognizes an antigenic determinant on the inside surface of synaptic vesicles, purified from the electric organ of Narcine brasiliensis. The antigenic determinant appears to be unique to vesicles since it co-purifies with vesicle content and is blocked by an antiserum specific for synaptic vesicle antigens. Immunoblotting of vesicle proteins after sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows that the antigen has a low heterogeneous electrophoretic mobility and corresponds to a major protein component of pure synaptic vesicles. Synaptic vesicles contain a proteoglycan-like material since proteolytic digestion yields a ruthenium red-binding material that migrates during electrophoresis with a mammalian heparin standard. The only major vesicle component with which the proteoglycan-like material co-elutes during chromatography on Sepharose 6B is the material recognized by tor70. The antigen adsorbs specifically to beads coated with the lectin wheat germ agglutinin. Isolation of the tor70 antigen by velocity sedimentation in sodium dodecyl sulfate-sucrose gradients shows it to contain glucosamine (0.75 nmol/microgram of protein) and uronic acid but no galactosamine. Earlier work has shown that specific antiserum to pure synaptic vesicles could be used to identify nerve terminals, quantitate vesicle components, purify membranes, and monitor exocytosis. We now know that one of the components recognized by the antiserum is a molecule with properties of a proteoglycan, attached to the inside surface of vesicle membranes.

Animals↗
PubMed1983

Expressing a human proinsulin cDNA in a mouse ACTH-secreting cell. Intracellular storage, proteolytic processing, and secretion on stimulation.

The AtT-20 cell line, derived from the mouse anterior pituitary, synthesizes an adrenocorticotropic hormone (ACTH) precursor, proteolytically processes it to mature ACTH, stores it in secretory granules, and releases mature ACTH on stimulation with a secretagogue. A cDNA for human proinsulin inserted downstream from the SV40 early promoter in an SV40-pBR322 recombinant vector was introduced into AtT-20 cells. The stably transformed cell line, AtT-20ins4b/1, stores immunoreactive insulin, proteolytically processes proinsulin to smaller fragments, and on stimulation with secretagogues releases insulin-like material, not proinsulin, into the medium. Similarly transformed fibroblast L-cells secrete only proinsulin; they do not store it, and their secretion rate is unaffected by secretagogues. The transport mechanism for precursor ACTH thus appears to recognize other prohormones.

Adrenocorticotropic Hormone↗
PubMed1983

A synaptic vesicle antigen is restricted to the junctional region of the presynaptic plasma membrane.

The plasma membrane of electric organ nerve terminals has two domains that can be distinguished by monoclonal antibodies. A library of 111 mouse monoclonal antibodies raised to nerve terminals from Torpedo californica contains 4 antibodies that bind specifically to the outside of intact synaptosomes. The distribution of the binding sites of these monoclonal antibodies on the outside of intact nerve terminals was examined by immunofluorescence and immunoelectron microscopy. The binding sites of 3 (tor23, 25, and 132) are distributed uniformly over nerve trunks and fine terminal branches. The binding site of the fourth (tor70) is restricted to synaptic junctional regions. This antibody, but not the other 3, recognizes a major component of synaptic vesicles, a proteoglycan associated with the inner surface of the vesicle membrane. The difference in the pattern of binding of these monoclonal antibodies suggests that the region of the plasma membrane containing active zones is antigenically distinguishable from other nerve terminal plasma membrane. We suggest that the antigen recognized by tor70 is externalized by exocytosis of synaptic vesicles while other plasma antigens take a different route to the surface. The unexpected observation that the vesicle antigen remains on the surface after exocytosis and is prevented from diffusion from the synaptic junctional region would be consistent with an interaction between the vesicle proteoglycan and elements of the synaptic cleft.

Animals↗
PubMed1983

Identification of three coated vesicle components as alpha- and beta-tubulin linked to a phosphorylated 50,000-dalton polypeptide.

Coated vesicles are involved in the intracellular transport of membrane proteins between a variety of membrane compartments. The coats of bovine brain coated vesicles contain at least six polypeptides in addition to an 180,000-dalton polypeptide called clathrin. In this report we show that the 54,000- and 56,000-dalton coated vesicle polypeptides are alpha- and beta-tubulin, determined by immunoblotting and two-dimensional gel electrophoresis. An affinity-purified tubulin antiserum can precipitate coated vesicles. The tubulin polypeptides are tightly associated with a 50,000-dalton coated vesicle polypeptide, which is phosphorylated. The phosphorylated 50,000-dalton polypeptide appears to be related to brain microtubule-associated tau proteins since it can be specifically immunoprecipitated by an affinity-purified antiserum directed against these proteins. In addition, gel filtration experiments indicate that at least a fraction of the 50,000-dalton polypeptide may associate with the 100,000-dalton coated vesicle polypeptide. Since brain is a tissue rich in tubulins, liver coated vesicles were analyzed for the presence of alpha- and beta-tubulin. Like brain coated vesicles, liver coated vesicles also contain an endogenous kinase activity, which phosphorylates polypeptides of the same molecular weights and isoelectric points as the brain coated vesicle 50,000-dalton, tau-like polypeptide, and alpha- and beta-tubulin. The phosphorylated 50,000-dalton polypeptide may link the membrane and contents of coated vesicles with components of the cytoskeleton.

Adaptor Proteins, Vesicular Transport↗
PubMed1983

A subclass of proteins and sulfated macromolecules secreted by AtT-20 (mouse pituitary tumor) cells is sorted with adrenocorticotropin into dense secretory granules.

The AtT-20 cell, a mouse pituitary tumor line that secretes adrenocorticotropin and beta-endorphin, sorts the proteins it externalizes into two exocytotic pathways. Cells that are labeled with [35S]methionine or [35S]sulfate can be shown to transport three acidic polypeptides (65,000, 60,000, and 37,000 mol wt) and at least two sulfated macromolecules into storage secretory granules. When the cells are stimulated by the secretagogue 8-bromo-cAMP, these polypeptides are coordinately secreted with mature adrenocorticotropin into the culture medium. In contrast, a completely different set of secreted polypeptides and sulfated macromolecules does not enter a storage form and is transported to the cell surface more rapidly. Their secretion from the cells is constitutive and does not require the presence of secretagogues. These molecules, like a viral membrane glycoprotein described previously (Gumbiner, B., and R. B. Kelly, 1982, Cell, 28:51-59) are not found in isolated secretory granules and therefore must reach the cell surface in a different exocytotic vesicle. The segregation of a subclass of secretory macromolecules into the secretory granules, despite the existence of another potential secretory pathway, suggests that these molecules have specific functions related to regulated hormone secretion or storage. Presumably all of the proteins secreted by the regulated secretory granule pathway share some common property that targets them to the secretory granule.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1982

Two distinct intracellular pathways transport secretory and membrane glycoproteins to the surface of pituitary tumor cells.

The pituitary cell line, AtT-20, synthesizes adrenocorticotropic hormone (ACTH) as a glycoprotein precursor that is cleaved into mature hormones during packaging into secretory granules. The cells also produce an endogenous leukemia virus (MuLV) that is glycosylated after translation similar to the glycosylation of the ACTH precursor. Our evidence suggests that the envelope glycoprotein and some precursor ACTH get to the cell surface in a vesicle different from the mature ACTH secretory granule. Viral glycoproteins and ACTH precursor are released from the cells much sooner after synthesis than mature ACTH. Isolated secretory granules do not contain significant amounts of the envelope glycoprotein or ACTH precursor. Exposing cells to 8Br-cAMP stimulates release of mature ACTH four to five fold, but has little effect on the release of the ACTH precursor or the viral glycoproteins. We propose that the viral glycoproteins and some of the ACTH precursor are transported by a constitutive pathway, while mature ACTH is stored in secretory granules where its release is enhanced by stimulation.

Adrenocorticotropic Hormone↗
PubMed1982

Partial purification of presynaptic plasma membrane by immunoadsorption.

During transmitter release, synaptic vesicle membrane is specifically inserted into the nerve terminal plasma membrane only at specialized sites or "active zones." In an attempt to obtain a membrane fraction enriched in active zones, we have utilized the electric organ of the marine ray. From this organ, a fraction enriched in nerve terminals (synaptosomes) was prepared by conventional means. These synaptosomes were bound to microscopic beads by an antiserum to purified electric organ synaptic vesicles (anti-SV). The success of this immunoadsorption procedure was demonstrated by increased specific activities of bead-bound nerve terminal cytoplasmic markers and decreased specific activities of markers for contaminating membranes. To obtain a presynaptic plasma membrane (PSPM) fraction, we lysed the bead-bound synaptosomes by hypoosmotic shock and sonication, resulting in complete release of cytoplasmic markers. When the synaptosomal fraction was surface-labeled with iodine before immunoadsorption, 10% of this label remained bead-bound after lysis, compared with 2% of the total protein, indicating an approximately fivefold enrichment of bead-bound plasma membrane. Concomitantly, the specific activity of bead-bound anti-SV increased approximately 30-fold, indicating an enrichment of plasma membrane which contained inserted synaptic vesicle components. This PSPM preparation is not simply synaptic vesicle membrane since two-dimensional electrophoresis revealed that the polypeptides of the surface-iodinated PSPM preparation include both vesicle and numerous nonvesicle components. Secondly, antiserum to the PSPM fraction is markedly different from anti-SV and binds to external, nonvesicle, nerve terminal components.

Acetylcholine↗
PubMed1982

Nerve terminal components from normal and denervated Narcine electric organ.

Exocytosis at the nerve terminal will only be fully understood when purified synaptic vesicles can be made to fuse with presynaptic plasma membrane in vitro. While the purification of synaptic vesicles from electric organ is now straightforward, isolation of the presynaptic plasma membrane presents a greater difficulty because of lack of specific markers. We have utilized pure synaptic vesicles in a novel way to overcome this difficulty. Antibodies raised to pure synaptic vesicles can be used to detect the presence of vesicle antigens. Thus, for example, we can show that synaptosome preparations isolated from electric organ by conventional procedures have about 5% of their protein in synaptic vesicles. The synaptic vesicle antigens and choline acetyltransferase both disappear from the synaptosome fraction after denervation of the electric organ, verifying that they are nerve terminal specific. Some of the synaptic vesicle antigens can be detected on the outside of the intact synaptosomes by binding rabbit anti-synaptic vesicle antibodies. Such antibody-coated synaptosomes will absorb specifically to goat anti-rabbit IgG attached to polyacrylamide beads. Lysis of such bead-bound synaptosomes leaves a plasma membrane fraction firmly attached to the beads. By raising antibodies to this membrane fraction an antiserum has been generated that binds to antigenic determinants in the electric organ. Some of these antibodies bind to the frog neuromuscular junction. The antigens recognized by this antiserum are, like synaptic vesicle antigens, lost on denervation. Unlike synaptic vesicle antigens, however, the antigens recognized by the anti-plasma membrane serum are present on the outside of resting frog nerve terminals.

Acetylcholinesterase↗
PubMed1981

Lipids of synaptic vesicles: relevance to the mechanism of membrane fusion.

Synaptic vesicles from the electric organ of the marine ray Narcine brasiliensis, purified to at least 90% homogeneity, were analyzed for the lipid and fatty acid content of their membranes. The major lipids (mol %) were phosphatidylcholine (32.3%), phosphatidylethanolamine (20.5%), phosphatidylserine (6.1%), sphingomyelin (3.0%), and cholesterol (33.3%), a composition which did not differ greatly from that of the parent electric organ. While the number of double bonds per fatty acid molecule was similar for both synaptic vesicle and whole electric organ phospholipids, the vesicles were highly enriched in docosahexenoic acid (22:6). Reaction with the amine labeling reagents isethionylacetimidate and trinitrobenzenesulfonic acid indicated that 40% of the phosphatidylserine and 60% of the phosphatidylethanolamine are present on the external (cytoplasmic) surface of the synaptic vesicle. These data on a natural fusing membrane have relevance to models of membrane fusion, which have been based largely on studies of in vitro fusion using synthetic membranes.

Animals↗
PubMed1981

Secretory granules of an anterior pituitary cell line, AtT-20, contain only mature forms of corticotropin and beta-lipotropin.

The pituitary cell line, AtT-20, synthesizes the precursor to corticotropin (adrenocorticotropic hormone; ACTH) and beta-endorphin and correctly glycosylates and cleaves it to make the mature forms of the hormones before they are secreted. This cell line was used to study the intracellular transport, packaging, and secretion of these hormones. Secretory granules from the cells were isolated by homogenization and differential centrifugation and isopycnic sedimentation on a 2H2O-Ficoll gradient to give a preparation having a specific activity of 90micrograms ACTH per mg of protein, which is 30- to 90-fold greater than that of whole cells. The granules have density characteristics and a sedimentation coefficient that are appropriate for spheres of 1000 A radius. They contain all of the fragments of the initial ACTH/endorphin precursor but almost undetectable amounts of the intact precursor. The fragments constitute about 50% of the protein in the secretory granule fraction and, from density measurements, we estimate that they are present in approximately 60,000 copies per vesicle. The cell line secretory granules appear, therefore, to be similar to mature secretory granules in normal differentiated tissues. ACTH first appears in the secretory granule at 30-45 min after synthesis. Cleavage of the precursor to mature ACTH occurs at about the same time in the whole cell. Therefore, proteolysis of the prohormone to ACTH and to beta-lipotropin is a metabolic event that can be correlated with the packaging of the hormone into a mature secretory granule. Cleavage of beta-lipotropin to beta-endorphin occurs later, probably in the secretory granule.

Adrenocorticotropic Hormone↗
PubMed1981

Transfer of synaptic vesicle antigens to the presynaptic plasma membrane during exocytosis.

We have utilized immunofluorescence techniques to look for synaptic vesicle antigens on the plasma membrane of resting and active nerve terminals. Rabbit antiserum was raised against purified cholinergic synaptic vesicles from the electric organ of Narcine brasiliensis, a marine electric ray. Antibodies to synaptic vesicles were shown to bind selectively to nerve terminals in cryostat sections of frog nerve-muscle preparations. Binding was demonstrated indirectly by using fluorescein-labeled goat anti-rabbit antibodies. Structures in cross sections that bound antiserum were identified as nerve terminals because of their size, shape, and position and because they coincided with sites that bound rhodamine-conjugated alpha-bungarotoxin and had acetylcholine esterase activity. Presumably, sectioning gave antibodies access to binding sites within the nerve terminal. However, when antibodies to synaptic vesicles were added to the bathing medium of intact neuromuscular preparations prior to sectioning, antibody binding was marginal or undetectable, suggesting that few vesicle antigens were normally accessible on the outer surface of resting nerve terminals. When intact preparations were stimulated to release their vesicular acetylcholine by the addition of 1 mM LaCl3, antibody binding to the intact nerve terminals became striking. These findings suggest that the synaptic vesicle membrane and the synaptic terminal plasma membrane differ in composition. They also provide further support for the exocytotic hypothesis of neurotransmitter release, which predicts that vesicle markers should be exposed on the outside of nerve terminals when vesicles fuse with the plasma membrane during stimulation.

Animals↗