Constitutive and basal secretion from the endocrine cell line, AtT-20.

Matsuuchi, Linda; Kelly, Regis B.

doi:10.1083/jcb.112.5.843

Cited by 95 publications

(64 citation statements)

References 51 publications

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“…In contrast, ACTH product had ϳ10-fold increase in secretion after stimulation with 8-Br-cAMP. These results are consistent with other studies (13,18,28) and indicate that the ABI is secreted via a constitutive-like process, while ACTH is secreted in a regulated manner (28).As shown in Table II, all the stable cell lines displayed A, endogenous expression of TRIP8b in AtT20 was determined by indirect immunofluorescence using a TRIP8b-specific antibody. Arrowheads indicate the localization of TRIP8b at the cell tips.…”

supporting

confidence: 80%

Rab8b and Its Interacting Partner TRIP8b Are Involved in Regulated Secretion in AtT20 Cells

Chen¹,

Liang²,

Chia³

et al. 2001

Journal of Biological Chemistry

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Rab proteins are a family of small GTPases that regulate intracellular vesicle traffic. Rab8b, because of its homology with Rab8, has been suggested to function in vesicle transport to the plasma membrane. Using the yeast two-hybrid system, we identified a Rab8b interacting clone, termed TRIP8b, from a rat brain cDNA library. The gene encodes a 66-kDa protein with homology to the peroxisomal targeting signal 1 receptor. The interaction between Rab8b and TRIP8b was further verified by in vitro binding assays and co-immunoprecipitation studies. Additional experiments with Rab8b mutants demonstrated that Rab8b requires a guanine nucleotide but not prenylation for its interaction with TRIP8b. Western immunoblot analysis showed that TRIP8b was primarily expressed in brain. Subcellular fractionation of AtT20 cells revealed that TRIP8b was present in both cytosolic and membrane fractions. To investigate the function of Rab8b and TRIP8b in secretion, we examined the release of ACTH from AtT20 cells. Results from stable cell lines expressing Rab8b or TRIP8b indicated that both proteins had a stimulatory effect on cAMP-induced secretion of ACTH. In summary, these data suggest that Rab8b and TRIP8b interact with each other and are involved in the regulated secretory pathway in AtT20 cells.Rab proteins are a family of small GTP-binding proteins that are important regulators of vesicle transport (1-3). They undergo nucleotide exchange to establish the active GTP-bound form and are incorporated onto transport vesicles either during or after vesicle formation. The GTP-bound form of Rab proteins recruit effectors, either directly or indirectly, to target vesicles to the appropriate sites on acceptor membranes. These effectors include motor proteins which link the vesicles to the cytoskeleton (4, 5), docking complexes which are recruited from the cytosol or tethering factors that mediate the initial contact of membranes that are destined to fuse (6, 7).To date, about 40 distinct Rab proteins have been identified and each is believed to be specifically associated with a particular vesicle transport pathway (2,8). However, only a fraction of known Rab proteins have their functions characterized in detail. Among these, Rab8 (which we term Rab8a) has been shown to be a key regulator of constitutive polarized vesicle transport to the dendrites in the neurons or to the basolateral membrane in epithelial cells (9 -11). Using immunofluorescence and electron microscopy, Rab8a is localized to the Golgi region, cytoplasmic vesicular structures, and the plasma membrane of Madine-Darby canine kidney epithelial cells. When the wild type and dominant active mutant forms of Rab8a are overexpressed in baby hamster kidney fibroblast cells, a dramatic change in cell morphology occurs. The cells form elongated processes as a result of a reorganization of both their actin filaments and microtubules. In this case, newly synthesized vesicular stomatitis virus, a basolateral marker protein is preferentially delivered into these cell outgrowths.While th...

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supporting

confidence: 80%

Rab8b and Its Interacting Partner TRIP8b Are Involved in Regulated Secretion in AtT20 Cells

Chen¹,

Liang²,

Chia³

et al. 2001

Journal of Biological Chemistry

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“…Nonetheless, this direct release mechanism could represent a "basal secretion pathway" of the resting cell described by Matsuuchi and Kelly. 24 The sequence of events in vivo reported here shows many similarities but also some differences in either the description or interpretation of those that occur during the release of vWF from endothelial cells in culture.…”

Section: Discussionmentioning

confidence: 95%

Morphological alterations in endothelial cells associated with the release of von Willebrand factor after thrombin generation in vivo.

Richardson

Tinlin

et al. 1994

Arterioscler Thromb

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von Willebrand factor (vWF) is synthesized by endothelial cells and stored in endothelium-specific granules, the Weibel-Palade (WP) bodies. The release of vWF from endothelial cells in vitro in response to secretagogues such as thrombin is considered to result in the loss of WP bodies through the fusion of the WP bodies with the plasma membrane. Biochemical and morphological techniques, including transmission (TEM) and scanning (SEM) electron microscopy, were used to examine the plasma profile of vWF in parallel with morphological alterations in endothelial cells associated with the generation of thrombin in vivo. There was a rapid loss of high-molecular-weight multimers of the circulating vWF, with full recovery within 1 hour. Simultaneously, TEM demonstrated that the endothelial cells lost WP bodies and became V on Willebrand factor (vWF) is a large multimeric plasma protein that plays a central role in hemostasis. 12 It is synthesized in megakaryocytes 34 and endothelial cells 56 and stored in the a-granules of platelets 7 and in the endothelial cell storage organelles, the Weibel-Palade (WP) bodies. 8 P-selectin is a component of both the platelet a-granule and WP body membrane, 9 but other components of the a-granules have not been identified in WP bodies. 10 vWF is released from endothelial cells both constitutively and via the regulated pathway from WP bodies after endothelial cell stimulation." Release from WP bodies makes available the highly platelet-interactive, high-to abnormally high-molecular-weight multimeric forms of vWF. 2 vWF is released from endothelial cells in culture in response to stimulation by a variety of secretagogues, including thrombin.1112 It is generally considered that the mechanism of release involves the fusion of the limiting membrane of the WP bodies with either the luminal or abluminal membranes of the endothelial cell. This process has been inferred from observation in two settings. In vitro studies, using immunofluorescence 8 " 13 or electron mi- 10 demonstrated that the release of vWF was associated with a reduction in the numbers of WP bodies and the appearance of vWF in the supernatant. In studies in vivo, WP bodies appeared to release their contents into the circulation after fusing with the luminal membrane of endothelial cells.14 Although this observation was reported after the recognition that WP bodies stored vWF, 8 this was not appreciated by the authors, who considered the observation to indicate that WP bodies were secretory in nature but of unknown function.14 To our knowledge, there has been no in vivo study in which plasma vWF levels have been quantified in parallel with alterations in the WP bodies and vWF distribution within endothelial cells.We have developed a model for the induction of thrombin generation in vivo in various animal species. This is achieved through the infusion of two defined components of the prothrombinase complex 15 : factor Xa (activated factor X [FXa]) and a coagulant active phospholipid surface provided by phosphatidylchol...

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“…The transfection experiments were extended to a nonlymphoid cell line to determine whether B-cell-specific components other than monomer IgM and J chain contributed to IgM polymerization and secretion. The murine AtT20 pituitary line was selected for these experiments because the assembly and intracellular trafficking of immunoglobulin in these cells had been shown to resemble that in B cells (13). An AtT20 clone, WT,A5, cotransfected with plasmids containing p,-and A-chain sequences under the control of a Rous sarcoma virus (RSV) long terminal repeat (LTR) (13) was used to introduce a J-chain cDNA also under the control of a RSV LTR.…”

Section: Resultsmentioning

confidence: 99%

“…WT,uA 5, a G418-resistant variant of the corticotropinsecreting pituitary cell line AtT-20, was transfected with pSVJneoEp. and the hygromycin selection plasmid pHS-53 by calcium phosphate precipitation (13). Transfectants were maintained in medium containing hygromycin (175 ,ug/ml; Calbiochem) and/or G418 sulfate (500 Ag/ml; GIBCO/BRL).…”

Section: Methodsmentioning

confidence: 99%

Polymer IgM assembly and secretion in lymphoid and nonlymphoid cell lines: evidence that J chain is required for pentamer IgM synthesis.

Niles

Matsuuchi

Koshland

1995

Proc. Natl. Acad. Sci. U.S.A.

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The requirements for IgM assembly and secretion were evaluated by introducing a constitutively expressed J-chain cDNA into lymphoid and nonlymphoid cell lines expressing the secretory form of monomer IgM. Assays of cell lysates and supernatants showed that only secretory monomer IgM is required for the synthesis and secretion of hexamer IgM, whereas J chain, as well as the secreted form of monomer, is required for the synthesis and secretion of pentamer IgM. Moreover, J chain facilitates the polymerization process so that pentamer IgM is preferentially synthesized. Other components of the polymerization process were found to be shared by all the cell lines examined, whether the cells were of lymphoid or nonlymphoid origin and had a rudimentary or developed secretory apparatus. These results identify monomer IgM and J chain as the two components that determine the B-cell-specific expression of IgM antibodies and, thus, as the appropriate targets for therapeutic regulation of IgM responses.In a primary immune response, a resting B cell is triggered by antigen and successive cytokine signals to differentiate into a blast cell that secretes pentamer IgM antibody. Analyses of the response have identified the de novo synthesis of the pentamer joining protein, the J chain, as a critical step in the differentiative process (1). The evidence is based in part on structural studies which revealed that J chain is incorporated exclusively into the pentameric form of IgM (2). Within the pentamer it links two monomer subunits through disulfide bonds between cysteines at positions 14 and 68 of the J chain and the penultimate cysteines of opposing p. heavy chains (3, 4).Evidence for the role of J chain is also based on the responses of B-cell lines to J-chain gene expression. B lymphomas expressing receptors for interleukin 2 or 5 can be induced by cytokine treatment to amplify J-chain gene transcription and, as a consequence, to markedly increase secretion of IgM (5-7). Moreover, B-cell lymphoma lines can be converted to IgM secretors by transfection with a J-chain cDNA (2) or by fusion with an IgG-secreting myeloma that expresses the J chain (8, 9).Although the expression of J chain correlates strongly with pentamer IgM synthesis, several questions remain concerning the actual function of the J polypeptide in the polymerization process. First, is J chain required for pentamer assembly, or does it simply facilitate closing of the polymer at the pentamer stage? Second, does the expression of J chain in turn induce changes in other B-cell components that contribute to IgM assembly and secretion? These questions were addressed in the present study by transfecting the murine J-chain cDNA into a series of B lymphomas representing successive stages in B-cell differentiation and transfecting all three pentamer components into a nonlymphoid cell line. Here we present evidence that (i) J chain is required for the synthesis of the pentamer, but not the hexamer form of IgM, (ii) its expression is regulated independently of the oth...

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Constitutive and basal secretion from the endocrine cell line, AtT-20.

Cited by 95 publications

References 51 publications

Rab8b and Its Interacting Partner TRIP8b Are Involved in Regulated Secretion in AtT20 Cells

Rab8b and Its Interacting Partner TRIP8b Are Involved in Regulated Secretion in AtT20 Cells

Morphological alterations in endothelial cells associated with the release of von Willebrand factor after thrombin generation in vivo.

Polymer IgM assembly and secretion in lymphoid and nonlymphoid cell lines: evidence that J chain is required for pentamer IgM synthesis.

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