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Hsiaoping H Moore - One of the best experts on this subject based on the ideXlab platform.
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Biosynthesis and Secretion of Pituitary Hormones: Dynamics and Regulation
Archives of physiology and biochemistry, 2002Co-Authors: Hsiaoping H Moore, J. M. Andresen, Benjamin A. Eaton, Michael Grabe, Michael Haugwitz, Terry E. MachenAbstract:Production and secretion of hormones by the pituitary involve highly orchestrated intraCellular transport and sorting steps. Hormone precursors are routed through a series of compartments before being packaged in secretory granules. These highly dynamic carriers play crucial roles in both prohormone processing and peptide exocytosis. We have employed the ACTH-secreting AtT-20 Cell Line to study the membrane sorting events that confer functionality (prohormone activation and regulated exocytosis) to these secretory carriers. The unique ability of granules to promote prohormone processing is attributed to their acidic interior. Using a novel avidin-targeted fluorescence ratio imaging technique, we have found that the trans-Golgi of live AtT-20 Cells maintains a mildly acidic (approximately pH 6.2) interior. Budding of secretory granules causes the lumen to acidify to
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Biogenesis of Regulated Exocytotic Carriers in Neuroendocrine Cells
The Journal of neuroscience : the official journal of the Society for Neuroscience, 2000Co-Authors: Benjamin A. Eaton, Michael Haugwitz, Dana Lau, Hsiaoping H MooreAbstract:Ca2+-triggered exocytosis is a hallmark of neurosecretory granules, but the Cellular pathway leading to the assembly of these regulated exocytotic carriers is poorly understood. Here we used the pituitary AtT-20 Cell Line to study the biogenesis of regulated exocytotic carriers involved in peptide hormone secretion. We show that immature secretory granules (ISGs) freshly budded from the trans -Golgi network (TGN) exhibit characteristics of unregulated exocytotic carriers. During a subsequent maturation period they undergo an important switch to become regulated exocytotic carriers. We have identified a novel sorting pathway responsible for this transition. The SNARE proteins, VAMP4 and synaptotagmin IV (Syt IV), enter ISGs initially but are sorted away during maturation. Sorting is achieved by vesicle budding from the ISGs, because it can be inhibited by brefeldin A (BFA). Inhibition of this sorting pathway with BFA arrested the maturing granules in a state that responded poorly to stimuli, suggesting that the transition to regulated exocytotic carriers requires the removal of a putative inhibitor. In support of this, we found that overexpression of Syt IV reduced the stimulus-responsiveness of maturing granules. We conclude that secretory granules undergo a switch from unregulated to regulated secretory carriers during biogenesis. The existence of such a switch may provide a mechanism for Cells to modulate their secretory activities under different physiological conditions.
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Analysis of Constitutive and Constitutive-like Secretion in Semi-intact Pituitary Cells
Methods (San Diego Calif.), 1998Co-Authors: Eric Dumermuth, Hsiaoping H MooreAbstract:To study biosynthetic transport through the constitutive and regulated secretory pathways, we have designed a semi-intact mammalian Cell system that restores the transport of secretory proteins from the trans-Golgi/trans-Golgi network (TGN) to the Cell surface. The mouse pituitary AtT-20 Cell Line is a suitable model to biochemically analyze molecular sorting in the secretory pathway. The prohormone proopiomelanocortin is sulfated on N-linked carbohydrate chains in the trans-Golgi prior to proteolytic processing in the secretory granule. Radiolabeling with [35S]sulfate therefore provides a convenient tool to selectively follow molecular events in the regulated secretory pathway without interference from earlier steps. Likewise, transport through the constitutive secretory pathway may be monitored using sulfate-labeled glycosaminoglycan chains. We show that export from the TGN is efficiently reconstituted in Cells made semi-intact with streptolysin O, and is dependent on temperature, ATP and GTP hydrolysis, and cytosol. Packaging of proopiomelanocortin into immature secretory granules also activates the proteolytic processing machinery which eventually converts the prohormone to its bioactive mature product, adrenocorticotropic hormone. In addition, a large fraction of incompletely processed proopiomelanocortin is secreted as the processing intermediates from immature secretory granules. This process of constitutive-like secretion can be clearly distinguished from direct constitutive secretion from the trans-Golgi network by kinetic and compositional criteria. Furthermore, we have found that specific inhibitors of different protein phosphatases and kinases are potent blockers of constitutive and constitutive-like secretion. This experimental model should provide a valuable system to elucidate the molecular mechanism regulating post-Golgi traffic during secretory granule biogenesis.
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ionic milieu controls the compartment specific activation of pro opiomelanocortin processing in att 20 Cells
Molecular Biology of the Cell, 1995Co-Authors: Walter K Schmidt, Hsiaoping H MooreAbstract:Newly synthesized prohormones and their processing enzymes transit through the same compartments before being packaged into regulated secretory granules. Despite this coordinated intraCellular transport, prohormone processing does not occur until late in the secretory pathway. In the mouse pituitary AtT-20 Cell Line, conversion of pro-opiomelanocortin (POMC) to mature adrenocorticotropic hormone involves the prohormone convertase PC1. The mechanism by which this proteolytic processing is restricted to late secretory compartments is unknown; PC1 activity could be regulated by compartment-specific activators/inhibitors, or through changes in the ionic milieu that influence its activity. By arresting transport in a semi-intact Cell system, we have addressed whether metabolically labeled POMC trapped in early secretory compartments can be induced to undergo conversion if the ionic milieu in these compartments is experimentally manipulated. Prolonged incubation of labeled POMC trapped in the endoplasmic reticulum or Golgi/trans-Golgi network did not result in processing, thereby supporting the theory that processing is normally a post-Golgi/trans-Golgi network event. However, acidification of these compartments allowed effective processing of POMC to the intermediate and mature forms. The observed processing increased sharply at a pH below 6.0 and required millimolar calcium, regardless of the compartment in which labeled POMC resided. These conditions also resulted in the coordinate conversion of PC1 from the 84/87 kDa into the 74-kDa and 66-kDa forms. We propose that POMC processing is predominantly restricted to acidifying secretory granules, and that a change in pH within these granules is both necessary and sufficient to activate POMC processing.
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Synthesis and Targeting of Insulin-like Growth Factor-I to the Hormone Storage Granules in an Endocrine Cell Line*
The Journal of biological chemistry, 1994Co-Authors: Walter K Schmidt, Hsiaoping H MooreAbstract:Abstract Export of growth factors is generally believed to be restricted to the constitutive secretory pathway, whereas peptide hormones are typically secreted in a regulated manner. Here we show that insulin-like growth factor (IGF)-I, a growth factor released constitutively from the liver, is synthesized and secreted from the mouse pituitary AtT-20 Cell Line via the regulated pathway. IGF-I production is 1500-fold less than the peptide hormone ACTH. Secretagogue induces IGF-I secretion in a manner similar to ACTH. Like ACTH, IGF-I is sorted into the regulated pathway >35-fold more efficiently than a constitutively secreted protein. Dense core granules isolated from Cells transfected with a human IGF-I cDNA contain both ACTH and human IGF-I. AtT-20 Cells also synthesize IGF-binding proteins, and at least one of these is secreted by the regulated pathway. Human IGF-I does not exhibit milieu-induced, concentration-dependent aggregation, in contrast to secretogranin II which sorts by a proposed aggregation mechanism. These data suggest that 1) growth factors are not solely released from tissues via the constitutive pathway, 2) IGF-I may contain information for correct granular targeting, and 3) IGF-I may be sorted by a mechanism distinct from that proposed for the secretogranins.
Walter K Schmidt - One of the best experts on this subject based on the ideXlab platform.
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ionic milieu controls the compartment specific activation of pro opiomelanocortin processing in att 20 Cells
Molecular Biology of the Cell, 1995Co-Authors: Walter K Schmidt, Hsiaoping H MooreAbstract:Newly synthesized prohormones and their processing enzymes transit through the same compartments before being packaged into regulated secretory granules. Despite this coordinated intraCellular transport, prohormone processing does not occur until late in the secretory pathway. In the mouse pituitary AtT-20 Cell Line, conversion of pro-opiomelanocortin (POMC) to mature adrenocorticotropic hormone involves the prohormone convertase PC1. The mechanism by which this proteolytic processing is restricted to late secretory compartments is unknown; PC1 activity could be regulated by compartment-specific activators/inhibitors, or through changes in the ionic milieu that influence its activity. By arresting transport in a semi-intact Cell system, we have addressed whether metabolically labeled POMC trapped in early secretory compartments can be induced to undergo conversion if the ionic milieu in these compartments is experimentally manipulated. Prolonged incubation of labeled POMC trapped in the endoplasmic reticulum or Golgi/trans-Golgi network did not result in processing, thereby supporting the theory that processing is normally a post-Golgi/trans-Golgi network event. However, acidification of these compartments allowed effective processing of POMC to the intermediate and mature forms. The observed processing increased sharply at a pH below 6.0 and required millimolar calcium, regardless of the compartment in which labeled POMC resided. These conditions also resulted in the coordinate conversion of PC1 from the 84/87 kDa into the 74-kDa and 66-kDa forms. We propose that POMC processing is predominantly restricted to acidifying secretory granules, and that a change in pH within these granules is both necessary and sufficient to activate POMC processing.
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Synthesis and Targeting of Insulin-like Growth Factor-I to the Hormone Storage Granules in an Endocrine Cell Line*
The Journal of biological chemistry, 1994Co-Authors: Walter K Schmidt, Hsiaoping H MooreAbstract:Abstract Export of growth factors is generally believed to be restricted to the constitutive secretory pathway, whereas peptide hormones are typically secreted in a regulated manner. Here we show that insulin-like growth factor (IGF)-I, a growth factor released constitutively from the liver, is synthesized and secreted from the mouse pituitary AtT-20 Cell Line via the regulated pathway. IGF-I production is 1500-fold less than the peptide hormone ACTH. Secretagogue induces IGF-I secretion in a manner similar to ACTH. Like ACTH, IGF-I is sorted into the regulated pathway >35-fold more efficiently than a constitutively secreted protein. Dense core granules isolated from Cells transfected with a human IGF-I cDNA contain both ACTH and human IGF-I. AtT-20 Cells also synthesize IGF-binding proteins, and at least one of these is secreted by the regulated pathway. Human IGF-I does not exhibit milieu-induced, concentration-dependent aggregation, in contrast to secretogranin II which sorts by a proposed aggregation mechanism. These data suggest that 1) growth factors are not solely released from tissues via the constitutive pathway, 2) IGF-I may contain information for correct granular targeting, and 3) IGF-I may be sorted by a mechanism distinct from that proposed for the secretogranins.
Shuyang Wang - One of the best experts on this subject based on the ideXlab platform.
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Role of Vasopressin Receptor 2 and 3 in ACTH-Secreting Tumors and their Potential Therapeutic Implications.
Experimental and Clinical Endocrinology & Diabetes, 2019Co-Authors: Jia Yang, Yehong Yang, Yongfei Wang, Shuo Zhang, Hai-xia Cheng, Shuyang WangAbstract:Purpose We investigated the expression of vasopressin receptor 2 and 3 on corticotrophin tumor Cells, their role in regulating ACTH secretion, and their potential therapeutic implications. Methods We retrospectively assessed 52 hospitalized patients with pathologically confirmed ACTH-secreting tumors. The expression of vasopressin receptor 2 and 3 was explored via qualitative and quantitative immunohistochemistry analyses. The role of vasopressin receptors in regulating ACTH secretion was further studied in the AtT-20 Cell Line. Results Among 50 cases of pituitary corticotrophin adenoma, 31 were vasopressin receptor 2 positive, 38 were vasopressin receptor 3 positive, and 24 were both vasopressin receptor 2 and 3 positive. Two patients with ectopic ACTH syndrome were vasopressin receptor 3 positive, and one was also vasopressin receptor 2 positive. In 12 patients who underwent bilateral inferior petrosal sinus sampling before surgery, the central ACTH increment ratio after desmopressin stimulation was correlated with vasopressin receptor 2 but not with vasopressin receptor 3 staining intensity. In an in vitro study, the expression of both vasopressin receptor 2 and 3 on AtT-20 Cells was confirmed. The vasopressin receptor 2 antagonist Tolvaptan inhibited desmopressin-induced ACTH secretion in a dose-dependent manner. Conclusions Both vasopressin receptor 2 and 3 are expressed in ACTH-secreting tumors. Vasopressin receptor 2 rather than vasopressin receptor 3 is the primary receptor that seems to mediate the ACTH response in corticotrophin tumors. A vasopressin receptor 2 antagonist can inhibit ACTH secretion induced by desmopressin in AtT-20 Cells.
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Role of Vasopressin Receptor 2 and 3 in ACTH-Secreting Tumors and their Potential Therapeutic Implications.
Experimental and clinical endocrinology & diabetes : official journal German Society of Endocrinology [and] German Diabetes Association, 2019Co-Authors: Jia Yang, Yehong Yang, Yongfei Wang, Shuo Zhang, Hai-xia Cheng, Shuyang WangAbstract:We investigated the expression of vasopressin receptor 2 and 3 on corticotrophin tumor Cells, their role in regulating ACTH secretion, and their potential therapeutic implications. We retrospectively assessed 52 hospitalized patients with pathologically confirmed ACTH-secreting tumors. The expression of vasopressin receptor 2 and 3 was explored via qualitative and quantitative immunohistochemistry analyses. The role of vasopressin receptors in regulating ACTH secretion was further studied in the AtT-20 Cell Line. Among 50 cases of pituitary corticotrophin adenoma, 31 were vasopressin receptor 2 positive, 38 were vasopressin receptor 3 positive, and 24 were both vasopressin receptor 2 and 3 positive. Two patients with ectopic ACTH syndrome were vasopressin receptor 3 positive, and one was also vasopressin receptor 2 positive. In 12 patients who underwent bilateral inferior petrosal sinus sampling before surgery, the central ACTH increment ratio after desmopressin stimulation was correlated with vasopressin receptor 2 but not with vasopressin receptor 3 staining intensity. In an in vitro study, the expression of both vasopressin receptor 2 and 3 on AtT-20 Cells was confirmed. The vasopressin receptor 2 antagonist Tolvaptan inhibited desmopressin-induced ACTH secretion in a dose-dependent manner. Both vasopressin receptor 2 and 3 are expressed in ACTH-secreting tumors. Vasopressin receptor 2 rather than vasopressin receptor 3 is the primary receptor that seems to mediate the ACTH response in corticotrophin tumors. A vasopressin receptor 2 antagonist can inhibit ACTH secretion induced by desmopressin in AtT-20 Cells. © Georg Thieme Verlag KG Stuttgart · New York.
R. Verhoeven - One of the best experts on this subject based on the ideXlab platform.
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Selection of Glucocorticoid-Resistant Mutations from an AtT-20 Cell Line Containing a Glucocorticoid-Regulated Selectable Transgene
Biochemical and biophysical research communications, 1995Co-Authors: R.w. Harrison, S.s. Lippman, R. VerhoevenAbstract:AtT-20/IDG8 Cells contain the stably transfected, selectable gene, neomycin phosphotransferase, under negative glucocorticoid regulation. Thus, when cultured in the simultaneous presence of the neomycin analogue, G418, and dexamethasone, AtT-20/IDG8 Cells fail to grow. Our hypothesis was that mutated AtT-20/IDG8 Cells capable of growth in such medium would have a defect in the glucocorticoid-mediated regulation of the neor gene. AtT-20/IDG8 Cells were chemically mutagenized using ethyl-methane sulfonate and cloned in the presence of G418 and dexamethasone. Fourteen clones were obtained and loss of glucocorticoid control of neor expression was confirmed in them all. The naturally occurring gene, pro-opiomelanocortin, which is down-regulated by glucocorticoids in parent AtT-20/IDG8 Cells, was down-regulated by dexamethasone in ten of the mutant Lines, indicating that in those Cells the receptor was functional in spite of aberrant regulation of neor. In the other four Lines, pro-opiomelanocortin regulation was lost, also suggesting that a general transcription factor, such as the receptor, had been altered. These results indicate that multiple factors are involved in glucocorticoid-mediated gene regulation and that new, informative mutations can be produced after insertion of a regulated, selectable gene into a previously non-selectable Cell Line.
Lloyd D. Fricker - One of the best experts on this subject based on the ideXlab platform.
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Examination of the rate of peptide biosynthesis in neuroendocrine Cell Lines using a stable isotopic label and mass spectrometry.
Journal of neurochemistry, 2004Co-Authors: Fa Yun Che, Elena Kalinina, Quan Yuan, Lloyd D. FrickerAbstract:The biosynthesis of neuroendocrine peptides is typically examined by following the rate of appearance of a radioactive amino acid into mature forms of peptides. In the present study, we labeled Cell Lines with L-leucine containing 10 deuterium residues (d10-Leu) and used mass spectrometry to measure the biosynthetic rate of c-lipotropin in the AtT-20 Cell Line and insulin in the INS-1 Cell Line. After 3 h of labeling, both peptides show detectable levels of the d-labeled form in the Cells and media. The relative levels of the d-labeled forms are greater in the media than in the Cells, consistent with previous studies that found that newly synthesized peptides are secreted at a higher rate than older peptides under basal conditions. When AtT-20 Cells were stimulated with KCl or forskolin, the ratio of d- to H-labeled c-lipotropin in the medium decreased, suggesting that the older peptide was in a compartment that could be released upon the appropriate stimulation. Overexpression of proSAAS in AtT-20 Cells reduced the ratio of d- to H-labeled c-lipotropin, consistent with the proposed role of proSAAS as an endogenous inhibitor of prohormone convertase-1. Labeling with d10-Leu was also used to test whether altering the pH of the secretory pathway with chloroquine affected the rate of peptide biosynthesis. In AtT-20 Cells, 30 lM chloroquine for 3 or 6 h significantly reduced the rate of formation of c-lipotropin in both Cells and media. Similarly, INS-1 Cells treated with 10, 30, or 60 lM chloroquine for 6 h showed a significant decrease in the rate offormation ofinsulininbothCellsandmedia. Theseresultsare consistent with the acidic pH optima for peptide processing enzymes. Stable isotopic labeling with d10-Leu provides a sensitive method to examine the rate of peptide formation in
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Analysis of the carboxypeptidase D cytoplasmic domain: Implications in intraCellular trafficking.
Journal of cellular biochemistry, 2002Co-Authors: Elena Kalinina, Oleg Varlamov, Lloyd D. FrickerAbstract:Metallocarboxypeptidase D (CPD) is a type 1 transmembrane protein that functions in the processing of proteins that transit the secretory pathway. Previously, CPD was found to be enriched in the trans Golgi network (TGN) and to cycle between this compartment and the Cell surface. In the present study, the roles of specific regions of the CPD cytosolic tail in intraCellular trafficking were investigated in the AtT-20 Cell Line. When the CPD transmembrane region and cytosolic tail are attached to the C-terminus of albumin, this protein is retained in the TGN and cycles to the Cell surface. Deletion analysis indicates that a C-terminal region functions in TGN-retention; removal of 10 amino acids from the C-terminus greatly increases the amount of fusion protein that enters nascent vesicles, which bud from the Golgi, but does not affect the half-life of the fusion protein or the ability of Cell surface protein to return to the TGN. Because the 10-residue deletion disrupts a casein kinase 2 (CK2) consensus site, the two Thr in this site (TDT) were mutated to either Ala (ADA) or Glu (EDE). Neither mutation has an increased rate of budding from the TGN, although the ADA mutant has a shorter half-life than either the wild type sequence or the EDE mutant. Adaptor protein-1 and -2 bind to most of the deletion mutants, the EDE point mutant, and the CK2-phosphorylated CPD tail, but not to the wild type tail. Taken together, these results suggest that CPD localization to the TGN requires both static retention involving the C-terminal domain and phosphorylation at a CK2 site, which regulates the binding of adaptor proteins.