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

1983 ◽  
Vol 97 (3) ◽  
pp. 810-817 ◽  
Author(s):  
H P Moore ◽  
B Gumbiner ◽  
R B Kelly
Keyword(s):  
Cell Surface ◽  
Secretory Granule ◽  
Pituitary Tumor ◽  
Common Property ◽  
Secretory Pathway ◽  
Culture Medium ◽  
Secretory Granules ◽  
Hormone Secretion ◽  
Tumor Line ◽  
Mouse Pituitary

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.

scholarly journals Differential trafficking of soluble and integral membrane secretory granule-associated proteins

1994 ◽  
Vol 124 (1) ◽  
pp. 33-41 ◽  
Author(s):  
SL Milgram ◽  
BA Eipper ◽  
RE Mains
Keyword(s):  
Cell Surface ◽  
Secretory Granule ◽  
Secretory Pathway ◽  
Secretory Granules ◽  
Posttranslational Processing ◽  
Endoproteolytic Cleavage ◽  
Associated Proteins ◽  
Storage Granules ◽  
Regulated Secretory Pathway ◽  
And Storage

The posttranslational processing enzyme peptidylglycine alpha-amidating monooxygenase (PAM) occurs naturally in integral membrane and soluble forms. With the goal of understanding the targeting of these proteins to secretory granules, we have compared the maturation, processing, secretion, and storage of PAM proteins in stably transfected AtT-20 cells. Integral membrane and soluble PAM proteins exit the ER and reach the Golgi apparatus with similar kinetics. Biosynthetic labeling experiments demonstrated that soluble PAM proteins were endoproteolytically processed to a greater extent than integral membrane PAM; this processing occurred in the regulated secretory pathway and was blocked by incubation of cells at 20 degrees C. 16 h after a biosynthetic pulse, a larger proportion of soluble PAM proteins remained cell-associated compared with integral membrane PAM, suggesting that soluble PAM proteins were more efficiently targeted to storage granules. The nonstimulated secretion of soluble PAM proteins peaked 1-2 h after a biosynthetic pulse, suggesting that release was from vesicles which bud from immature granules during the maturation process. In contrast, soluble PAM proteins derived through endoproteolytic cleavage of integral membrane PAM were secreted in highest amount during later times of chase. Furthermore, immunoprecipitation of cell surface-associated integral membrane PAM demonstrated that very little integral membrane PAM reached the cell surface during early times of chase. However, when a truncated PAM protein lacking the cytoplasmic tail was expressed in AtT-20 cells, > 50% of the truncated PAM-1 protein reached the cell surface within 3 h. We conclude that the trafficking of integral membrane and soluble secretory granule-associated enzymes differs, and that integral membrane PAM proteins are less efficiently retained in maturing secretory granules.

scholarly journals Targeted Ablation of the Chromogranin A (Chga) Gene: Normal Neuroendocrine Dense-Core Secretory Granules and Increased Expression of Other Granins

2006 ◽  
Vol 20 (8) ◽  
pp. 1935-1947 ◽  
Author(s):  
Geoffrey N. Hendy ◽  
Tong Li ◽  
Martine Girard ◽  
Richard C. Feldstein ◽  
Shree Mulay ◽  
...  
Keyword(s):  
Secretory Granule ◽  
Chromogranin A ◽  
Chromaffin Cells ◽  
Secretory Granules ◽  
Hormone Secretion ◽  
Proteolytic Processing ◽  
Dense Core ◽  
Secretory Process ◽  
Chromogranin B ◽  
Developmental Abnormalities

Abstract Chromogranin A (CgA), originally identified in adrenal chromaffin cells, is a member of the granin family of acidic secretory glycoproteins that are expressed in endocrine cells and neurons. CgA has been proposed to play multiple roles in the secretory process. Intracellularly, CgA may control secretory granule biogenesis and target neurotransmitters and peptide hormones to granules of the regulated pathway. Extracellularly, peptides formed as a result of proteolytic processing of CgA may regulate hormone secretion. To investigate the role of CgA in the whole animal, we created a mouse mutant null for the Chga gene. These mice are viable and fertile and have no obvious developmental abnormalities, and their neural and endocrine functions are not grossly impaired. Their adrenal glands were structurally unremarkable, and morphometric analyses of chromaffin cells showed vesicle size and number to be normal. However, the excretion of epinephrine, norepinephrine, and dopamine was significantly elevated in the Chga null mutants. Adrenal medullary mRNA and protein levels of other dense-core secretory granule proteins including chromogranin B, and secretogranins II to VI were up-regulated 2- to 3-fold in the Chga null mutant mice. Hence, the increased expression of the other granin family members is likely to compensate for the Chga deficiency.

scholarly journals Coordinate, equimolar secretion of smaller peptide products derived from pro-ACTH/endorphin by mouse pituitary tumor cells.

1981 ◽  
Vol 89 (1) ◽  
pp. 21-28 ◽  
Author(s):  
R E Mains ◽  
B A Eipper
Keyword(s):  
Pituitary Tumor ◽  
Culture Medium ◽  
Tumor Cell Line ◽  
Molecular Forms ◽  
Beta Endorphin ◽  
Calcium Dependent ◽  
Common Precursor ◽  
Mouse Pituitary ◽  
Secreted Peptides ◽  
Stimulation Of

The secretion of peptide products derived from pro-ACTH/endorphin was examined with several radioimmunoassays and with polyacrylamide gel analyses of immunoprecipitates of radioactively labeled peptides. In studies using a mouse pituitary tumor cell line the accumulation of each of the four molecular forms of adrenocorticotropic hormone (ACTH) in tissue culture medium was shown to be a linear function of time. No evidence for self inhibition of secretion by accumulated, secreted peptides (i.e., ultra-short feedback) was found. Furthermore, synthetic human ACTH and synthetic camel beta-endorphin did not alter secretion of peptides when added to the culture medium at levels up to 10,000 times physiological. Stimulation of the release of ACTH-, endorphin-, lipotropin-, and 16k fragment immunoreactive material by norepinephrine was fully blocked by cobalt; by this criterion, stimulated release was calcium dependent. All the smaller molecules derived from the pro-ACTH/endorphin common precursor were secreted in equimolar amounts under all circumstances tested, within the precision of these studies (+/- 11%). Norepinephrine and cobalt did not significantly alter the secretion of pro-ACTH/endorphin and ACTH biosynthetic intermediate. The stimulation of secretion by norepinephrine and inhibition of secretion by cobalt was restricted to the lower molecular weight products derived from pro-ACTH/endorphin: glycosylated and nonglycosylated ACTH(1-39); beta-lipotropin, beta-endorphin, and gamma-lipotropin; and 16k fragment.

Mouse Pituitary Tumor Line Secreting Only the α-Subunit of the Glycoprotein Hormones: Development from a Thyrotropic Tumor*

Endocrinology ◽  
1983 ◽  
Vol 113 (5) ◽  
pp. 1587-1591 ◽  
Author(s):  
E. CHESTER RIDGWAY ◽  
J. DAVID KIEFFER ◽  
DOUGLAS S ROSS ◽  
MAUREEN DOWNING ◽  
HEIDI MOVER ◽  
...  
Keyword(s):  
Pituitary Tumor ◽  
Glycoprotein Hormones ◽  
Tumor Line ◽  
Α Subunit ◽  
Mouse Pituitary

Two proteins associated with secretory granule membranes identified in chicken regulated secretory cells

1994 ◽  
Vol 107 (5) ◽  
pp. 1297-1308
Author(s):  
J.L. Thomas ◽  
A. Stieber ◽  
N. Gonatas
Keyword(s):  
Secretory Granule ◽  
Secretory Pathway ◽  
Secretory Granules ◽  
Cell Types ◽  
Pancreatic Acinar Cells ◽  
Secretory Cells ◽  
Electrophoretic Patterns ◽  
Granule Membrane ◽  
The Central Nervous System ◽  
Ultrathin Frozen Sections

Lately, we have identified two polypeptides of 92–94 kDa (GRL1) and 45–60 kDa (GRL2), expressed in cytoplasmic granules of chicken granulocytes and thrombocytes. Here, we report that GRL1 and GRL2 are widely distributed in all exocrine and several endocrine cell types, but not in neurons of the central nervous system, during late stages of embryonic development, as well as in newly hatched and two-month-old chickens. Immunogold studies in ultrathin frozen sections of pancreatic acinar cells show that GRL1 and GRL2 are co-localized at the periphery of zymogen granules, in granules fused with apical acinar membranes and on apical membranes of acini, while the pregranular compartments of the secretory pathway are weakly or not labeled. Semiquantitative morphometric studies indicate that GRL1 and GRL2 are equally distributed in secretory granules. A variety of physical and metabolic studies reveal that GRL2, a highly N-glycosylated polypeptide, is an intrinsic membrane protein, while GRL1 is a peripheral membrane polypeptide released by Na2CO3 treatment of granulocyte membranes. In all hematopoietic, exocrine or endocrine cells examinated, GRL1 shows identical electrophoretic patterns, while GRL2 is identified as a diffuse band, at 40–65 kDa, in hematopoietic and pancreatic cells. Taken together, the morphological and biochemical studies indicate that GRL1 and GRL2 are components of the secretory granule membrane in chicken exocrine, endocrine and hemopoietic cell types.

scholarly journals Effect of Zinc Binding Residues in Growth Hormone (GH) and Altered Intracellular Zinc Content on Regulated GH Secretion

Endocrinology ◽  
2013 ◽  
Vol 154 (11) ◽  
pp. 4215-4225 ◽  
Author(s):  
Vibor Petkovic ◽  
Maria Consolata Miletta ◽  
Andrée Eblé ◽  
Daniel I. Iliev ◽  
Gerhard Binder ◽  
...  
Keyword(s):  
Secretory Pathway ◽  
Culture Medium ◽  
Secretory Granules ◽  
Gene Mutations ◽  
Zinc Content ◽  
Gh Secretion ◽  
Extracellular Secretion ◽  
Mutant Variant ◽  
Regulated Secretory Pathway ◽  
And Storage

Endocrine cells store hormones in concentrated forms (aggregates) in dense-core secretory granules that are released upon appropriate stimulation. Zn2+ binding to GH through amino acid residues His18, His21, and Glu174 are essential for GH dimerization and might mediate its aggregation and storage in secretory granules. To investigate whether GH-1 gene mutations at these positions interfere with this process, GH secretion and intracellular production were analyzed in GC cells (rat pituitary cell line) transiently expressing wt-GH and/or GH Zn mutant (GH-H18A-H21A-E174A) in forskolin-stimulated vs nonstimulated conditions. Reduced secretion of the mutant variant (alone or coexpressed with wt-GH) compared with wt-GH after forskolin stimulation was observed, whereas an increased intracellular accumulation of GH Zn mutant vs wt-GH correlates with its altered extracellular secretion. Depleting Zn2+ from culture medium using N,N,N′,N′-tetrakis(2-pyridylemethyl)ethylenediamine, a high-affinity Zn2+ chelator, led to a significant reduction of the stimulated wt-GH secretion. Furthermore, externally added Zn2+ to culture medium increased intracellular free Zn2+ levels and recovered wt-GH secretion, suggesting its direct dependence on free Zn2+ levels after forskolin stimulation. Confocal microscopy analysis of the intracellular secretory pathway of wt-GH and GH Zn mutant indicated that both variants pass through the regulated secretory pathway in a similar manner. Taken together, our data support the hypothesis that loss of affinity of GH to Zn2+ as well as altering intracellular free Zn2+ content may interfere with normal GH dimerization (aggregation) and storage of the mutant variant (alone or with wt-GH), which could possibly explain impaired GH secretion.

scholarly journals Intracellular transport and sorting of mutant human proinsulins that fail to form hexamers.

1991 ◽  
Vol 113 (5) ◽  
pp. 987-996 ◽  
Author(s):  
D Quinn ◽  
L Orci ◽  
M Ravazzola ◽  
H P Moore
Keyword(s):  
Intracellular Transport ◽  
Secretory Pathway ◽  
Secretory Granules ◽  
Point Mutations ◽  
Wild Type ◽  
Sorting Process ◽  
Storage Granules ◽  
Self Association ◽  
Regulated Secretory Pathway ◽  
Mouse Pituitary

Human proinsulin and insulin oligomerize to form dimers and hexamers. It has been suggested that the ability of prohormones to self associate and form aggregates may be responsible for the sorting process at the trans-Golgi. To examine whether insulin oligomerization is required for proper sorting into regulated storage granules, we have constructed point mutations in human insulin B chain that have been previously shown to prevent formation of insulin hexamers (Brange, J., U. Ribel, J. F. Hansen, G. Dodson, M. T. Hansen, S. Havelund, S. G. Melberg, F. Norris, K. Norris, L. Snel, A. R. Sorensen, and H. O. Voight. 1988. Nature [Lond.]. 333:679-682). One mutant (B10His----Asp) allows formation of dimers but not hexamers and the other (B9Ser----Asp) prevents formation of both dimers and hexamers. The mutants were transfected into the mouse pituitary AtT-20 cells, and their ability to be sorted into regulated secretory granules was compared to wild-type insulin. We found that while B10His----Asp is sorted somewhat less efficiently than wild-type insulin as reported previously (Carroll, R. J., R. E. Hammer, S. J. Chan, H. H. Swift, A. H. Rubenstein, and D. F. Steiner. 1988. Proc. Natl. Acad. Sci. USA. 85:8943-8947; Gross, D. J., P. A. Halban, C. R. Kahn, G. C. Weir, and L. Villa-Kumaroff. 1989. Proc. Natl. Acad. Sci. USA. 86:4107-4111). B9Ser----Asp is targeted to granules as efficiently as wild-type insulin. These results indicate that self association of proinsulin into hexamers is not required for its targeting to the regulated secretory pathway.

Inhibition of ACTH secretion in mouse pituitary tumor cells by activation of muscarinic cholinergic receptors

1985 ◽  
Vol 63 (6) ◽  
pp. 723-730 ◽  
Author(s):  
Seymour Heisler
Keyword(s):  
Cyclic Amp ◽  
Tumor Cells ◽  
Pituitary Tumor ◽  
Secretory Pathway ◽  
Hormone Release ◽  
Inhibitory Effects ◽  
Acth Secretion ◽  
Mouse Pituitary ◽  
Muscarinic Agents ◽  
Cyclic Amp Synthesis

Hormonally stimulated secretion of ACTH from AtT-20 mouse pituitary tumor cells is a cyclic AMP-mediated process. The presence of inhibitory cholinergic muscarinic receptors on these cells was recently reported, and in this study, the relationship between the activation of these receptors and the consequent inhibition of cyclic AMP formation and ACTH secretion was investigated. The muscarinic agent, oxotremorine, antagonized both cyclic AMP synthesis and ACTH secretion in response to corticotropin-releasing factor (CRF), vasoactive intestinal peptide, a 27-amino acid peptide with an N-terminal histidine and a C-terminal isoleucine amide, and forskolin. Other muscarinic agents, carbachol and bethanechol, had similar inhibitory effects. The cholinomimetics reduced basal (unstimulated) ACTH secretion without decreasing basal cyclic AMP levels, and also antagonized hormone release in response to cyclic AMP-independent agonists such as K+, A-23187, and phorbol ester. Scopolamine reversed the inhibitory effects of the muscarinic agents on basal and stimulated ACTH secretion and cyclic AMP formation. Increasing the extracellular calcium concentration reversed the muscarinic antagonism of basal and CRF-stimulated hormone release without affecting the cyclic AMP response. Pertussis toxin pretreatment attenuated the inhibitory effects of the muscarinic agents on forskolin-stimulated cyclic AMP synthesis and ACTH secretion as well as the inhibitory effect of carbachol on basal ACTH release. The data suggest that cyclic AMP is an essential mediator in the ACTH secretory pathway, but that an alternate cyclic AMP-independent ACTH pathway also exists in the clonal cells, and that both pathways may be modulated by a common postcholinergic receptor mechanism.

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