Sorting of the Neuroendocrine Secretory Protein Secretogranin II into the Regulated Secretory Pathway

Intracellular associations indicate that granins may play a role in the regulatory mechanisms involved in differential secretion of gonadotrophins. The effect of GnRH on mRNA expression, storage and secretory patterns of granins and gonadotrophins was investigated in male mice. GnRH antiserum (G/A) was injected into mice in the treatment group (n = 15) at 12 h intervals for 2 days and a subset (n = 9) was killed. Buserelin (G/A + B) was administered to the remaining mice (n = 6), which were killed 2 h later; control mice (n = 6) were killed at the onset of the study. LHb mRNA content was lower in G/A and G/A + B mice compared with controls, whereas plasma LH concentrations were higher in G/A + B mice. FSHbeta mRNA content did not change, whereas plasma FSH concentrations were lower in G/A mice compared with controls, and higher in G/A + B mice compared with both G/A and control mice. Secretogranin II (SgII) and CgA mRNA contents were not different between experimental groups. There were more granules per gonadotroph in G/A mice, and considerably fewer after Buserelin treatment. Immunogold labelling of gonadotrophs revealed the presence of LH(+ve)/SgII(+ve) and LH(+ve)/SgII(-ve) granules, and negligible numbers of LH(-ve)/SgII(+ve) granules. Both the numbers of LH(+ve)/SgII(+ve) granules and overall granule antigenicity for SgII were higher in G/A mice compared with controls and G/A + B mice. In contrast, there were fewer LH(+ve)/SgII(-ve) granules per gonadotroph in G/A mice compared with controls. In conclusion, absence of GnRH input to the pituitary gland resulted in preferential storage of SgII and subsequently increased intragranular co-aggregation with LH. Administration of Buserelin to G/A mice resulted in the apparent release of LH(+ve)/SgII(+ve) granules that was reflected by an increase in plasma LH concentrations, indicating that these granules were in the regulated secretory pathway. In contrast, secretion of LH(+ve)/SgII(-ve) granules did not appear to be influenced by the actions of Buserelin and, therefore, may have been destined for constitutive release, possibly to maintain basal plasma LH concentrations.

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Prohormone transport through the secretory pathway of neuroendocrine cells

Biochemistry and Cell Biology ◽

10.1139/o00-020 ◽

2000 ◽

Vol 78 (3) ◽

pp. 289-298 ◽

Cited By ~ 12

Author(s):

Roland P Kuiper ◽

Gerard JM Martens

Keyword(s):

Secretory Pathway ◽

Potential Role ◽

Protein Transport ◽

Secretory Granules ◽

Secretory Protein ◽

Neuroendocrine Cells ◽

Secretory Proteins ◽

Regulated Secretory Pathway ◽

Made In

En route through the secretory pathway of neuroendocrine cells, prohormones pass a series of membrane-bounded compartments. During this transport, the prohormones are sorted to secretory granules and proteolytically cleaved to bioactive peptides. Recently, progress has been made in a number of aspects concerning secretory protein transport and sorting, particularly with respect to transport events in the early regions of the secretory pathway. In this review we will deal with some of these aspects, including: i) selective exit from the endoplasmic reticulum via COPII-coated vesicles and the potential role of p24 putative cargo receptors in this process, ii) cisternal maturation as an alternative model for protein transport through the Golgi complex, and iii) the mechanisms that may be involved in the sorting of regulated secretory proteins to secretory granules. Although much remains to be learned, interesting new insights into the functioning of the secretory pathway have been obtained.Key words: regulated secretory pathway, p24 family, vesicular transport, POMC, protein sorting, secretory granule, Xenopus laevis.

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Transport via the regulated secretory pathway in semi-intact PC12 cells: role of intra-cisternal calcium and pH in the transport and sorting of secretogranin II.

The Journal of Cell Biology ◽

10.1083/jcb.127.3.693 ◽

1994 ◽

Vol 127 (3) ◽

pp. 693-705 ◽

Cited By ~ 55

Author(s):

L Carnell ◽

H P Moore

Keyword(s):

Pc12 Cells ◽

Secretory Pathway ◽

Secretory Granules ◽

Ph Gradients ◽

Secretogranin Ii ◽

Prohormone Processing ◽

Endoproteolytic Cleavage ◽

Regulated Secretory Pathway ◽

Gamma S

To gain insight into the mechanisms governing protein sorting, we have developed a system that reconstitutes both the formation of immature secretory granules and their fusion with the plasma membrane. Semi-intact PC12 cells were incubated with ATP and cytosol for 15 min to allow immature granules to form, and then in a buffer containing 30 microM [Ca2+]free to induce exocytosis. Transport via the regulated pathway, as assayed by the release of secretogranin II (SgII) labeled in the TGN, was inhibited by depletion of ATP, or by the inclusion of 100 microM GTP gamma S, 50 microM AlF3-5 or 5 micrograms/ml BFA. When added after immature granules had formed, GTP gamma S stimulated rather than inhibited exocytosis. Thus, exocytosis of immature granules in this system resembles the characteristics of fully matured granules. Transport of SgII via the regulated pathway occurred at a fourfold higher efficiency than glycosaminoglycan chains, indicating that SgII is sorted to some extent upon exit from the TGN. Addition of A23187 to release Ca2+ from the TGN had no significant effect on sorting of SgII into immature granules. In contrast, depletion of lumenal calcium inhibited the endoproteolytic cleavage of POMC and proinsulin. These results establish the importance of intra-cisternal Ca2+ in prohormone processing, but raise the question whether lumenal calcium is required for proper sorting of SgII into immature granules. Disruption of organelle pH gradients with an ionophore or a weak base resulted in the inhibition of transport via both the constitutive and the regulated pathways.

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Reconstitution in vitro of the pH-dependent aggregation of pancreatic zymogens en route to the secretory granule: implication of GP-2

Biochemical Journal ◽

10.1042/bj2910289 ◽

1993 ◽

Vol 291 (1) ◽

pp. 289-296 ◽

Cited By ~ 49

Author(s):

F A Leblond ◽

G Viau ◽

J Lainé ◽

D Lebel

Keyword(s):

Secretory Granule ◽

Secretory Pathway ◽

Relative Proportion ◽

Secretory Protein ◽

Secretory Proteins ◽

Zymogen Granule ◽

Granule Membrane ◽

Regulated Secretory Pathway ◽

Golgi Network

Regulated secretory proteins are thought to be sorted in the trans-Golgi network (TGN) via selective aggregation. To elucidate the biogenesis of the secretory granule in the exocrine pancreas, we reconstituted in vitro the conditions of pH and ions believed to exist in the TGN using the end product of this sorting process, the zymogen granule contents. Protein aggregation was dependent on pH (acidic) and on the presence of cations (10 mM Ca2+, 150 mM K+) to reproduce the pattern of proteins found in the granule. The constitutive secretory protein IgG was excluded from these aggregates. Zymogen aggregation correlated with the relative proportion of the major granule membrane protein GP-2 in the assay. These results show that the glycosylphosphatidylinositol-anchored protein GP-2 co-aggregates with zymogens in the acidic environment believed to exist in the pancreatic TGN, and thus suggest that GP-2 would function as a membrane anchor for zymogen aggregates, facilitating their entrapment in budding vesicles directed towards the regulated secretory pathway.

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Salivary glands as a potential gene transfer target for gene therapeutics of some monogenetic endocrine disorders

Journal of Endocrinology ◽

10.1677/joe.1.06171 ◽

2005 ◽

Vol 185 (3) ◽

pp. 363-372 ◽

Cited By ~ 58

Author(s):

Antonis Voutetakis ◽

Ioannis Bossis ◽

Marc R Kok ◽

Weitian Zhang ◽

Jianghua Wang ◽

...

Keyword(s):

Gene Transfer ◽

Salivary Glands ◽

Viral Vectors ◽

Secretory Pathway ◽

Protein Localization ◽

Secretory Protein ◽

Stable Gene ◽

Endocrine Disorders ◽

Target Tissue ◽

Regulated Secretory Pathway

Salivary glands (SGs) exhibit several important features which are also common to endocrine glands: self-containment due to a surrounding capsule, highly efficient protein production and the ability to secrete proteins into the bloodstream. We have hypothesized that SGs are potentially useful as gene transfer targets for the correction of inherited monogenetic endocrine disorders. In the present communication, we extend our studies and attempt to test our hypothesis by comparing the efficacy of two commonly used viral vectors and the resulting serum and salivary distribution of transgene encoded hormones. A low dose (5 ×109 particles) of either an adenoviral serotype 5 (Ad5) vector encoding the human erythropoietin (hEPO) cDNA or an adeno-associated virus sero-type 2 (AAV2) vector encoding either the hEPO or human growth hormone (hGH) cDNA was administered to individual submandibular SGs of Balb/c mice. Serum and salivary hEPO and hGH levels were determined at defined time points. Two additional recombinant viruses encoding enhanced green fluorescence protein (GFP) were also used (AdGFP and AAVGFP) in order to perform immunohistochemical analyses of transgenic protein localization in SG sections post-administration. AAV2 vectors led to stable gene transfer unlike the results with the Ad5 vectors. Indeed, in one mouse we observed hEPO production for a period of two years after administration of AAVhEPO to SGs. hEPO, which is a constitutive pathway secretory protein, was readily secreted into the bloodstream from the SGs, yielding therapeutically adequate serum levels. Conversely, hGH, a regulated secretory pathway protein, was preferentially secreted into saliva. SGs may be an attractive candidate target tissue for gene therapeutics of some monogenetic endocrine deficiency disorders. At present, AAV2 vectors seem particularly useful for such applications, and transgenes encoding constitutive secretory pathway hormones are more suitable for this application with SGs than those encoding regulated secretory pathway hormones.

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

The Journal of Cell Biology ◽

10.1083/jcb.112.5.843 ◽

1991 ◽

Vol 112 (5) ◽

pp. 843-852 ◽

Cited By ~ 63

Author(s):

L Matsuuchi ◽

R B Kelly

Keyword(s):

Cell Line ◽

Secretory Pathway ◽

Secretory Granules ◽

Secretory Protein ◽

Dense Core ◽

Wild Type ◽

Dna Encoding ◽

Basal Secretion ◽

Constitutive Secretion ◽

Regulated Secretory Pathway

A variant of the ACTH-secreting pituitary cell line, AtT-20, has been isolated that does not make ACTH, sulfated proteins characteristic of the regulated secretory pathway, or dense-core secretory granules but retains constitutive secretion. Unlike wild type AtT-20 cells, the variant cannot store or release on stimulation, free glycosaminoglycan (GAG) chains. In addition, the variant cells cannot store trypsinogen or proinsulin, proteins that are targeted to dense core secretory granules in wild type cells. The regulated pathway could not be restored by transfecting with DNA encoding trypsinogen, a soluble regulated secretory protein targeted to secretory granules. A comparison of secretion from variant and wild type cells allows a distinction to be made between constitutive secretion and basal secretion, the spontaneous release of regulated proteins that occurs in the absence of stimulation.

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