Mammalian prosomatostatin (PSS) is cleaved at an Arg-Lys doublet in order to produce somatostatin-14 (SS-14), and at two singlets, Arg and Lys, in order to release SS-28 and PSS$ sb{ lbrack 1{-}10 rbrack}$ respectively. Furin, PC1, and PC2 are three recently cloned mammalian endoproteases, associated with the Golgi (furin) or targeted towards the secretory granules of the regulated pathway (PC1 and PC2). In order to determine the enzymes involved in the proteolytic processing of PSS and localize the endoproteolytic reactions in specific compartments of the secretory pathway, I compared PSS processing in endocrine (AtT-20, GH3, and GH4C1 pituitary cells) or nonendocrine tumor cell lines (COS-7 kidney cells, PC 12 pheochromocytoma, LoVo colon adenocarcinoma cells). The efficiency of PSS processing to SS-14, SS-28, and PSS$ sb{ lbrack 1{-}10 rbrack}$ was correlated with: (i) secretion through the constitutive or regulated pathway, (ii) mRNA expression for furin, PC1 and PC2, and (iii) exogenous expression of furin, PC1, and PC2 in cells deficient in these proteinases. Coexpression of PC1 and PC2 with rat PSS was accomplished by transient (COS-7 cells) or stable (GH4C1 cells) cotransfections of their cDNAs in the respective cell lines. In the case of furin, recombinant vaccinia viruses expressing human furin or rat PSS were used to coinfect COS-7 or LoVo cells. Furthermore, I examined the effect of granules on the expression of PC2 and on PSS processing, by incubating GH4C1 cells with insulin, EGF, and $ beta$-estradiol, a treatment known to induce granule formation. Cell extracts and secretion media were further analyzed by HPLC and somatostatin specific RIAs. / Conclusions. (i) PSS is capable of monobasic processing within the constitutive secretory pathway. (ii) Such cleavages may be effected by furin or related endoproteases but are relatively inefficient. (iii) PC1 is capable of dibasic cleavage of PSS to SS-14 in both constitutive or regulated secretory cells. (iv) PC2 mediates SS-14 conversion only if expressed in regulated secretory cells. (v) The milieu of secretory cells, and not the granules, is required for PC2 activity. (vi) Furin is a mammalian SS-28 convertase, but not the unique one. (vii) A yet unknown endoprotease is responsible for PSS$ sb{ lbrack 1{-}10 rbrack}$ cleavage.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.29022 |
| Date | January 1995 |
| Creators | Galanopoulou, Aristea S. |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Doctor of Philosophy (Division of Experimental Medicine.) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | alephsysno: 001468321, proquestno: NN08100, Theses scanned by UMI/ProQuest. |
Page generated in 0.0015 seconds