The biosynthesis of low molecular weight polypeptide hormones is dependent upon a complex sequence of posttranslational processing events. The processing enzymes that mediate these events are found within distinct subcompartments of the ER/golgi/secretory granule network. Understanding the expression, regulation and intracellular routing of these processing enzymes within organelles is central to establishing a clear understanding of the cell biology of secretory cells. Studies on non-mammalian endocrine tissues reveal the array of strategies that have evolved to mediate the biosynthesis of polypeptide hormones. Amphibian model systems will be used to analyzed two aspects of the Proopiomelanocortin (POMC) biosynthetic pathway that are relevant to polypeptide biosynthetic pathways in general: the segregation of processing events within cells, and the developmental regulation of processing enzymes. The toad, Bufo marinus, provides a unique model system to study the N- acetylation of the POMC products, alpha-MSH and beta-endorphin. In mammals, these reactions are performed in parallel in secretory granules. In the intermediate pituitary of anuran amphibians, these reactions are separated both temporally and spatially. A series of studies involving pulse/chase paradigms, immunoelectron microscopy, subcellular fractionation and kinetics analyses will attempt to explain how this amphibian has succeeded in segregating the N-acetylation of these POMC end products. These projects will provide insights into the strategies for routing and regulating processing enzymes within vesicles. Studies on the anterior pituitary of larval Ambystoma tigrinum and neotenic Ambystoma mexicanum will investigate developmental changes in the proteolytic cleavage of ACTH. The developmental shift in ACTH processing observed in amphibian appears to be a common feature of tetrapod corticotrope ontogeny. The amphibian pituitary studies provide convenient models for studying ACTH posttranslational mechanisms, and circumvent many of the limitations of the fetal rat pituitary model system. The ACTH- related cleavage products generated by larval corticotropes will be characterized, and the effects of these products on steroid synthesis by larval adrenal cells will be determined.

Agency
National Institute of Health (NIH)
Institute
National Center for Research Resources (NCRR)
Type
Research Project (R01)
Project #
5R01RR006565-02
Application #
3421585
Study Section
Special Emphasis Panel (SRC (BM))
Project Start
1990-09-30
Project End
1993-09-29
Budget Start
1991-09-30
Budget End
1992-09-29
Support Year
2
Fiscal Year
1991
Total Cost
Indirect Cost
Name
University of Denver
Department
Type
Schools of Arts and Sciences
DUNS #
City
Denver
State
CO
Country
United States
Zip Code
80208
Dores, R M; Wasinger, H; Vaudry, D et al. (1994) Melanotropes of the lizard, Anolis carolinensis, lack N-acetylating mechanisms for both alpha-melanocyte-stimulating hormone and beta-endorphin. Neuroendocrinology 59:603-9
Lancha, A; Batista, M A; Dores, R M (1994) An analysis of the proopiomelanocortin systems in the pituitary of the squamate reptile Lacerta galloti. Gen Comp Endocrinol 93:438-47
Dores, R M; Gieseker, K; Steveson, T C (1994) The posttranslational modification of beta-endorphin in the intermediate pituitary of the toad, Bufo marinus, includes processing at a monobasic cleavage site. Peptides 15:1497-504
Dores, R M; Harris, S (1993) Differential N-acetylation of alpha-MSH and beta-endorphin in the intermediate pituitary of the turtle, Pseudemys scripta. Peptides 14:849-55
Dores, R M; Steveson, T C; Price, M L (1993) A view of the N-acetylation of alpha-melanocyte-stimulating hormone and beta-endorphin from a phylogenetic perspective. Ann N Y Acad Sci 680:161-74
Dores, R M; Sandoval, F L; McDonald, L K (1993) Proteolytic cleavage of ACTH in corticotropes of sexually mature axolotls (Ambystoma mexicanum). Peptides 14:1029-35
Dores, R M; McDonald, L K; Purdom, L C et al. (1993) Reversed phase HPLC analysis of proenkephalin-related and prodynorphin-related end-products in the brain of a urodele amphibian, Ambystoma tigrinum. Brain Behav Evol 42:69-76
Goldsmith, A M; Sei, C A; Lance, V et al. (1992) Detection of prodynorphin end products in lizard, turtle, and alligator brain extracts. Peptides 13:435-40
Dores, R M; Truong, T; Steveson, T C (1992) Detection and partial characterization of proopiomelanocortin-related end-products from the pars intermedia of the toad, Bombina orientalis. Gen Comp Endocrinol 87:197-207
Dores, R M; Lancha, A; Rand-Weaver, M et al. (1991) Detection of a novel sequence change in the major form of alpha-MSH isolated from the intermediate pituitary of the reptile, Anolis carolinensis. Peptides 12:1261-6

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