POLYPEPTIDE HORMONE BIOSYNTHESIS--AMPHIBIAN MODELS

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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.