This is a proposal to study polyprotein processing during development. Pro-opiomelanocortin (POMC) is a polyprotein that contains the amino acid sequences of several pituitary hormones including corticotropin (ACTH) and the opioid peptide beta-endorphin. POMC processing serves as a particularly informative model system to investigate because adult anterior and intermediate pituitary cells (derived from the same embryonic epithelium) process POMC to different sets of end-product hormones. Furthermore, these cell types are under the control of different regulators. Our long range goals are to determine when and how the lobe-specific patterns of POMC processing are established during pituitary development, and to establish when during development POMC producing cells become responsive to regulation by neurotransmitters, hypothalamic-releasing-factors and glucosteroids. In our studies we will compare and contrast POMC processing in timed rhesus monkey and rat fetal pituitaries. This is significant because we have shown that the monkey processes POMC differently than the rat. This may be due to important differences in the amino acid sequences of POMC in monkey and rat. Also, relative gestational dates and developmental stages are different between monkey and the rodent. First, we will determine the forms of POMC-derived peptides present in the fetal monkey anterior and intermediate pituitary of different gestational ages by combining electrophoretic fractionation and radioimmunoassays specific for six different POMC domains. These results will be compared with our studies in the rat which have shown that the relative intracellular distribution of POMC-derived peptides changes during late stage gestation and early neonatal pituitary development. Secondly, we will determine the time course of the lobe-specific chemical modification processes for POMC-derived products during monkey and rat pituitary development. Radiolabeled POMC peptides from cultures of anterior and intermediate pituitary of different developmental stages will be isolated by immunoprecipitation, followed by fractionation and further biochemical analyses. This will allow us to determine when the lobe-specific N-acetylation, amidation, and glycosylation enzyme activities are expressed during pituitary development. Finally, we will determine the extent of the regulatory differentiation of POMC producing cells at various stages of development by culturing monkey and rat fetal pituitary cells in the presence of regulators known to control full differentiated POMC producing cells. Information derived from these studies would contribute greatly to the understanding of polyprotein expression, pituitary development, and to the understanding of the development of the hypothalamic-pituitary-adrenal axis at the molecular level.
