This application, entitled """"""""Structural Determinants in Regulation of POMC Processing"""""""" proposes funding for the NIDA Scientist Development Award. The applicant has had excellent clinical and research training, including time spent at the National Institutes of Health and has now accepted a junior faculty position in the Department of Medicine, UCLA School of Medicine, in the Division of Endocrinology of Cedars-Sinai Medical Center. The NIDA Scientist Development Award will allow the principal investigator to develop molecular biology and X-ray crystallography skills, in addition to his well-recognized biochemistry expertise and allow him to develop an independent research program at the conclusion of the granting period. This grant will allow the applicant to answer basic questions in the field of opiate biosynthesis, leading to elucidation of the molecular mechanisms of drug addiction. Pro-opiomelanocortin (POMC) is the precursor of the biologically active peptides, ACTH and beta-endorphin. Heroin addicts have decreased levels of beta-endorphin. An understanding of the processing of POMC to the natural opiate, beta-endorphin, may provide insight into the subcellular mechanisms of opiate dependency. Recently PC1 and PC2, members of a new family of subtilisin-like serine proteases, the prohormone convertases, have been cloned and shown to cleave POMC in co-transfection experiments, and the principal investigator has shown that recombinant PC1 can cleave POMC in vitro. POMC appears to be differentially processed by PC1 and PC2. I am interested in understanding the substrate specificity of this differential processing and the regulation of this processing by opiate agonists and antagonists. AtT-20 cells, with endogenous PC1 diminished by antisense treatment; and GH3 cells, with endogenous PC2 diminished by antisense treatment, will be used to characterize the processing of POMC by PC1 and PC2. Particular emphasis will be placed on measuring the generation of beta-endorphin. The role of the primary and secondary structure of POMC will be analyzed by generating mutants of POMC by mutation and expression techniques. These POMC mutants will then be transiently transfected into the antisense cDNA- containing cell lines, and the processing of POMC will be examined. Additionally, chronic treatment with morphine and the opiate antagonist, naltrexone, as well as manipulation of the hypothalamic- pituitary-adrenal axis will be used to assess the in vivo regulation of PC1 and PC2 in the brain and pituitary by opioids, IL6, CRH and glucocorticoids. Finally, in the later years of the project, the role of the tertiary structure of POMC in its processing will be assessed by generating milligram quantities of POMC and screening the x-ray grade crystals to determine the three dimensional structure of POMC. These studies will provide valuable information on the subcellular mechanisms subserving the post- translational generation of beta-endorphin and related peptides. Thus, the biological basis for addiction may begin to be elucidated.
