REGULATION OF PROCESSING OF OPIOID PEPTIDE PRECURSORS

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Neuropeptides are synthesized as large precursor proteins which undergo specific proteolytic cleavages and modifications to produce bioactive peptides. The initial cleavages are thought to occur at pairs or basic amino acid residues to produce peptides with C-terminal Lys of Arg residues. The Lys or Arg residues are removed by carboxypeptidase-like enzymes to produce bioactive peptides. Although the proteolytic reactions are critical steps in activating neuropeptides, we know very little about the endopeptidases and exopeptidase that catalyze these reactions. Recently, a member of the kallikrein family of serine proteases has been implicated in the endoproteolytic cleavage of pro-opiomelanocortin (POMC) in the pituitary. By use of a mouse kallikrein cDNA probe we have been able to demonstrate that a single kallikrein protease is present in the mouse pituitary tumor cell line that produces POMC. A carboxypeptidase enzyme has been implicated in proenkephalin processing in the bovine adrenal medulla. This enzyme has been purified and partially sequenced. Several approaches will be used to determine whether the kallikrein and carboxypeptidase enzymes are required for POMC and proenkephalin processing. We will first determine the effect of selectively removing these proteins from the cellular protein pool on processing of the two precursors. This will be done by a gene transfer technique. Second, the subcellular and cellular distribution of the enzyme will be determined. Third, we will test the ability of specific inhibitors of these enzymes to alter processing of the precursors. Fourth, we plan to determine the structure of the genes that code for these enzymes. Control of expression of these genes at the transcriptional level will also be analyzed to determine whether activity of these genes is regulated coordinately with the activity of precursor genes. A final objective will be to determine when the enzyme genes are turned on relative to the precursor genes during embryonic development.