Small peptide hormones are usually synthesized as large precursor proteins which undergo tissue specific post-translational modifications and proteolytic cleavages to produce mature bioactive peptides. It is not known what accounts for the tissue specificity of the processing reactions. A number of factors which may be involved include: selective expression of processing enzymes, tissue specific modification of the precursor proteins which would govern cleavage site accessability, and differential compartmentalization of either the protease or the precursor protein. We have sought to study many of these questions with gene transfer techniques. Using recombinant vaccinia virus as a mammalian cell expression vector, we have recently established that the yeast alpha-factor endopeptidase, KEX2, processes mouse proopiomelanocortin (mPOMC) in the mammalian cell. The mPOMC/KEX2 reconstitution system will be fully characterized regarding both the processed peptides as well as the subcellular location of processing. The ability of KEX2 to recognize and cleave another neuroendocrine precursor, human proenkephalin (hPE), will also be studied. The processed peptides generated by KEX2 in vivo will be compared to those generated in vitro. This novel complementation methodology will be used to authenticate the role of a candidate POMC endopeptidase, AtT-20 kallikrein. Secondly, using oligonucleotide-directed site specific mutagenesis, we will determine what role specific sequences and/or post-translational modifications have in the transport, endoproteolytic maturation and secretion of mPOMC in heterologous cell types. Following characterization of the hPE side group modifications, the mutagenesis approach will also be used to determine their role in hPE maturation and secretion. Finally, we will exploit unique features of vaccinia biology to express mutant POMC proteins in the homologous cell environment to determine what role specific sequences and/or post-translational modifications have in the tissue specific processing of POMC. Taken together, this information will provide the foundation for our long term goals of defining the functions of specific protein domains as well as cell factors responsible for the tissue specific generation of different sets of mature hormones from single precursor.
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