A pituitary tumor cell line (AcT-20) from the mouse packages mature ACTH into secretory vesicles but also leaks the longer precursor form, proopiomelanocortin (POMC) into the medium by a non-regulated or constitutive pathway. Human proinsulin, human growth hormone and rat trypsinogen, encoded by transfected DNA also take two pathways to the surface but other proteins such as the endogenous laminin or the truncated viral G protein, encoded by transfected DNA, only exit constitutively. Since the truncated viral G protein can be targeted to the secretory vesicle by fusing it with human growth hormone, we propose that peptide hormones, and trypsinogen, share a common sorting domain responsible for correct targeting to the secretory vesicle. To identify such domains we plan to delete by oligonucleotide mutagenesis candidate domains from rat trypsinogen, taking advantage of the known three-dimensional structure of rat trypsinogen and other proteases. To examine the properties of the constitutive pathway, DNA encoding immunoglobulin light and heavy chains will be transfected into AtT-20 cells. Since immunoglobulins must assemble before secretion, we hope to gain insight into rules of protein assembly that will facilitate the engineering of proteins capable of secretion by mammalian cells. The immediate goal is to link fragments of peptide hormone to immunoglobulin chains, and re-direct them to secretory vesicles. To allow us to begin a molecular explanation of protein secretion we have developed an AtT-20 variant into the cytoplasm of which macromolecules can be delivered with high efficiency using red blood cell fusion. Antibodies to vesicle and cytoskeletal components will be introduced to try to inhibit secretory vesicle formation, and vesicle movement to the cell surface. Evidence that secretory vesicles show a selective association with cytoskeletal elements will be sought. The ability to study secretory vesicle movement, secretory vesicle formation and exocytosis in a cell line that can be readily transfected with appropriate expression vectors, and whose cytoplasm can be readily modified by red blood cell microinjection gives an unusually rich opportunity to study the nature of protein secretion and its disorders.
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