The long term objective of this proposal is to understand how prolactin (PRL) regulates cell proliferation and differentiation. To study multiple components along the PRL-stimulated mitogenic pathway, we cloned and characterized the PRL receptor (PRL-R), a member of the hematopoietin/cytokine receptor superfamily, and a panel of early response genes (transcription factors, cytokines, cytokine receptors) and delayed early response genes, such as RNUDC. The structure and function of two of these genes will be analyzed: the PRL-R which mediates the mitogenic effects of PRL, and RNUDC, whose synthesis is a result of PRL-stimulated cellular proliferation.
AIM number 1 analyzes PRL-R domains involved in interacting with signaling proteins. How the PRL-R intracellular domain tyrosine residues serve as docking sites for recruiting the transcription factors Stat1 and Stat5 will be examined by mutagenesis, transfection, gel sift and in vitro interaction assays. The significance of PRL-R interaction with the enzyme 2',5- oligoadenylate synthetase (OAS), which was identified as a PRL-R interacting protein by yeast two-hybrid genetics, will be addressed. PRL-R truncations will be used to determine the site of PRL-R/OAS interaction, and OAS sense and anti-sense constructs will be used to address potential OAS function in PRL-R signal transduction.
AIM number 2 addresses RNUDC structure and function. Genetic complementation studies in Aspergillus nidulans have shown that rat RNUDC is a functional homologue of a nuclear movement protein in the fungus; however, its function in mammalian cells is unclear. Our localization of RNUDC staining to the centrosome/Golgi region suggests the exciting possibility that RNUDC may play a role in centrosome/Golgi structure and/or function. A series of biochemical, pharmacological, genetic and immunofluorescence microscopy studies are outlined to determine the cellular localization of RNUDC, and to address potential RNUDC function by sense and anti-sense strategies in mammalian cells. Finally, the identity of a 50 kDa RNUDC interacting protein will be determined by microsequencing. These combined studies should provide new insights into the signaling mechanisms, signaling molecules, and gene sequences involved in PRL regulation of cell proliferation. On a broader scale, these studies may elucidate important steps in cellular growth controls and provide insights into neuroendocrine-immune system interactions.
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