Evidence indicates that prolactin profoundly influences rat liver growth via receptor-mediated stimulation of PKC in the hepatocyte nucleus. Therefore, the hypothesis to be challenged by the experiments described in this proposal, is that prolactin, internalized by receptor-mediated endocytosis, is channelled to the nucleus, where it interacts with specific nuclear prolactin receptors coupled to PKC activation, which thereby evokes transcription of specific messenger RNAs requisite for a trophic response. This hypothesis will be tested in two distinctly different experimental cell systems, one transformed (Nb2 lymphoma cells) and one normal (rat hepatocytes) , in which prolactin stimulates growth. In both systems we will identify the nuclear prolactin receptor and investigate its coupling to mRNA transcription. By investigating two distinctly different paradigms of prolactin-induced mitogenesis, we will be able to determine whether the direct interaction of prolactin within the nucleus is phenomenon common to multiple systems. These studies will establish a direct nuclear site of action for growth factors such as prolactin and extend our understanding of the mechanism by which growth factors stimulate normal and tumor cell growth.
The specific aims are as follows: 1.) To determine whether prolactin, internalized by receptor-mediated endocytosis, is targeted to the hepatocyte and Nb2 lymphoma cell nucleus; 2.) To identify and characterize the prolactin receptor present within hepatocyte and Nb2 lymphoma cell nuclei; 3.) To determine whether nuclear prolactin receptor occupation leads to transcription of """"""""rapid response genes"""""""". We will study the intracellular fate of prolactin in intact cells Specific Aim 1) by indirect immunofluorescence and by subcellular fractionation of cells incubated with radiolabeled prolactin. The nuclear prolactin receptor will be identified and characterized (Specific Aim 2) by immunoprecipitation and ligand-cross-linking techniques and gel electrophoresis. Nuclear runoff and northern blot analysis will be utilized to determine which specific genes are transcribed in response to prolactin treatment in verifiably pure rat liver and Nb2 node lymphoma cell nuclei (Specific Aim 3). The elucidation of the mechanism(s) by which growth factors and hormones modulate cell proliferation will significantly broaden our understanding of normal and aberrant growth regulation. Since many transformed cells either synthesize and/or require growth factors for proliferation, the demonstration of a receptor-mediated nuclear site of action for these substances will suggest alternative cellular targets for possible therapeutic intervention.
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