The first part of this proposal addresses the mechanism of action of dopamine in the regulation of prolactin secretion. Dr. Weiner has described phosphorylation of an 80 kD microtubule-associated protein that is regulated by dopamine removal and TRH addition. He will attempt to identify this phosphoprotein and look for others associated with microtubules and secretory granules whose phosphorylation changes. To address more proximal events in the action of dopamine, he will develop dopamine-responsive GH3 cell lines by gene transfer of the recently cloned anterior pituitary D2-dopamine receptor cDNA that he has obtained elsewhere. Receptor expression will be confirmed by RNA analysis, ligand binding and functional coupling of the receptor to prolactin secretion and second messenger activation (adenylate cyclase and phospholipase C). This approach will permit analysis of the D2 dopamine receptor by site-directed mutagenesis. The second part will characterize the neuroendocrine regulation of a GnRH-producing cell line that was produced by creating transgenic mice with the oncogene, SV40 T antigen, targeted to the GnRH-producing cells with the GnRH promoter. a) He will determine the optimum culture conditions for expressing a neuron-like differentiated state, by measuring GnRH production and neurite outgrowth. Immunocytochemical and ultrastructural analyses will be used to study the expression of neuron-specific markers and peptide processing. b) He will test whether cultured GnRH neurons spontaneously exhibit pulsatility of peptide release or can be synchronized by K+ depolarization. The effect of GnRH on its own release will be determined by a short loop feedback. c) Many transmitters are involved in the regulation of GnRH release, but it is unclear whether they act directly on GnRH neurons or via proximal synapses. He will study the regulation of GnRH and GAP release from GT-1 cells by dopamine, norepinephrine, serotonin and GABA. Relevant receptors will be characterized by ligand binding studies.
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