The long term goal of this project is the biochemical and molecular characterization of dopaminergic receptor-mediated information transduction, and its regulation, across neuronal membranes. Three major interelated areas of research on D-1 and D-2 dopamine receptors are currently under investigation. 1. Solubilization, purification and biochemical characterization of dopamine receptors. Solubilization protocols for both D1 and D2 receptors were developed using various tissues and detergents. Affinity chromatography matrices were synthesized and evaluated for use in the purification of each receptor subtype. Other more conventional methods of purification were investigated which, when coupled with an affinity chromatography step, should yield highly purified receptor preparations. Several potential affinity probes of D-1 and D-2 receptors were synthesized. 2. Cell biology, pharmacology, and regulation of dopamine receptors. The D-1 dopamine receptor in the bovine parathyroid gland was directly identified and characterized using radioligland binding techniques. A wide variety of cultured mammalian cell lines were screened, via ligand binding, for their ability to express dopamine receptors. One murine neuroblastoma cell line was found to posses D-1 receptors while one human neuroblastoma and several human retinoblastoma cell lines were found to contain D-2 receptors. These cells will be used to extensively investigate the function and regulation of both D-1 and D-2 receptor subtype. Several fluorescent probes of D-1 and D-2 receptors were synthesized and shown to retain high receptor affinity. 3. Molecular cloning of dopamine receptors. In order to initiate molecular cloning efforts for the human D-2 dopamine receptor, a cDNA library was constructed from one of our retinoblastoma cell lines which express the D-2 receptor in high abundance. Oligonucleotide probes to potentially conserved regions among catecholamine receptors were synthesized for use in screening the cDNA library. Oligonucleotide probes to partial amino acid sequence of the D-2 receptor will be utilized when such sequence is available.
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