The major objective of this application is to provide the applicant with new and intensive training in molecular and cellular biology by characterizing new receptors within the dopamine receptor family and to study their roles in modulating ion transport. The applicant will clone and sequence the cDNAs encoding the different peripheral dopamine receptors and will characterize these receptors. In the initial studies, the known cDNA sequences of the rat central dopamine receptors (D1A, D1B, D2 short, D2long, D3, and D4) will be used in combination with advanced recombinant DNA techniques and strategies such as the polymerase chain reaction (CR), restriction mapping and sequencing, and screening under low and high stringency conditions to clone and sequence the peripheral dopamine receptors in the rat kidney. The cloned receptors will then be characterized at the mRNA, protein and signal transduction levels by inducing expression of the CDNAS of the different receptors in COS-7 cells, a cell line which does not exhibit native expression of dopamine receptors. Characterization at the protein level will include ligand binding and competition experiments, as well as studies on the cell surface localization, stability, biosynthesis and signal transduction of the newly expressed receptors. In these studies, techniques routinely used in the sponsors laboratories such as fluorescent microscopy, immunoprecipitation with antibodies raised by the fusion protein technique, and measurements of intracellular cAMP, polyphosphoinositides (Pipins), diacylglycerol (DAG), inositol phosphates (IP) and cytosolic Ca++ will aid to characterize the newly expressed receptors. The information obtained and the skills learned during this comprehensive training will then be used in later, independent research to identify the renal dopamine receptors(s), the second messenger system(s), and the molecular mechanism(s) mediating the inhibition of Na+,K+-ATPase by dopamine. Completion of these studies will provide expertise over a spectrum of approaches to study the molecular basis of the dopamine-regulated renal tubular transport processes, and will enable the applicant to pursue an independent research career in the field of developmental cellular physiology.
