Decreased activity of the renal dopaminergic system has been linked to the development and/or maintenance of hypertension. Exogenously administered or endogenously generated dopamine (DA) increases sodium and water excretion, actions that are mediated primarily through stimulation of renal D1 receptors. A great deal of evidence indicates that renal D1 receptors are concentrated on kidney proximal tubules and that D1 stimulation inhibits proximal tubule sodium reabsorption. We have recently cloned the porcine D1A receptor gene and have shown that it is expressed in the proximal tubule-like porcine renal epithelial cell line, LLC-PK1. The porcine D1A receptor gene is the first cloned D1A receptor gene known to be expressed homologously in renal cells. Nothing is known, however, about how the renal D1A receptor is regulated at the level of gene transcription or translation. LLC-PK1 cells are thus an ideal model system to study the regulation of the renal D1A receptor gene at both the transcriptional and translational levels. There are 5 Specific Aims. We propose to: 1. Study the regulation of transcription of the native D1A receptor gene expressed in LLC-PK1 cells by exposing cells to a variety of factors known to influence gene transcription and monitoring changes in the steady state levels of D1A receptor mRNA. 2. Characterize the regulatory domains of the 5' flanking region of the porcine D1A receptor gene by constructing a series of successively truncated D1A receptor 5' flanking region-chloramphenicol acetyltransferase (CAT) reporter gene fusion constructs and measuring transcriptional activity in transiently transfected LLC-PK1 cells under basal and stimulated conditions (Sp.
Aim #1). 3. Characterize the positive and negative modulatory regions of the D1A receptor gene promoter by DNase I footprinting, gel mobility shift and supershift assays. 4. Determine the basis for cell-specific transcription of the D1A receptor gene by comparing transcriptional activity of D1A receptor promoter-CAT constructs expressed in renal (LLC-PK1, HEK-293, COS-7 and OK) and non- renal (NS20Y and NB41A3 mouse neuroblastoma) cells and by determining if nuclear extracts from cells known to express the D1A receptor (LLC-PK1, NS20Y, OK) yield different patterns of binding to the D1A receptor promoter. 5. Determine the extent to which D1A receptor 5' leader sequence polypeptides are translated and whether they play a role in regulating translation of the receptor protein. These studies will provide important new insights into the regulation of the physiologically significant kidney D1A receptor at the transcriptional and translational levels.
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