GRK4 and D3R regulation of NHE3 and NCC expression

长期目标是确定五种多巴胺受体和其他G蛋白偶联蛋白之间的相互作用 受体(GPCRs)在调节肾脏电解质转运和血压中的作用。D3多巴胺受体(D3R)， 单独或通过与其他多巴胺受体(如DIR)和其他GPCRs(如血管紧张素1型受体)相互作用，调节肾脏钠转运和血压。D3R基因缺失(D3R-/-)导致盐敏感型高血压，与肾脏钠/氢交换器3型表达增加相关 (NHE3[SLC9A3])、氯化钠交换器(NCC[SLC12A3])和上皮钠通道α亚单位(ENaC[SCNN11A])。初步数据表明，D3R泛素化NHE3，NHE3泛素化是由于D3R介导的对泛素酶USP48的抑制。项目3的总体假设是高血压 在D3R-/-小鼠中，NHE3和NCC的活性和表达增加；它们的表达增加是由于USP48去泛素化导致降解减少所致。特殊目标1将测试这样一种假设，即由于表达减少(D3-/-)或由于人类GRK4伽马变异体(例如GRK4 Gammpl42V)的结构性脱敏而导致D3R功能受损，导致在NaCI摄入量正常时肾脏NHE3和NCC的表达增加，而当NaCI摄入量增加时NCC和ENaC的肾脏表达增加。D3-/-小鼠排钠能力受损导致高血压的发生。特定目标2将检验这一假设，即D3R抑制USP4S活性，防止NHE3和NCC的去泛素化；这保留了它们的泛素化，导致降解增加，从而减少表达。人GRK4Gamma 142V使D3R表达减少或D3R功能受损，增加USP48的表达和活性，促进去泛素化，阻止NHE3和NCC的降解，从而提高它们的表达水平。高血压是一种复杂的多基因疾病。然而，根据我们的研究结果，GRK4对有限数量的GPCR的监管，以及 GPCRs对基因/蛋白质的下游调控，如D3R，使单个基因GRK4在高血压的发病机制中起关键作用。

lXhe long-term objective is to determine the interaction among the five dopamine receptors and other G protein-coupled receptors (GPCRs) in the regulation of renal electrolyte transport and blood pressure. The D3 dopamine receptor (D3R), by itself, or via its interaction, with other dopamine receptors (e.g., DIR) and other GPCRs (e.g., angiotensin type 1 receptor), regulates renal sodium transport and blood pressure. Deletion of the D3R gene (D3R-/-) results in saltsensitive hypertension that is associated with increased renal expression of sodium/hydrogen exchanger type 3 (NHE3[SLC9A3]), sodium chloride exchanger (NCC[SLC12A3]) and alpha subunit of the epithelial sodium channel (ENaC [SCNNl A ]). Preliminary data show that the D3R ubiquitinates NHE3 and that the ubiquitination of NHE3 is due to D3R-mediated inhibition of USP48, an ubiquitinase. The overall hypothesis of project 3 is that the hypertension in D3R-/- mice is caused by increased activity and expression of NHE3 and NCC; their increased expression is caused by decreased degradation due to deubiquitination by USP48. Specific aim 1 will test the hypothesis that impaired D3R function, because of decreased expression (D3-/-) or because of constitutive desensitization by human GRK4 gamma variants (e.g., GRK4 gammpl42V), results in increased renal expression of NHE3 and NCC when NaCI intake is normal and increased renal expression of NCC and ENaC when NaCI intake is increased. The impaired ability of D3-/-mice to excrete sodium contributes to the development of hypertension. Specific aim 2 will test the hypothesis that D3R inhibits USP4S activity, preventing the deubiquitination of NHE3 and NCC; this preserves their ubiquitination, resulting in increased degradation and therefore, decreased expression. Decreased expression of D3R, or impairment of D3R function by human GRK4gamma 142V, increases USP48 expression and activity, promotes the deubiquitination and prevents the degradation of NHE3 and NCC, thus, increasing their expression levels. Hypertension is a complex polygenic disease. However, based on our findings, GRK4 regulation of a limited number of GPCRs, and the downstream regulation of genes/proteins by GPCRs, e.g., D3R, makes a single gene, GRK4, a key contributor in the pathogenesis of essential hypertension.