Abstract 653: G-protein-coupled Receptor Kinase 4 Binds Preferentially To The Caveolin-1α Isoform In Rat Renal Proximal Tubule Cells
Caveolins, localized in lipid rafts of plasma membranes, tether and regulate signaling complexes into functional units and have been shown to inhibit g-protein-coupled receptor kinases GRK2 and GRK5 via the caveolin scaffolding domain. In human renal proximal tubule cells (RPTC), the dopamine-1-receptor (D1R) is phosphorylated and inhibited by G protein-coupled receptor kinase 4 (GRK4). Defects in D1R coupling leads to sodium-retention and a rise in blood pressure. We showed earlier that chronic disruption of lipid rafts or Caveolin-1 (Cav1) protein expression in the kidney induces hypertension in rats by relieving the steric inhibition of Cav1 on GRK4. Here we extend these studies by examining caveolin expression in renal proximal tubule cells microdissected from Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR). We discovered a significant decrease in Cav1 and Cav2 expression in SHR compared to WKY (98 ±1.2% reduction; n=3, P<0.001). In order to determine if the D1R coupling defect found in these SHR renal proximal tubule cells is due to loss of either of these proteins, we made stable SHR cell lines expressing either Cav1α, Cav1β or Cav2. Using co-immunoprecipitiation and western blot analysis, we demonstrate for the first time that GRK4 and Cav1 physically interact in rat cells, and that it is the Cav1α isoform that binds predominantly to GRK4 (Cav1β, Cav2 and vector control show a 90 ±1.5%, 84±0.8% and 87±2.1% reduction, respectively; n=3, P<0.001). Similar to an effect found in mouse Cav1 knockout animals, we find that Cav2 is not stable without the co-expression of Cav1. Our data suggest that Cav1α. but not Cav1β, is important for normal D1R function by binding to and inhibiting GRK4, implicating a domain other than the caveolin scaffolding domain in this interaction. These findings might be used in designing selective GRK4 inhibitors as novel antihypertensive compounds.
- © 2012 by American Heart Association, Inc.