Abstract 616: Caveolin-1 (CAV1) inhibits G Protein Coupled Receptor Kinase Type 4 (GRK4) kinase activity
In human renal proximal tubule cells, the dopamine-1 receptor (D1R) has been shown to increase cAMP production and negatively regulate sodium transport. Defects in D1R signaling have been associated with SNPs in G-protein-coupled receptor kinase type 4 (GRK4) which hyperphosphorylates and decreases D1R function, and the D1R uncoupling phenotype is fully reverted by reducing GRK4 expression with siRNA. We have also shown that CAV1 co-immunoprecipitates with GRK4, and that the normal D1R inhibition of the sodium transporter, NaKATPase alpha, can be blocked using siRNA to CAV1. We therefore hypothesized that CAV1 directly physically interacts with GRK4 and inhibits its kinase activity. Using purified human GRK4 and CAV1 proteins and an in-vitro peptide homogeneous kinase assay, we investigated the effects of caveolin-1 (CAV1) on the kinase activity of GRK4. By overexpressing and isolating a tandem affinity purified protein (TAP tagged) functional version of human CAV1 isolated from a human renal proximal tubule cell line, we were able to ensure that all of the post-translational modifications of the protein were present. Utilizing a peptide Ser/Thr kinase activity assay, we demonstrated that with an excess of CAV1, the kinase activity of GRK4 is inhibited by 37.8±1.3% (n=4, p<0.05). Using this same assay, we showed that purified human calcium calmodulin 1 (CALM1), a known inhibitor of GRK4 when using a complete cell lysate, does not significantly decrease the kinase activity of purified GRK4 using a CALM1 concentration over 100 times the published Kd value. This suggests that there is a missing co-factor or factors required for CALM1 to inhibit GRK4. In summary, we report for the first time that CAV1 inhibits the kinase activity of GRK4, and that a known inhibitor of GRK4, CALM1, does not directly inhibit GRK4 kinase activity using purified proteins in an in-vitro kinase activity assay.
- © 2012 by American Heart Association, Inc.