Abstract 517: Ubiquitylation of G Protein Coupled Receptor Kinase 4 (GRK4) Regulates its Localization and Abundance

Abstract

The dopamine-1 receptor (D 1 R) is responsible for regulating up to 60% of natriuresis in the kidney under sodium loaded conditions. G protein coupled receptor kinase type 4 (GRK4) phosphorylates the D 1 R and reduces its membrane expression, but little is known about regulation of GRK4. We hypothesized that GRK4 is targeted for elimination through ubiquitylation. Using D 1 R and GRK4γ stably transfected human embryonic kidney cells (HEK293), we demonstrated GRK4 and ubiquitin coimmunoprecipitation. Addition of clasto-lactacystin beta-lactone (CLBL, an inhibitor of the 26S proteasome) increased GRK4 expression levels (western blot) using two different GRK4 fusion protein constructs. The addition of CLBL (10 μmol/L, 24 hrs) increased the expression of both a tandem affinity tagged (tap-tag) GRK4 fusion construct (2.65±0.19 fold over vehicle (VEH): VEH 4,010±404 RFU; CLBL 10,626±744 RFU, P<0.05, N=3) as well as a mCherry GRK4 fusion construct (6.98±0.50 fold increase over VEH: VEH 5,044±3229 RFU; CLBL 35,210±2547 RFU, P<0.05, N=3). The ubiquitin binding sites (AA 216,217; MYACK Ub K Ub LQKK) are located near the nuclear localization signal (AA 219-228; QKKRIKKRK) in GRK4. We therefore examined the subcellular localization of GRK4 after CLBL treatment. Nuclear accumulation of the GRK4-mCherry fusion protein fluorescence decreased markedly while cytoplasmic GRK4-mCherry fluorescence increased (nuclear to cytoplasmic GRK4 ratio: VEH 1.18±0.09 RFU; CLBL 0.78±0.03 RFU, P<0.01, N=12). In summary, GRK4 expression levels are negatively regulated by the ubiquitin proteasome system and ubiquitylation of GRK4 prevents nuclear import. We hypothesize that ubiquitylation is sterically hindering the nuclear localization sequence in GRK4 from interacting with the nuclear import machinery of the cell. Through manipulation of GRK4 ubiquitylation, one may be able to separate nuclear vs. cytoplasmic activity of GRK4 and reverse the negative impact overactive GRK4 has on dopaminergic signaling.