N‐Terminal Targeting of RGS2 for FBXO44‐Mediated Proteasomal Degradation

G‐protein Coupled Receptors (GPCRs) are common targets for therapeutic drugs; however, unwanted side effects arise from the variety of functions GPCRs can serve in different tissues. Regulator of G protein Signaling (RGS) proteins are tissue‐selective negative modulators of the initial effector of GPCR activation, the Gα subunit of heterotrimeric G‐proteins. Targeting RGS proteins instead of GPCRs provides a novel way to address disease while minimize off‐target effects. RGS2 is a Gα q ‐specific GTP‐ase activating protein, reduced levels of which are implicated in cardiovascular disease, anxiety, and various types of cancers. A key regulatory mechanism for RGS2 is its rapid degradation via the ubiquitin‐proteasomal pathway. We previously demonstrated that RGS2 is targeted for proteasomal degradation via a novel Cullin‐RING E3 ligase complex. The substrate recognition component of this E3 ligase is F‐box Only Protein 44 (FBXO44) and inhibiting the interaction with FBXO44 would represent a novel strategy to stabilize RGS2 levels. For this study, we hypothesized that the N‐terminal of RGS2 binds FBXO44. To investigate this hypothesis we utilized western blotting, siRNA knock‐down, co‐immunoprecipitation, and a β‐galactosidase‐dependent chemiluminescent assay. We examined N‐terminally truncated mutants of RGS2 based on previously reported alternative translation initiation sites at methionine 5, 16 and 33 (M5, M16 and M33, respectively). We found that truncating the N‐terminal of RGS2 resulted in reduced ubiquitination when treated with the proteasome inhibitor MG‐132 in addition to preventing co‐immunoprecipitation with FBXO44. In addition, siRNA knockdown of FBXO44 only stabilized full‐length RGS2 as well. However, MG‐132 treatment stabilized full‐length RGS2 as well as the M5 variant, but not M16 or M33, as demonstrated by both western blotting and a chemiluminescent enzyme complementation assay. To explain the differences between full‐length and M5 RGS2, we mutated Serine 3 to Alanine (S3A, phospho‐dead) or Aspartic acid (S3D, phospho‐mimetic), hypothesizing that loss of a phosphorylation site would destabilize M5 if it retained the FBXO44‐interaction site. We found that WT and S3A RGS2 were stabilized by MG‐132 treatment, but S3D was not. In addition, less S3D RGS2 co‐immunoprecipitated with FBXO44 compared to WT or S3A indicating that phosphorylation at Ser3 prevents RGS2 from binding FBXO44. Altogether our data indicate that RGS2 binds FBXO44 through an N‐terminal motif located between residues 5 and 16, and that phosphorylation at Ser3 can inhibit this interaction and prevent RGS2 degradation. Ongoing studies aims to further define the interaction site between RGS2 and FBXO44, as well as developing high‐throughput screening strategies to identify small molecule RGS2‐FBXO44 interaction inhibitors. These would be promising leads in disease states associated with low RGS2 protein levels. Support or Funding Information American Heart Association (15SDG21630002) Ralph W. and Grace M. Showalter Research Trust