Abstract
Hypertension and heart failure are major health issues with a need to identify more effective treatments. Regulator of G protein Signaling 2 (RGS2) is highly expressed throughout the cardiovascular system and negatively regulates signaling via several G protein-coupled receptors known to mediate vasoconstriction and hypertrophic responses. As a result, RGS2 KO mice are hypertensive, show enhanced responses to vasoconstrictors and lower tolerability to pressure overload. We have shown that pharmacologically enhanced RGS2 protein expression has functional effects on G protein signaling. Thus, stabilizing RGS2 would be novel route for cardiovascular therapies.
RGS2 is rapidly degraded through the ubiquitin-proteasomal pathway and identifying the specific molecular mechanisms involved in RGS protein degradation will enable rational drug discovery efforts for stabilizers of RGS2 protein. Using high-throughput siRNA screening we identified a novel Cullin 4B/DNA Damage Binding protein 1/F-box protein 44 (CUL4B/DDB1/FBXO44) E3 ligase that mediates RGS protein degradation. FBXO44 is a poorly characterized member of over 50 F-box proteins that confer substrate recognition in Cullin-RING E3 ligases. Despite high homology with a family of glycoprotein-binding F-box proteins (e.g. FBXO2), FBXO44 does not bind these substrates. Furthermore, although FBXO44 also associates with CUL1/Skp1, this complex is unable to mediate RGS2 degradation. Thus, the way FBXO44 recognizes its substrates seems to occur via unique molecular mechanisms.
Our current work is focused on the molecular characteristics underlying FBXO44 substrate recognition. FBXO44 associates with CUL4B and DDB1 through its conserved F-box domain, whereas association with RGS2 occurs through its F-box Associated (FBA) domain, which is much more variable. Based on the homology between FBXO44 and FBXO2 we are using site-directed mutagenesis to identify substrate recognition sites for RGS2 in FBXO44. Mutations in non-conserved regions of the FBA domain are revealing key interaction sites with RGS2. These studies will aid in rational design of small molecule stabilizers of RGS2 protein that could serve as novel therapeutics in disease states associated with low RGS2 protein levels.
