Project Summary Regulator of G protein Signaling (RGS) proteins play a key modulatory role in G Protein-coupled receptor (GPCR) signaling. Through both G protein-dependent and -independent mechanisms, RGS proteins play important roles in disease, and this has driven numerous efforts to pharmacologically target their function. These efforts are, however, hindered by the fact that RGS proteins are considered difficult drug targets, and identifying mechanisms that control RGS protein activity, expression and/or subcellular localization has therefore become an important area of investigation. Our long-term goal is to determine how levels and activity of RGS proteins is regulated, with a view of identifying druggable “soft-spots” within the regulatory network. Our rationale for the current proposal is that identification of specific mechanisms regulating RGS2 will uncover therapeutic points of intervention to increase RGS protein levels and activity. Low RGS2 protein levels or activity are associated with a wide range of pathologies, including hypertension, heart failure and asthma, and our central hypothesis is that pharmacologically enhancing RGS2 protein levels would have broad clinical implications. Our objective in this proposal is to decipher posttranslational mechanisms regulating expression, activity and subcellular localization of RGS2. We will focus on two crucial, but understudied, mechanisms regulating RGS2 protein levels and activity; proteasomal degradation and phosphorylation. We identified a Cullin-RING E3 ligase (CRL) targeting RGS2 for proteasomal degradation, and recently identified the degron in RGS2 that is recognized by F-box only protein 44 (FBXO44), the substrate-recognizing component of the CRL. In Aim 1 we will use biochemical and structural approaches to determine how FBXO44 interacts with RGS2. We will also determine whether FBXO44-RGS2 binding is determined by the associated CRL. FBXO44 degrades RGS2 only in the context of a CUL4B/DDB1, but not a CUL1/Skp1 complex in cells suggesting that FBXO44 RGS2 specificity may depend on the nature of the CRL. In Aim 2 we will determine the role of phosphorylation for RGS2 function. Phosphorylation plays a central role in signal transduction cascades, however there is a lack of comprehensive information on the global role of phosphorylation for RGS2 protein function. Our previous studies identified potential importance for PKC and Src kinase in regulating RGS2. We will determine which residues are phosphorylated, as well as the consequence for RGS2 protein stability, using a wide range of techniques, including PhosTag gel electrophoresis, in vitro kinase activity assays, as well as LC-MS. In Aim 3 we will determine the functional consequences of altered posttranslational RGS2 regulation. We will determine effects on G protein-dependent and -independent RGS2 functions in both transfected cells, physiologically relevant cell lines and ex vivo models. The expected outcome of these studi...