RESUMO
G protein-coupled receptors (GPCRs) convert extracellular stimuli in the form of hormones, odorants and light into profound changes in cell homeostasis. Their timely desensitization is critical for cells to rapidly respond to changes in their environment and to avoid damage from sustained signaling. Seven GPCR kinases (GRKs) phosphorylate and regulate the activity of most of the ~800 GPCRs in the human genome. Although GRKs normally play an adaptive role, in conditions such as chronic heart failure they are overexpressed and linked to disease progression. GRK2 and GRK5 have thus become important targets for the treatment of heart failure and pathological cardiac hypertrophy, respectively. Our lab has determined atomic structures representing all three vertebrate GRK subfamilies, and is now in the midst of a campaign to develop selective inhibitors of these enzymes using structure-based rational design. We have identified the FDA approved drug paroxetine as a selective GRK2 inhibitor, determined the crystal structure of the GRK2·paroxetine complex and, in collaboration with the Koch lab, showed that the drug improves contractility in myocytes and, most impressively, recovery in post-myocardial infarcted mice. Since then, we have identified additional chemical scaffolds that exhibit even higher potency and/or selectivity for GRK5. Using a ″hybrid″ inhibitor design approach we have generated GRK selective chemical probes that exhibit improved potency and stability and are able to increase inotropy and dampen the hypertrophic response in cardiomyocytes and small animal models. Structural analysis has revealed the molecular basis for selectivity and potency in many of these compounds, allowing for the design of future generations of GRK chemical probes.
RESUMO
FOXC1 and FOXC2 are forkhead transcription factors that play essential roles during development and physiology. Despite their critical role, the mechanisms that regulate the function of these factors remain poorly understood. We have identified conserved motifs within a previously defined N-terminal negative regulatory region of FOXC1/C2 that conforms to the definition of synergy control or SC motifs. Because such motifs inhibit the activity of transcription factors by serving as sites of post-translational modification by small ubiquitin-like modifier (SUMO), we have examined whether FOXC1/C2 are targets of SUMOylation and probed the functional significance of this modification. We find that endogenous FOXC1 forms modified by SUMO2/3 can be detected. Moreover, in cell culture, all three SUMO isoforms are readily conjugated to FOXC1 and FOXC2. The modification can be reconstituted in vitro with purified components and can be reversed in vitro by treatment with the SUMO protease SENP2. SUMOylation of FOXC1 and FOXC2 occurs primarily on one consensus synergy control motif with minor contributions of a second, more degenerate site. Notably, although FOXC1 is also phosphorylated at multiple sites, disruption of sites immediately downstream of the SC motifs does not influence SUMOylation. Consistent with a negative functional role, SUMOylation-deficient mutants displayed higher transcriptional activity when compared with wild type forms despite comparable protein levels and subcellular localization. Thus, the findings demonstrate that SC motifs mediate the inhibitory function of this region by serving as sites for SUMOylation and reveal a novel mechanism for acute and reversible regulation of FOXC1/C2 function.
