Structure, function, and inhibition of G protein-coupled receptor kinases

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Project Summary G protein-coupled receptor (GPCR) kinases (GRKs) phosphorylate activated GPCRs at multiple sites within their cytoplasmic loops and C-terminal tails, leading to the recruitment of arrestins, uncoupling of the receptors from heterotrimeric G proteins, and subsequently their internalization. Although GRK activity in healthy cells allows them to adapt and avoid damage from sustained signaling, maladaptive overexpression of GRKs is strongly associated with different pathologies including cardiovascular diseases such as heart failure and maladaptive cardiac hypertrophy. A critical gap in our understanding of these processes is how GRKs recognize activated GPCRs and how these GPCRs in turn activate the GRKs. Although we have been successful at generating selective and potent inhibitors of GRK2, another critical gap is the lack of validated chemical probes for GRK5, which plays a distinct role from GRK2 in cardiovascular disease although both have been shown to be up-regulated in failing human myocardium. During the last funding cycle, our lab developed derivatives of paroxetine, an FDA approved drug, that potently and selectively inhibit GRK2 activity in vitro and in vivo, and that improve heart failure outcomes relative to paroxetine alone in myocardial infarcted mice. We have now also identified a class of covalent inhibitors based on a different scaffold that are specific for GRK5. Furthermore, we developed methodology to trap an agonist and GRK-ligand dependent GRK– GPCR complex that is suitable for structural analysis by cryo electron microscopy (cryo-EM). In the first aim of this competitive renewal, we will further develop and characterize analogs of the GRK2 and GRK5 subfamilies based on the structure-activity relationships we have previously established. The best inhibitors will be evaluated in cell-based and animal models relevant to human disease and will be used assess the relative importance of GRK2 and GRK5 in cardiac physiology and disease. In the second aim, we will perform a cryo- EM single particle reconstruction of the GRK1-rhodopsin complex, use this complex to select for nanobodies that specifically recognize the complex, and extend our methodology to other GRK–receptor assemblies. Collectively, our studies will contribute to a chemical “tool box” that can be used to help decipher the function of specific GRKs in living cells and disease states, and that can be exploited to achieve a better understanding of how GRKs interact with cellular targets using biophysical approaches.