tt is becoming increasingly clear that G protein-coupled receptor (GPCR) kinases (GRKs} can play a critical role in modulating myocardial signaling and function. GRKs phosphorylate agonist-occupied GPCRs triggering the process of desensitization, leading to the toss of down-stream signaling. The actions of GRKs appear to become especially important during conditions of compromised heart function such as heart failure (HF). The most abundant GRK in the heart is GRK2, which is commonly known as the beta-adrenergic receptor kinase (betaARK1). GRK5 is a second GRK expressed in the heart and both GRK5 and betaARK1 have been shown to be up-regulated in cardiac hypertrophy and HF models including increased mRNA, protein and GRK activity of betaARK1 in human HF. We have previously shown that genetic manipulation of betaARK1 and GRK5 in the heart can have profound effects on cardiac function. This includes cardiac overexpression of these two GRKs in the hearts of transgenic mice that has led to cardiac contractile dysfunction manifested by the loss of betaAR inotropic reserve. Alternatively, there is an enhancement of cardiac function, including in HF, when a peptide inhibitor of betaARKt (betaARKct) is expressed in the heart. The betaARKct is the last 194 carboxyl-terminal amino acids of this GRK and acts by inhibiting the activation of betaARK1 through binding to the beta-gamma-subunits of heterotrimeric G proteins (Gbeta-gamma). These studies have shown that the betaARKct region is critical for in vivo regulation of betaARK1 and this GRK appears to be a key element in HF pathogenesis. However, other regions of GRKs aside from the carboxyl terminus of betaARK1 and GRK5 may also have important roles in GPCR regulation, indeed, the amino terminus of these two GRKs have domains that can interact with proteins such as Galpha-q, caveolin and specific protein phosphatase 2A (PP2A) subunits that could have novel GPCR implications in cardiac signaling and function. This includes a putative Regulator of G protein Signaling (RGS) domain and this region of betaARK1 has been shown to have RGS function against the G protein, Gq, which interestingly, is the triggering signal of pressure overload cardiac hypertrophy. Therefore, targeting non-Gbeta-gamma domains within GRKs (i.e. the amino terminus) may lead to a more specific mechanistic understanding of GRK activity in the heart. Our Central Hypothesis is that specific domains within GRKs play a key role in the signaling and physiology of the heart that becomes critical in hypertrophied and failing myocardium and dissecting functional regions of these kinases involving novel GPCR (or non-GPCR) interactions in vivo will lead to a broader understanding of the pathogenesis of HF.
Specific Aims are (1) To generate and characterize transgenic mice with cardiac-targeted expression of the RGS domains of barkARK1 and GRK5 to determine their in vivo role in myocardial signaling and function; (2) To determine if these RGS peptides expressed in the hearts of mice can alter abnormal G protein signaling and cardiac phenotypes in murine models of hypertrophy and HF; (3) To determine the cardiac consequences of amino terminal GRK peptides when delivered exogenously to larger animal models of hypertrophy and HF via intracoronary adenoviral-mediated gene transfer; (4) To investigate the novel interaction between betaARK1 and a specific regulatory subunit of PP2A, and to determine its potential rote in cardiac GPCR signaling.
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