Nuclear translocation of cardiac G protein-Coupled Receptor kinase 5 downstream of select Gq-activating hypertrophic ligands is a calmodulin-dependent process.

G protein-Coupled Receptors (GPCRs) kinases (GRKs) play a crucial role in regulating cardiac hypertrophy. Recent data from our lab has shown that, following ventricular pressure overload, GRK5, a primary cardiac GRK, facilitates maladaptive myocyte growth via novel nuclear localization. In the nucleus, GRK5\u27s newly discovered kinase activity on histone deacetylase 5 induces hypertrophic gene transcription. The mechanisms governing the nuclear targeting of GRK5 are unknown. We report here that GRK5 nuclear accumulation is dependent on Ca(2+)/calmodulin (CaM) binding to a specific site within the amino terminus of GRK5 and this interaction occurs after selective activation of hypertrophic Gq-coupled receptors. Stimulation of myocytes with phenylephrine or angiotensinII causes GRK5 to leave the sarcolemmal membrane and accumulate in the nucleus, while the endothelin-1 does not cause nuclear GRK5 localization. A mutation within the amino-terminus of GRK5 negating CaM binding attenuates GRK5 movement from the sarcolemma to the nucleus and, importantly, overexpression of this mutant does not facilitate cardiac hypertrophy and related gene transcription in vitro and in vivo. Our data reveal that CaM binding to GRK5 is a physiologically relevant event that is absolutely required for nuclear GRK5 localization downstream of hypertrophic stimuli, thus facilitating GRK5-dependent regulation of maladaptive hypertrophy

Protein-coupled receptor kinase 5 exacerbates cardiac pathology through activation of the nuclear factor of activated T cells pathway

G protein-coupled receptor (GPCR) kinases (GRKs) serve to regulate the contractile function of the heart. The upregulation of both GRK2 and GRK5, the predominant GRKs expressed in the heart, has been shown in failing human myocardium. While the canonical role of GRKs is to desensitize receptors via phosphorylation, it has been demonstrated that GRK5, unlike GRK2, can reside in the nucleus of cardiomyocytes and exert GPCR-independent effects that promote maladaptive cardiac hypertrophy and heart failure (HF). We find that cardiac-specific NFAT luciferase reporter mice crossed with mice that overexpress wild-type GRK5 in a cardiomyocyte specific manner exhibit an increase in NFAT activity both in the basal state as well as after the hypertrophic stressors transaortic constriction and phenylephrine administration. Complimentary to this, GRK5 null mice exhibit less NFAT transcriptional activity after left ventricular pressure-overload as shown by the expression of the NFAT target gene RCAN. Importantly, loss of NFATc3 expression protected GRK5 over-expressing mice from the exaggerated hypertrophy and early progression to HF seen after TAC. Molecular studies suggest that GRK5 acts in concert with NFAT to increase hypertrophic gene transcription in the nucleus and this is a kinase-independent action of GRK5 that may involve its known property of DNA binding. This study provides another non-canonical role for GRK5 in activating the hypertrophic NFAT pathway in cardiomyocytes and may serve as a building block for the development of novel therapeutics which are capable of inhibiting and reversing the progression of HF

Differential role of g protein-coupled receptor Kinase 5 in physiological versus pathological cardiac hypertrophy

RATIONALE: G protein-coupled receptor (GPCR) kinases (GRKs) are dynamic regulators of cellular signaling. GRK5 is highly expressed within myocardium and is up-regulated in heart failure (HF). Although GRK5 is a critical regulator of cardiac GPCR signaling, recent data has uncovered non-canonical activity of GRK5 within nuclei that plays a key role in pathological hypertrophy. Targeted cardiac elevation of GRK5 in mice leads to exaggerated hypertrophy and early HF after transverse aortic constriction (TAC) due to GRK5 nuclear accumulation. OBJECTIVE: In this study we investigated the role of GRK5 in physiological, swimming induced hypertrophy (SIH). METHODS AND RESULTS: Cardiac-specific GRK5 transgenic mice (TgGRK5) and non-transgenic littermate control (NLC) mice were subjected to a 21-day high intensity swim protocol (or no swim sham controls). SIH and specific molecular and genetic indices of physiological hypertrophy were assessed including nuclear localization of GRK5 and compared to TAC. Unlike after TAC, swim-trained TgGRK5 and NLC mice exhibited similar increases in cardiac growth. Mechanistically, SIH did not lead to GRK5 nuclear accumulation, which was confirmed in vitro as insulin-like growth factor-1, a known mediator of physiological hypertrophy, was unable to induce GRK5 nuclear translocation in myocytes. We found specific patterns of altered gene expression between TAC and SIH with GRK5 overexpression. Further, SIH in post-TAC TgGRK5 mice was able to preserve cardiac function. CONCLUSIONS: These data suggest that while nuclear-localized GRK5 is a pathological mediator after stress, this non-canonical nuclear activity of GRK5 is not induced during physiological hypertrophy

Differential role of g protein-coupled receptor Kinase 5 in physiological versus pathological cardiac hypertrophy

Corals in the Eastern Pacific extend south from the Gulf of California to Ecuador and oceanic Chile, and west from Colombia to Clipperton Atoll. Nevertheless, large stretches of the Mexican Pacific remain fundamentally unstudied. Therefore, to assess the current conditions of coral communities, a coastal fringe ?300km long (17�40?N, 101�39?W to 16�46?N, 99�49?W) was surveyed within the Southern Mexican Pacific, between 2005 and 2009. Fifteen stony coral species were identified at 13 coral communities and six Pocillopora-dominated fringing reefs, with Pocillopora verrucosa and Pocillopora damicornis the primary contributing taxa. Reef development was identified in embayments or behind rocks or islands that offered shelter from northern and northwestern winds. Observations of Pocillopora effusus, Pocillopora inflata, Porites lobata, Pavona clavus, and Pavonavarians expanded the species known geographic ranges by several degrees of latitude, suggesting reef building fauna comprised a mixture of widespread and relatively rare Eastern Pacific corals. Results indicated greater live coral cover in the Ixtapa-Zihuatanejo area (15-73%) than in the Acapulco localities, which had high algal dominance; the reefs in the latter region exhibited high erosion. Regional differences are likely the result of long-standing anthropogenic pressures around Acapulco since 1950, when it became an important tourist destination. This paper is the first detailed report of ecologically stressed corals and coral reefs from the state of Guerrero on the Mexican Southern Pacific coast. � 2012 Blackwell Verlag GmbH

GRK5W30A displays increased plasma membrane association following agonist treatment and differential ability to desensitize GPCRs compared to WT.

<p>(<b>A</b>) AdRbM were infected with an adenovirus expressing GRK5-GFP or GRK5W30A-GFP and cultured overnight. Using TIRFM cells were imaged at 10 sec intervals for 700 sec. Baseline myocytes were untreated while stimulated myocytes were treated with either AngII (10 µM) (A) or PE (50µM) (<b>B</b>) at 120 sec. Fluorescence was normalized and reported to fold change versus baseline. n = 4 (<b>C</b>) Changes in GRK5 activity at the membrane was measured using an IP₁ ELISA to determine changes in desensitization. NRVM were infected with Ad-LacZ, Ad-GRK5 or Ad-GRK5W30A. After 48 hours, cells were stimulated with PE or ET-1 for 2 hr, then assayed for IP₃ generation via IP₁ ELISA. *p<0.01 v. LacZ PE and WT PE, #p<0.01 v. LacZ ET-1, one-way ANOVA with a Bonferroni correction, n = 3, done in duplicate.</p

GRK5 nuclear accumulation is diminished after treatment with a CaM inhibitor.

<p>(<b>A</b>) NRVM were infected with Ad-GRK5 and either Ad-LacZ or Ad-Gq-CAM. 48 hr after infection, cells were treated with DMSO or inhibitor: BIM1 (10 µM), Gö6976 (10 µM), CDZ (10 µM) and KN93 (10 µM) for 1 hr. The cells were harvested using subcellular fractionation and immunoblotted for GRK5. (<b>B</b>) Immunoblots were quantitated by densitometry, normalized to fibrillarin, and reported as fold change over baseline. * p<0.05 v. untreated baseline, # p<0.05 v. CDZ, one-way ANOVA with a Bonferroni correction, n = 4. (<b>C</b>) NRVM were infected with Ad-LacZ, Ad-GRK5 and Ad-Gq-CAM. 48 hr after infection, cells were treated with DMSO or CDZ (10 µM) for 1 hr. The cells were harvested using subcellular fractionation, and immunoblotted for GRK5. (<b>D</b>) Densitometric analysis for (<b>C</b>) with GRK5 normalized to fibrillarin and calculated as fold change over baseline. *p<0.01 v. DMSO GRK5, #p<0.01 v. DMSO GRK5+ Gq, one-way ANOVA with a Bonferroni correction, n = 4. (<b>E</b>) NRVM were infected with either Ad-LacZ or Ad-Gq-CAM. 48 hr after infection, cells were treated with DMSO or CDZ (10 µM). Immunofluorescence was detected using a polyclonal GRK5 antibody. (<b>F</b>) TIRF analysis of AdRbM infected with an adenovirus expressing GRK5-GFP and cultured overnight. Cells were imaged at 10 sec intervals for 700 sec. Cells were pre-treated with CDZ or DMSO for 30 min at 37°C prior to imaging. Baseline myocytes were untreated while stimulated myocytes were treated with PE (50 µM) at 120 sec. Fluorescence was normalized and reported to fold change versus baseline. n = 4. (<b>G</b>) Same experimental design as (<b>F</b>) except cells were stimulated with AngII (10 µM) at 120 s. n = 4.</p

Cartoon depicting the select Gq-coupled receptor CaM-mediated translocation of GRK5 into the nucleus of cardiomyocytes.

<p>Gq activation due to catecholamines or AngII binding at the α₁AR or AT-1R, respectively, causes CaM to bind GRK5 at its N-terminus, dislodging GRK5 from the plasma membrane. Via its NLS, GRK5 is directed to the nucleus where its interaction with CaM is stabilized by IP₃R-regulated Ca²⁺ release. Once in the nucleus, GRK5 can act as an HDAC5 kinase, relieving repression of MEF2 and inducing hypertrophic gene transcription. In contrast, endothelin-1 binding leads to a selective interaction between the ET-1R substrate and the desensitizing GRK5. CaM cannot bind the kinase in this state, thus keeping GRK5 at the plasma membrane.</p

PE and AngII induce translocation of GRK5 from the membrane to the nucleus.

<p>(<b>A</b>) Representative immunofluorescence staining of endogenous GRK5 in AdRbM shows increased nuclear GRK5 following PE (50 µM) and AngII (10 µM) treatment, but not ET-1 (100 nM). (<b>B</b>) NRVM were infected with Ad-GRK5 (50 MOI). After 48 hr, cells were treated with 50 µM PE for 5 different time points, harvested by subcellular fractionation. Nuclear were fractions immunoblotted for GRK5 and fibrillarin. (<b>C</b>) The amount of GRK5 in the nucleus was calculated by denistometry, normalized to fibrillarin, and reported as Fold Change over baseline. *p<0.05, one-way ANOVA with a Bonferroni correction, n = 4. (<b>D</b>) Rabbit myocytes were infected with an adenovirus expressing GRK5-GFP and cultured overnight. Using TIRFM cells were imaged at 10 sec intervals for 700 sec. Baseline myocytes were untreated while stimulated myocytes were treated with either PE (50 µM), AngII (10 µM), or Et-1 (100 nM) at 120 sec. Fluorescence was normalized and reported as fold change versus baseline. n = 4. (<b>E</b>) Representative TIRF images for each agonist at the beginning and end of imaging.</p

A mutant GRK5 (W30AK31Q) unable to bind CaM at its N-terminal CaM binding site displays less nuclear accumulation following Gq or PE stimulation.

<p>(<b>A</b>) Cartoon of GRK5’s structure illustrating pertinent domains and regulatory sites. (<b>B</b>) NRVM infected with Ad-GRK5 or Ad-GRK5W30A were stimulated with Ad-Gq-CAM (48 hr) or PE (1hr). Cells were then harvested by subcellular fractionation and immunoblotted for GRK5. (C) Quantitative analysis of (<b>B</b>) normalized to fibrillarin and reported as fold change over baseline. *p<0.001 v. WT GRK5, one-way ANOVA with a Bonferroni correction, n = 4. (<b>D</b>) AdRbM were co-infected with an adenovirus expressing either WT GRK5 tagged with GFP or GRK5 W30A tagged with GFP and Ad-Gq-CAM. Following an overnight culture, cells are imaged by confocal microscopy. Fluorescence within the nucleus was measured and normalized to cytoplasmic fluorescence. *p<0.001 v. WT GRK5+ Gq, one-way ANOVA with a Bonferroni correction, n = 4 (<b>E</b>) Images of representative myocytes showing WT GRK5-GFP (left) and GRK5W30A-GFP (right). (<b>F</b>) MEF2 activity in NRVM was measured using a luciferase assay system. Cells were co-infected with an adenovirus expressing a MEF2-luciferase reporter construct, Ad-LacZ, Ad-GRK5 or Ad-GRK5W30A and stimulated for 48 hr with the Ad-Gq-CAM virus. *p<0.001 v. WT GRK5, one-way ANOVA with a Bonferroni correction, n = 4, done in triplicate. Inset shows whole cell lysate of NRVM used in this experiment.</p