Originally cloned in 1993 from human heart cDNA, G protein-coupled receptor kinase 5 (GRK5) is a serine/threonine kinase and the fifth member of the GRK family to be discovered (Kunapuli and Benovic 1993). Proteins of the GRK family are categorized into subgroups that are based upon kinase homology, amino acid sequence similarity, and tissue expression patterns. These subgroups are: the rhodopsin kinase subfamily (GRK1 and GRK7), the β-adrenergic receptor kinase (β-ARK) subfamily (GRK2 and GRK3), and the GRK4-like subfamily (GRK4, GRK5, and GRK6) (Nagayama et al. 1996; Xu et al. 2014).
Structure, Function, and Regulation
Many regulatory mechanisms exist to augment or attenuate the catalytic activity of GRK5. It has been demonstrated that dimerization and autophosphorylation positively regulate the activity of GRK5 (Kunapuli et al. 1994; Xu et al. 2014). Dimerization utilizing the short-RGS domain facilitates plasma membrane localization and markedly enhances the catalytic activity of GRK5 (Xu et al. 2014). Similarly, autophosphorylation at two conserved residues (Ser-484 and Thr-485) augments the kinase activity of GRK5 towards receptor substrates (Kunapuli et al. 1994). GRK5 is also positively regulated by the phospholipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), which promotes membrane localization through binding to a small conserved region of the N-terminus (Pitcher et al. 1996). In contrast, both Ca2+/CaM binding, CaM-dependent autophosphorylation, and phosphorylation by protein kinase C (PKC) can negatively regulate GRK5 function (Chuang et al. 1996; Pronin and Benovic 1997; Pronin et al. 1998). CaM directly associates with GRK5 in a Ca2+-dependent fashion at its CaM-binding domains and inhibits its membrane localization and phosphorylation of receptors (Chuang et al. 1996). CaM-dependent autophosphorylation of GRK5 at the autoinhibitory domain attenuates the kinase activity of GRK5 towards receptor substrates (Pronin et al. 1998). PKC-mediated phosphorylation of GRK5 at two C-terminal regions attenuates the catalytic activity of GRK5 for both receptor and nonreceptor substrates (Pronin and Benovic 1997). Lastly, physical association with actin negatively regulates the kinase activity of GRK5 (Freeman et al. 1998). Actin binds to basic residues found within the N-terminal region of GRK5, which contains an overlapping Ca2+/CaM binding domain (Freeman et al. 1998). Ca2+/CaM competes with actin for binding to the N-terminal region and permits the phosphorylation of nonreceptor, but not receptor, substrates by GRK5 (Freeman et al. 1998). Overall, the substrate specificity of GRK5 is likely determined by the interplay of regulatory mechanisms, including actin, PKC, and Ca2+/CaM, that act on GRK5 under a given cellular context (Chuang et al. 1996; Pronin and Benovic 1997; Freeman et al. 1998).
The GRK5 protein also carries out many noncanonical functions, including kinase-dependent and kinase-independent regulation of non-GPCR receptor and nonreceptor substrates. Some examples of well-characterized noncanonical targets include: the low-density lipoprotein receptor-related protein 6 (LRP6) essential in the canonical Wnt signaling pathway, the tumor protein 53 (p53), and the cytoskeletal protein tubulin (Carman et al. 1998; Chen et al. 2009, 2010) (Fig. 2). Additionally, GRK5 can translocate to the nucleus, where it can carry out other noncanonical functions (Johnson et al. 2013). Nuclear GRK5 can phosphorylate histone deacetylase 5 (HDAC5) and stabilize IκBα, the inhibitory protein of the nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) signaling complex, in the nucleus through protein-protein interaction (Martini et al. 2008; Sorriento et al. 2008) (Fig. 2). GRK5 has also been shown to directly bind DNA in vitro (Johnson et al. 2013). A functional NLS sequence and the N-terminal CaM-binding site appear to be integral to DNA binding (Johnson et al. 2013). In contrast, CaM binding to the C-terminal site and autophosphorylation attenuate the DNA-binding capacity of GRK5 (Johnson et al. 2013). Lastly, CaM binding to the N-terminal site exposes the NES, promoting the export of GRK5 from the nucleus to the cytoplasm (Johnson et al. 2004, 2013).
GRK5 in Cancer
Recent literature within the past two decades has highlighted a putative role of GRK5 in the pathophysiology of various cancers including prostate cancer and glioblastoma (Kim et al. 2012; Kaur et al. 2013; Chakraborty et al. 2014). As a potential oncogene, GRK5 has the capacity to phosphorylate the tumor suppressor protein p53 and inhibit p53-mediated DNA damage–induced apoptosis (Chen et al. 2010). GRK5-mediated phosphorylation of p53 promotes binding to mouse double minute 2 homolog (MDM2), leading to p53 ubiquitination and degradation (Chen et al. 2010). GRK5 can also phosphorylate nucleophosmin 1 (NPM1), a protein that contributes to the regulation of apoptosis and the cell cycle (So et al. 2012). GRK5 phosphorylates NPM1 at Ser-4, a site that is also phosphorylated by polo-like kinase 1 (PLK1), a therapeutic target for various cancers (So et al. 2012). High-GRK5 expression renders cells less sensitive to apoptosis induced by PLK1 inhibition (So et al. 2012). Lastly, GRK5 has been demonstrated to augment Wnt signaling, which has been implicated in tumor growth and metastasis (Chen et al. 2009). LRP6, a downstream signaling component of the canonical Wnt pathway, is activated upon phosphorylation by GRK5 at the PPPSP motifs, ultimately resulting in stabilization of β-catenin and activation of Wnt target genes (Chen et al. 2009).
In terms of prostate cancer, GRK5 has been shown to promote proliferation of PC3 prostate cancer cells in vitro as well as in xenograft models (Kim et al. 2012). Mechanistically, GRK5 affects cell cycle progression by increasing the expression of cyclin D1 and the phosphorylation of retinoblastoma protein (Rb), both of which are necessary for regulating the G1-to-S transition (Kim et al. 2012). GRK5 also affects the distribution of moesin, an actin-remodeling protein implicated in the metastasis of prostate cancer (Chakraborty et al. 2014). In vivo knockdown of GRK5 hinders tumor growth and reduces the probability of cancer metastasis (Chakraborty et al. 2014). Overall, the oncogene nature of GRK5 highlights the importance in further researching GRK5 as a potential therapeutic target in the treatment of cancer.
GRK5 in the Brain
A number of studies using mouse models have implicated GRK5 in the pathogenesis of Alzheimer’s disease (AD). In APP(swe) transgenic mice, which overexpress the Aβ precursor protein (APP) carrying the Swedish mutation found in some early-onset AD patients, ablating one copy of the grk5 gene alters the processing of APP such that β-amyloidogenesis and Aβ plaque formation are favored (Cheng et al. 2010). APP(swe) mice also display reduced membrane localization of GRK5, suggesting that membrane proximal functions of GRK5 may be compromised (Zhang et al. 2014). Similar reductions in membrane localized GRK5 were also found in Tg-CRND8 mice, another transgenic model of early-onset AD, and precede the decline in cognitive function (Suo et al. 2004). The authors suggested that the accumulation of Aβ can feedback to reduce expression of membrane-localized GRK5, resulting in attenuation of GPCR desensitization (Suo et al. 2004). On its own, grk5 gene deficiency in mice leads to age-dependent AD-like pathology including abnormal axonal morphology and high levels of intracellular Aβ and microtubule-associated proteins within the hippocampus (Suo et al. 2007). Grk5-null mice also exhibit reduced expression of synaptic proteins and early signs of cholinergic neurodegeneration with functional impairments in working memory (Suo et al. 2007). Hypofunction of cholinergic neurons, which has been hypothesized to be the primary cause of cognitive decline in AD, may be the mechanistic link between GRK5 and AD. Indeed, deficient GRK5 signaling in cholinergic hippocampal neurons inhibits acetylcholine (ACh) release by interfering with the desensitization of presynaptic muscarinic 2/4 (M2/M4) autoreceptors (Liu et al. 2009). In line with these results, grk5-null mice carrying the APP(swe) transgene exhibit significant loss of cholinergic neurons in the basal forebrain compared with both of the single mutants (He et al. 2016). The involvement of GRK5 in the overall pathophysiology of AD, and the observation that deregulated GRK5 signaling occurs prior to cognitive decline, indicates a potential role of the GRK5 signaling pathway as a therapeutic target for delaying and preventing the progression of this disease.
GRK5 has also been implicated in regulating normal dendritic spine development and morphology within the hippocampus (Chen et al. 2011). GRK5 affects the formation and growth dynamics of filopodia by acting as a bundling protein for filamentous actin (F-actin) near the plasma membrane (Chen et al. 2011) (Fig. 2). GRK5-bundled F-actin is targeted to the plasma membrane by PI(4,5)P2-binding of GRK5 (Chen et al. 2011). The impact of grk5 ablation on neuronal morphogenesis may underlie the deficits in hippocampal-dependent learning and memory that are exhibited by grk5-null mice (Chen et al. 2011).
More recently, GRK5 has been shown to play a role in hippocampal neurogenesis (Zhang et al. 2015). Induction of NFκB signaling upregulates GRK5 expression in cultured neural stem cells (NSCs) and promotes neuronal differentiation (Zhang et al. 2015). Conversely, knockdown of GRK5 within NSCs inhibits both neuronal differentiation and axonal growth, while increasing apoptosis (Zhang et al. 2015).
Lastly, GRK5 has been implicated in the pathophysiology underlying morphine addiction (Glück et al. 2014). Morphine administration in mice results in the phosphorylation of Ser-375 of the mu opioid receptor through the recruitment of GRK5 (Glück et al. 2014). More notably, grk5-null mice exhibit a reduction in physical dependence to morphine compared with wild-type animals (Glück et al. 2014). These results suggest that GRK5 may be a promising target for the therapeutic treatment of morphine addiction (Glück et al. 2014).
GRK5 in Cardiac Pathophysiology
Multiple studies have pointed to a role of GRK5 in cardiovascular function and disease. GRK5 is expressed in healthy human heart but is significantly upregulated in the left ventricular chamber of dilated cardiomyopathy and volume overload patients (Dzimiri et al. 2004). In vascular smooth muscle cells, aortic hemodynamic stress markedly increases GRK5 expression at the mRNA and protein level in a calcium-dependent manner (Ishizaka et al. 1997). GRK5 can regulate the activity of myocardial β-adrenergic receptors (β-AR) in vivo, leading to uncoupling and desensitization of β-AR receptors (Rockman et al. 1996). However, GRK5 has also been detected in the nucleus of cardiomyocytes, where it may act in a noncanonical fashion (Johnson et al. 2004). Transgenic mice overexpressing cardiac GRK5 exhibit greater myocardial hypertrophy and a higher incidence of early heart failure (Martini et al. 2008). Induction of pressure-induced cardiac stress stimulates Gq activation, which initiates nuclear translocation and accumulation of GRK5 through CaM binding to the N-terminal domain (Martini et al. 2008; Gold et al. 2013). Myocardial stress-induced nuclear accumulation of GRK5 results in GRK5-dependent transcription of hypertrophic genes, including myocyte enhancer factor 2 (MEF2) (Martini et al. 2008). Mechanistically, GRK5 functions as an HDAC kinase downstream of Gq activation, initiating the expression of hypertrophy-related genes by inhibiting the transcriptional repressive activity of HDACs (Martini et al. 2008; Gold et al. 2013). Moreover, GRK5 cooperates with the transcription factor, nuclear factor of activated T-cells (NFAT), through direct DNA binding to induce the transcription of target hypertrophic genes (Hullmann et al. 2014) (Fig. 2). A third transcriptional pathway that controls hypertrophic gene expression is NFκB (Purcell et al. 2001; Islam et al. 2013). GRK5 is a positive regulator of the NFκB-associated proteins, p50 and p65, within cardiac myocytes (Islam et al. 2013). Furthermore, GRK5 was shown to phosphorylate p65, consequently increasing NFκB DNA binding and downstream transcription of target hypertrophic genes (Islam et al. 2013). Notably, a GRK5 polymorphism (GRK5-Leu41) that is common in African Americans has been associated with decreased mortality in individuals experiencing heart failure or cardiac ischemia (Liggett et al. 2008). Mechanistically, GRK5-Leu41 enhances desensitization of the β-AR, leading to a reduction in β-AR signaling (Liggett et al. 2008). Clearly, understanding how GRK5 mediates the physiological response to maladaptive hypertrophic stimuli is essential if one is to use GRK5 as a drug target to combat heart disease.
GRK5 in the Immune System
GRK5 has been implicated in signaling pathways that are critical for both innate and adaptive immunity. One of the signaling pathways that GRK5 can modulate is the Toll-like receptor 4 (TLR4)/NFκB pathway (Sorriento et al. 2008; Patial et al. 2011). GRK5 positively regulates TLR4-dependent phosphorylation and proteasomal degradation of IκBα, enabling the translocation of p65 and subsequent activation of the NFκB pathway (Patial et al. 2011; Packiriswamy et al. 2013). Patial et al. (2011) reported that grk5-null mice show reductions in neutrophil invasion and secretion of TLR4-dependent chemokines and cytokines following immune challenge (Patial et al. 2011). In contradiction to these findings, others have demonstrated that GRK5 facilitates the nuclear accumulation of IκBα through a kinase-independent, RGS domain-dependent physical association with IκBα (Sorriento et al. 2008). Furthermore, overexpression of GRK5 reduces the DNA binding and transcriptional activity of NFκB, resulting in an attenuated injury response, reduction in tumor necrosis factor alpha (TNFα) expression, and increase in apoptosis (Sorriento et al. 2008). GRK5-dependent changes in leukocyte migration and recruitment may depend on cross talk between TLR and chemokine signaling pathways (Fan and Malik 2003). TLR4 activation has been shown to dampen the expression of GRK5, lowering the desensitization of the chemokine receptor, C-X-C motif chemokine receptor 2 (CXCR2), and consequently enhancing neutrophil migration (Fan and Malik 2003).
Lastly, GRK5 has been implicated in the pathogenesis and progression of various bacterial infections. Research on polymicrobial sepsis demonstrated that in the absence of GRK5 in vivo, sepsis-induced inflammation, glucocorticoid secretion, and apoptosis of thymic cells are significantly reduced (Packiriswamy et al. 2013). Moreover, recent research has demonstrated a novel association between resistance to malaria infection and a known human GRK5 missense mutation (rs2230345) (Gupta et al. 2015). GRK5 has also been implicated in the clinical outcome of Escherichia coli (E. coli)-induced pneumonia (Packiriswamy et al. 2016). In E. coli–induced pneumonia, GRK5 negatively regulates the secretion of plasma chemokine ligand 1 (CXCL1)/keratinocyte chemoattractant (KC), thereby reducing neutrophil recruitment and migration and bacterial clearance (Packiriswamy et al. 2016). At nonlethal doses of E. coli, grk5 ablation is beneficial to the reduction of bacterial load and earlier resolution of the inflammation (Packiriswamy et al. 2016). In contrast, at lethal doses of E. coli, grk5-null mice exhibit greater mortality rates compared to wild-type mice (Packiriswamy et al. 2016).
GRK5 is a serine/threonine kinase belonging to the GRK family. The GRK family proteins are categorized into subgroups based on kinase homology, amino acid sequence, and expression patterns. These subgroups include: the rhodopsin kinase subfamily (GRK1 and GRK7), the β-ARK subfamily (GRK2 and GRK3), and the GRK4-like subfamily (GRK4, GRK5, and GRK6) (Nagayama et al. 1996; Xu et al. 2014). The canonical function of GRK5 is to phosphorylate agonist-bound GPCRs, facilitating internalization and desensitization of the receptor (Kunapuli and Benovic 1993). GRK5 can also function in a noncanonical manner, acting on non-GPCR and nonreceptor proteins in a kinase-dependent or kinase-independent fashion (Chen et al. 2009, 2010). Dimerization, autophosphorylation, and phospholipid binding all positively regulate the activity of GRK5 (Kunapuli et al. 1994; Pitcher et al. 1996; Xu et al. 2014). In contrast, Ca2+/CaM binding, CaM-dependent autophosphorylation, and phosphorylation by PKC can negatively regulate the canonical function of GRK5 (Chuang et al. 1996; Pronin and Benovic 1997; Pronin et al. 1998). GRK5 has recently been implicated in the pathophysiology of various cancers as an oncogene, negatively regulating genotoxicity-induced apoptosis driven by p53 and PLK1 (Chen et al. 2010; So et al. 2012). Furthermore, dysfunctional GRK5 signaling has been linked to the pathophysiology of AD (Suo et al. 2004, 2007). Lastly, GRK5 has been demonstrated to mediate the physiological response to hypertrophic signals associated with heart failure and hypertensive conditions (Martini et al. 2008). In the heart, GRK5 works in concert with HDACs, NFAT, and NFκB to initiate the transcription of hypertrophic genes (Martini et al. 2008; Gold et al. 2013; Hullmann et al. 2014).
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