GRK5 Knockout Mice Research Articles

G-protein coupled receptor kinase-5 mediates Toll-like receptor-4-induced NFκB activation in primary macrophages and in vivo in mice (94.11)

Abstract Toll-like receptor-4 (TLR4) activated by lipopolysaccharide (LPS) is involved in a number of inflammatory diseases, in part via activation of NFκB pathway. G-protein coupled receptor kinase-5 (GRK5), a serine-threonine kinase originally discovered for its role in desensitization of G-protein coupled receptors has recently been shown to regulate NFκB pathway stimulated by non-GPCRs. This study was undertaken to determine the potential role of GRK5 in inflammatory signaling in primary macrophages and in vivo using GRK5 knockout (KO) mice. We demonstrate here that TLR4-induced IκBα-NFκB pathway is selectively inhibited in primary macrophage from GRK5 KO mice compared to cells from GRK5 wild type (WT) mice. Consistent with the effects on the NFκB pathway, LPS-induced production of many inflammatory cytokines/chemokines (including Interleukin-2, -3, -4, -5, -12, -17, MCP-1, IFNγ, KC, GMCSF and Eotaxin) are inhibited in GRK5 KO macrophages. Confirming this in vitro data, LPS-induced cytokines/chemokines levels in the plasma are also significantly inhibited in the GRK5 KO mice in vivo. Associated with these effects LPS-mediated liver injury is markedly decreased in the GRK5 KO mice. Taken together, our findings demonstrate that GRK5 acts as a positive regulator of TLR4 signaling in vivo and further suggest that these findings could have potential implications for drug development in inflammatory diseases.

2009 Thomas W. Smith Memorial Lecture—GRK2 Inhibition as a Novel Therapeutic Target in Heart Failure

For more than 15 years, our laboratory has been researching the role of the G protein–coupled receptor kinases (GRKs) in myocardial function. This includes how GRKs are involved in the cardiovascular pathological processes of hypertension, ventricular hypertrophy, and heart failure. Studies with GRK2 (also known as β ARK1) have shown, from cell studies to whole-animal studies, that the increased activity of this kinase is pathological to the cardiomyocyte. Importantly, lowering its expression or activity has led to beneficial effects in several animal models of heart failure. Most recently, studies in myocyte-specific GRK2-knockout mice have revealed that the loss of GRK2 in heart cells prevents the development of heart failure after a myocardial infarction. In separate animal studies, gene therapy with a GRK2 inhibitor peptide has shown that lowering the activity of this kinase can reverse ventricular contractile failure and also adverse remodeling. Large animal preclinical studies are being done with gene therapy; however, development of small-molecule inhibitors of GRK2 is warranted. This lecture will focus on the translational continuum of research for the past 15+ years in the development of GRK2 as a definitive target for heart failure therapy. The lecture will also include new data supporting unique roles for GRK2 in myocardial signaling and function.

GRK5 Deficiency Leads to Reduced Hippocampal Acetylcholine Level via Impaired Presynaptic M2/M4 Autoreceptor Desensitization

G protein-coupled receptor kinase 5 (GRK5) deficiency has been linked recently to early Alzheimer disease (AD), but the mechanism by which GRK5 deficiency may contribute to AD pathogenesis remains elusive. Here we report that overexpression of dominant negative mutant of GRK5 (dnGRK5) in a cholinergic neuronal cell line led to decreased acetylcholine (ACh) release. This reduction was fully corrected by pertussis toxin, atropine (a nonselective muscarinic antagonist), or methoctramine (a selective M2/M4 muscarinic receptor antagonist). Consistent with results in cultured cells, high potassium-evoked ACh release in hippocampal slices from young GRK5 knock-out mice was significantly reduced compared with wild type littermates, and this reduced ACh release was also fully corrected by methoctramine. In addition, following treatment with the nonselective muscarinic agonist oxotremorine-M, M2, and M4 receptors underwent significantly reduced internalization in GRK5KO slices compared with wild type slices, as assessed by plasma membrane retention of receptor immunoreactivity, whereas M1 receptor internalization was not affected by loss of GRK5 expression. Moreover, Western blotting revealed no synaptic or cholinergic degenerative changes in young GRK5 knock-out mice. Altogether, these results suggest that GRK5 deficiency leads to a reduced hippocampal ACh release and cholinergic hypofunction by selective impairment of desensitization of presynaptic M2/M4 autoreceptors. Because this nonstructural cholinergic hypofunction precedes the hippocampal cholinergic hypofunction associated with structural cholinergic degeneration and cognitive decline in aged GRK5 knock-out mice, this nonstructural alteration may be an early event contributing to cholinergic degeneration in AD.

Augmented axonal defects and synaptic degenerative changes in female GRK5 deficient mice

Recent studies suggested that G protein-coupled receptor kinase 5 (GRK5) deficiency plays a significant role in early Alzheimer’s disease (AD) pathogenesis, and that the GRK5 knockout (GRK5KO) mouse displays an early Alzheimer-like cognitive deficit associated with increased hippocampal axonal defects and synaptic degenerative changes. Gender is known to play a role in AD, with females showing more extensive pathologic changes in brain compared to males. Although GRK5 deficiency is linked to AD, it is unknown whether the pathologic changes solely driven by the GRK5 deficiency are gender-dependent. To determine this, extent of the pathologic changes in aged GRK5KO mice was compared between genders. We find that female GRK5KO mice had a 2.5-fold increase in hippocampal swollen axonal clusters compared to male GRK5KO mice. Moreover, hippocampal levels of several synaptic proteins, including synaptophysin, were significantly lower in the female than the male. In addition, although increased Luteinizing hormone (LH) activity is believed to play a significant role in the gender phenomenon in AD, we found that desensitization of LH receptor is not affected by the GRK5 deficiency. Therefore, the worsened pathologic changes in the female mice cannot be attributed to an impaired LH receptor desensitization. Taken together, this study demonstrates a synergistic interaction between GRK5 deficiency and gender in promoting early AD-like pathologic changes in the female GRK5KO mice, although the underlying molecular mechanisms remain to be elucidated.

Regulation of the Platelet-derived Growth Factor Receptor-β by G Protein-coupled Receptor Kinase-5 in Vascular Smooth Muscle Cells Involves the Phosphatase Shp2

Smooth muscle cell (SMC) proliferation and migration are substantially controlled by the platelet-derived growth factor receptor-beta (PDGFRbeta), which can be regulated by the Ser/Thr kinase G protein-coupled receptor kinase-2 (GRK2). In mouse aortic SMCs, however, we found that prolonged PDGFRbeta activation engendered down-regulation of GRK5, but not GRK2; moreover, GRK5 and PDGFRbeta were coordinately up-regulated in SMCs from atherosclerotic arteries. With SMCs from GRK5 knock-out and cognate wild type mice (five of each), we found that physiologic expression of GRK5 increased PDGF-promoted PDGFRbeta seryl phosphorylation by 3-fold and reduced PDGFRbeta-promoted phosphoinositide hydrolysis, thymidine incorporation, and overall PDGFRbeta tyrosyl phosphorylation by approximately 35%. Physiologic SMC GRK5 activity also increased PDGFRbeta association with the phosphatase Shp2 (8-fold), enhanced phosphorylation of PDGFRbeta Tyr(1009) (the docking site for Shp2), and reduced phosphorylation of PDGFRbeta Tyr(1021). Consistent with having increased PDGFRbeta-associated Shp2 activity, GRK5-expressing SMCs demonstrated greater PDGF-induced Src activation than GRK5-null cells. GRK5-mediated desensitization of PDGFRbeta inositol phosphate signaling was diminished by Shp2 knock-down or impairment of PDGFRbeta/Shp2 association. In contrast to GRK5, physiologic GRK2 activity did not alter PDGFRbeta/Shp2 association. Finally, purified GRK5 effected agonist-dependent seryl phosphorylation of partially purified PDGFRbetas. We conclude that GRK5 mediates the preponderance of PDGF-promoted seryl phosphorylation of the PDGFRbeta in SMCs, and, through mechanisms involving Shp2, desensitizes PDGFRbeta inositol phosphate signaling and enhances PDGFRbeta-triggered Src activation.

β-Arrestin2-mediated inotropic effects of the angiotensin II type 1A receptor in isolated cardiac myocytes

The G protein-coupled receptor kinases (GRKs) and beta-arrestins, families of molecules essential to the desensitization of G protein-dependent signaling via seven-transmembrane receptors (7TMRs), have been recently shown to also transduce G protein-independent signals from receptors. However, the physiologic consequences of this G protein-independent, GRK/beta-arrestin-dependent signaling are largely unknown. Here, we establish that GRK/beta-arrestin-mediated signal transduction via the angiotensin II (ANG) type 1A receptor (AT(1A)R) results in positive inotropic and lusitropic effects in isolated adult mouse cardiomyocytes. We used the "biased" AT(1A)R agonist [Sar(1), Ile(4), Ile(8)]-angiotensin II (SII), which is unable to stimulate G(alpha)q-mediated signaling, but which has previously been shown to promote beta-arrestin interaction with the AT(1A)R. Cardiomyocytes from WT, but not AT(1A)R-deficient knockout (KO) mice, exhibited positive inotropic and lusitropic responses to both ANG and SII. Responses of WT cardiomyocytes to ANG were dramatically reduced by protein kinase C (PKC) inhibition, whereas those to SII were unaffected. In contrast, cardiomyocytes from beta-arrestin2 KO and GRK6 KO mice failed to respond to SII, but displayed preserved responses to ANG. Cardiomyocytes from GRK2 heterozygous knockout mice (GRK2(+/-)) exhibited augmented responses to SII in comparison to ANG, whereas those from GRK5 KO mice did not differ from those from WT mice. These findings indicate the existence of independent G(alpha)q/PKC- and GRK6/beta-arrestin2-dependent mechanisms by which stimulation of the AT(1A)R can modulate cardiomyocyte function, and which can be differentially activated by selective receptor ligands. Such ligands may have potential as a novel class of therapeutic agents.

GRK5 deficiency leads to early Alzheimer-like pathology and working memory impairment

G-protein coupled receptor kinase-5 (GRK5) deficiency has been linked to early Alzheimer's disease in humans and mouse models of the disease. To determine potential roles of GRK5 in the disease pathogenesis, the GRK5 knockout mouse was evaluated at pathological and behavioral levels. We found that these mice displayed an age-dependent increase in hippocampal axonal defects characterized by clusters of axonal swellings that accumulated abnormal amounts of molecular motor proteins, microtubule-associated proteins, intracellular β-amyloid, and subcellular organelles. In severe cases, extracellular β-amyloid fibrillar deposits were occasionally observed, along with degenerating axonal components, and were tightly surrounded by reactive astrocytes. Moreover, significant loss of synaptic proteins and early signs of cholinoceptive neurodegeneration were evident in the hippocampus as well. Consistent with the moderate level of pathologic change, aged GRK5 knockout mice displayed selective working memory impairment, with other cognitive domains unaffected. Taken together, these findings not only strongly support an important role of GRK5 deficiency in early Alzheimer's pathogenesis, but also promote the GRK5 knockout mouse as an additional model for early Alzheimer-related studies.