GRK5在高血压发病中的调控作用

原发性高血压(EH)及其并发症正成为影响人类健康的主要病因之一。血压的调控受到体内众多因子的调控。G蛋白偶联受体激酶(G protein-coupled receptor kinases,GRKs)属丝氨酸/苏氨酸蛋白激酶家族,GRK5属GRK4家族中的一员,在血管平滑肌、心脏表达丰富,它通过对G蛋白偶联受体及一些细胞因子的调控来实现对血压的调节。在去甲肾上腺素或血管紧张素Ⅱ诱导的高血压模型中,GRK5在血管平滑肌细胞中的表达明显增加,高表达的GRK5可能增加了血管平滑肌细胞对去甲肾上腺素或血管紧张素Ⅱ的敏感性,从而在高血压发病中发挥重要的作用。同时,GRK5的高表达很可能对心肌损伤、心肌肥大及心力衰竭也有一定作用。     

β-肾上腺素受体介导的G蛋白及β-arrestin依赖性通路

β-肾上腺素受体所介导的经典G蛋白通路在心功能的调节中发挥着重要的作用,参与调节心肌收缩以及心肌细胞肥大等过程。近年来的研究发现,β-arrestin与β-肾上腺素受体的脱敏、内化、再循环以及降解密切相关,并可以通过其桥梁作用,使受体与多条信号通路之间建立联系。此外,β-arrestin还可以进入细胞核或者通过NF-kB/ERK进而参与表达水平的调控。鉴于β-arrestin与β-肾上腺素受体的密切关系及其本身作用的多样性,对它的深入研究将有助于新一代G蛋白偶联受体药物的研发。     

异丙肾上腺素对大鼠心脏功能的动态影响

目的观察异丙肾上腺素(ISO)对在体大鼠心脏功能、心室肌细胞功能以及胞内相关下游蛋白表达的动态影响。方法采用血流动力学方法及乳鼠心肌细胞培养,观察ISO对在体及离体大鼠心肌收缩能力的影响;通过Fura-2荧光探针法观察ISO对成鼠心肌细胞内游离钙水平的影响;Western blot检测ISO下游胞内相关蛋白表达水平的变化。结果 (5~500)nmol/L ISO对成鼠在体心功能有一个剂量依赖性增强效应,100nmol/L为其最佳实验浓度:0.1μmoI/LISO作用大鼠心室肌细胞(1 000~1 200)s,胞内游离钙水平达高峰。0.1μmoI/LISO作用于乳鼠心肌细胞30min可使其跳动频率达高峰,维持至60min后开始下降,与胞内钙变化规律一致。心室肌细胞内钙调蛋白激酶Ⅱ(Calmodulin kinaseⅡ,CaMKⅡ)水平在ISO干预后3h达高峰;G蛋白偶联受体激酶2(GRK2)和β-arrestin也在ISO作用后48h达高峰。结论 ISO可通过兴奋胞内Ca2+-CaMKⅡ信号通路参与心肌细胞功能活动的动态调节;同时,ISO长期作用可升高胞内GRK2-β-arrestin水平,参与受体的脱敏调节。     

β3肾上腺素能受体与心力衰竭的研究进展

β3肾上腺素能受体(β3AR)属于G蛋白偶联受体超家族,研究显示,与β1AR和β2AR不同,β3AR激动可抑制心肌收缩力。心力衰竭时左室β3AR密度增高,心肌细胞β3AR mRNA及受体蛋白表达上调。β3AR与Gi/o蛋白偶联,参与NO-cGMP介导的负性肌力作用。β1／β2／β3AR阻滞剂布拉洛尔可以完全阻断其作用,而β1／β2AR阻滞剂无效。心力衰竭时随交感神经持续激活,出现β1AR下调、β2AR功能性脱偶联、β3AR持续上调,导致心功能的进展性恶化。本文拟对β3AR与充血性心力衰竭的研究进展作一综述。     

外周血GRK2和PLR对患者急性心肌梗死后心力衰竭及预后的预警价值分析

目的 探讨外周血G蛋白偶联受体激酶2(G-protein coupled receptor kinase 2,GRK2)和血小板/淋巴细胞计数比值(platelet/lymphocyte count ratio,PLR)对患者急性心肌梗死(acute myocardial infarction,AMI)后心力衰竭(he...     

β肾上腺素能受体信号转导通路对心肌肥大的调节

β肾上腺素能受体信号通路与心肌肥大和心力衰竭的发生密切相关.β肾上腺素能受体的长期激活可引起心功能异常、心肌肥大和心脏重塑.β肾上腺素能受体诱导的G蛋白偶联状态的改变、ERK信号通路激活、Epac和PKCε的活化等机制参与了心肌肥大的发生发展,阐明β肾上腺素能受体激活引起心肌肥大的信号转导机制,有助于更好地防治心肌肥大.     

心力衰竭β3肾上腺素能受体相关炎症机制的研究进展

心力衰竭为各种心脏疾病的终末期表现,而心力衰竭的发展亦是一个进展性的过程,即源于心脏重塑和功能恶化的进展.发生机制主要为长期神经激素激活和炎性细胞因子过度表达.心力衰竭时交感神经系统过度激活,诱导心肌细胞β肾上腺素能受体(AR)表达变化,β1-AR、β2-AR表达下调和脱敏,而由于β3-AR缺乏G蛋白耦联受体激酶的磷酸化位点等原因,心力衰竭时β3-AR的表达随着儿茶酚胺浓度增高而上调[1].交感神经过度兴奋刺激β3- AR,经多种途径介导心肌损害,对心室重塑、心肌细胞凋亡、氧化应激及免疫炎性反应产生重要影响.     

缬沙坦对自发性高血压大鼠左心室肥厚心肌细胞GRK2的表达及亚细胞分布的影响

目的观察缬沙坦对自发性高血压大鼠（SHR）左心室心肌细胞G蛋白偶联受体激酶2（GRK2）的表达及亚细胞分布的影响。方法18只SHR随机分为对照组（n=6）,低剂量缬沙坦组[SHRL组,10mg/（kg·d）,n=6]和高剂量缬沙坦组[SHRH组,30mg/（kg·d）,n=6],由6月龄喂养至8月龄,处死后分离心脏,通过免疫荧光标记、激光共聚焦显微镜及Werstern blot方法,观察左心室心肌细胞GRK2的表达及亚细胞分布的变化。结果与对照组比较,SHRL组及SHRH组左心室心肌组织总蛋白、浆蛋白及膜蛋白GRK2表达水平有明显减少（P＜0.05）,SHRH组较SHRL组减少更明显（P＜0.05）;与对照组比较, SHRL组及SHRH组GRK2在心肌细胞膜上分布减少,SHRH组较SHRL组进一步减少。结论缬沙坦能减少SHR左心室心肌细胞GRK2的表达及在细胞膜上的分布,这可能是逆转心肌肥大及心室重塑的机制之一。     

G蛋白偶联受体激酶的研究进展

G蛋白偶联受体激酶(GRKs)属丝氨酸／酪氨酸蛋白激酶家族,其亚型广泛存在于各种组织, 能特异地使活化的G蛋白偶联受体(GPCR)发生磷酸化及脱敏化,从而终止后者介导的信号转导通路．现就G蛋白偶联受体激酶的结构、种类及分布、生物学功能及与疾病关系的新进展进行总结与概括,并对其发展进行了展望．     

卡托普利对高血压左心室肥大心肌细胞中GRK_2表达和亚细胞分布的影响

目的:研究G蛋白偶联受体激酶2(GRK2)在高血压心肌肥大发生发展机制中的作用和卡托普利对心肌中GRK2表达水平、活性及亚细胞分布的影响,探讨卡托普利抑制心肌肥大的机制.方法:通过免疫荧光标记、共聚焦显微镜及Western blot等方法,检测6月龄的WKY(WKY组)、自发性高血压(SHR,SHRA组)大鼠,8月龄SHR(SHRB组)和卡托普利干预SHR(SHRC组)大鼠左心室心肌细胞中GRK2的表达及其分布.结果:各组大鼠左室心肌组织总蛋白中GRK2表达无明显变化(P>0.05).细胞质中GRK2表达SHRA组比WKY组表达减少(P<0.01);SHRB组比SHRC组GRK2表达进一步减少(P<0.01);而SHRC组比SHRB组GRK2表达增加( P<0.05).细胞膜蛋白中GRK2在SHRA组比WKY组表达增加(P<0.01);SHRB组比SHRA组GRK2表达进一步增加(P<0.05);而SHRC组比SHRB组GRK2表达减少(P<0.01).细胞核蛋白中无GRK2表达.共聚焦显微镜观察发现GRK2在SHR大鼠细胞膜特别是心肌细胞两端的闰盘聚集明显,卡托普利能减少GRK2在细胞膜分布.结论:GRK2与心肌细胞肥大发生发展关系密切,参与心肌肥大细胞信号转导的调控,卡托普利能通过调节GRK2亚细胞分布而发挥抑制心肌肥大作用.     

G protein coupled receptor kinases as therapeutic targets in cardiovascular disease

G protein-coupled receptors (GPCRs) represent the largest family of membrane receptors and are responsible for regulating a wide variety of physiological processes. This is accomplished via ligand binding to GPCRs, activating associated heterotrimeric G proteins and intracellular signaling pathways. G protein-coupled receptor kinases (GRKs), in concert with β-arrestins, classically desensitize receptor signal transduction, thus preventing hyperactivation of GPCR second-messenger cascades. As changes in GRK expression have featured prominently in many cardiovascular pathologies, including heart failure, myocardial infarction, hypertension, and cardiac hypertrophy, GRKs have been intensively studied as potential diagnostic or therapeutic targets. Herein, we review our evolving understanding of the role of GRKs in cardiovascular pathophysiology.     

G protein-coupled receptor kinase 2 ablation in cardiac myocytes before or after myocardial infarction prevents heart failure

Myocardial G protein-coupled receptor kinase (GRK)2 is a critical regulator of cardiac beta-adrenergic receptor (betaAR) signaling and cardiac function. Its upregulation in heart failure may further depress cardiac function and contribute to mortality in this syndrome. Preventing GRK2 translocation to activated betaAR with a GRK2-derived peptide that binds G(beta)gamma (betaARKct) has benefited some models of heart failure, but the precise mechanism is uncertain, because GRK2 is still present and betaARKct has other potential effects. We generated mice in which cardiac myocyte GRK2 expression was normal during embryonic development but was ablated after birth (alphaMHC-Cre x GRK2 fl/fl) or only after administration of tamoxifen (alphaMHC-MerCreMer x GRK2 fl/fl) and examined the consequences of GRK2 ablation before and after surgical coronary artery ligation on cardiac adaptation after myocardial infarction. Absence of GRK2 before coronary artery ligation prevented maladaptive postinfarction remodeling and preserved betaAR responsiveness. Strikingly, GRK2 ablation initiated 10 days after infarction increased survival, enhanced cardiac contractile performance, and halted ventricular remodeling. These results demonstrate a specific causal role for GRK2 in postinfarction cardiac remodeling and heart failure and support therapeutic approaches of targeting GRK2 or restoring betaAR signaling by other means to improve outcomes in heart failure.     

Gene-mediated inhibition of the b-adrenergic receptor kinase: a new therapeutic strategy for heart failure.

Molecular changes that take place during the evolution of heart failure (HF), especially the well characterized beta-adrenergic receptor (betaAR) signaling abnormalities, represent attractive targets for myocardial gene therapy. The beta-adrenergic receptor kinase (betaARK1 or GRK2) is a cytosolic enzyme that phosphorylates only agonist-occupied betaARs as well as other G protein-coupled receptors (GPCRs), leading to desensitization and functional uncoupling. betaARK1 levels and activity are elevated in the failing heart and therefore, it has recently been evaluated as a potential target for novel HF treatment. This review summarizes recent results obtained in transgenic mouse models as well as in animals where a betaARK1 inhibitor peptide (betaARKct) was delivered via the coronary arteries by exogenous gene transfer. These results strongly suggest that betaARK1 inhibition may represent a significant improvement in HF therapy.     

Heterotrimeric G protein beta1gamma2 subunits change orientation upon complex formation with G protein-coupled receptor kinase 2 (GRK2) on a model membrane

Few experimental techniques can assess the orientation of peripheral membrane proteins in their native environment. Sum Frequency Generation (SFG) vibrational spectroscopy was applied to study the formation of the complex between G protein-coupled receptor (GPCR) kinase 2 (GRK2) and heterotrimeric G protein β(1)γ(2) subunits (Gβγ) at a lipid bilayer, without any exogenous labels. The most likely membrane orientation of the GRK2-Gβγ complex differs from that predicted from the known protein crystal structure, and positions the predicted receptor docking site of GRK2 such that it would more optimally interact with GPCRs. Gβγ also appears to change its orientation after binding to GRK2. The developed methodology is widely applicable for the study of other membrane proteins in situ.     

Taking the heart failure battle inside the cell: small molecule targeting of Gβγ subunits

Heart failure (HF) is devastating disease with poor prognosis. Elevated sympathetic nervous system activity and outflow, leading to pathologic attenuation and desensitization of β-adrenergic receptors (β-ARs) signaling and responsiveness, are salient characteristic of HF progression. These pathologic effects on β-AR signaling and HF progression occur in part due to Gβγ-mediated signaling, including recruitment of receptor desensitizing kinases such as G-protein coupled receptor (GPCR) kinase 2 (GRK2) and phosphoinositide 3-kinase (PI3K), which subsequently phosphorylate agonistoccupied GPCRs. Additionally, chronic GPCR signaling signals chronically dissociated Gβγ subunits to interact with multiple effector molecules that activate various signaling cascades involved in HF pathophysiology. Importantly, targeting Gβγ signaling with large peptide inhibitors has proven a promising therapeutic paradigm in the treatment of HF. We recently described an approach to identify small molecule Gβγ inhibitors that selectively block particular Gβγ functions by specifically targeting a Gβγ protein-protein interaction "hot spot." Here we describe their effects on Gβγ downstream signaling pathways, including their role in HF pathophysiology. We suggest a promising therapeutic role for small molecule inhibition of pathologic Gβγ signaling in the treatment of HF. This article is part of a special issue entitled “Key Signaling Molecules in Hypertrophy and Heart Failure.”     

Regulation of G protein-coupled receptor kinases

G protein-coupled receptor kinases (GRKs) specifically interact with the agonist-activated form of G protein-coupled receptors (GPCRs) to effect receptor phosphorylation and desensitization. Recent studies demonstrate that GRK function is a highly regulated process, and it is perhaps in this manner that a handful of GRKs (7 have been identified to date) are able to regulate the responsiveness of numerous GPCRs in a given cell type in a coordinated manner. The mechanisms by which GRK activity is regulated can be divided into 3 categories: 1) subcellular localization; 2) alterations in intrinsic kinase activity; and 3) alterations in GRK expression levels. This review will summarize our current understanding of each of these regulatory processes, and offer explanations as to how such mechanisms influence GPCR regulation under various physiologic conditions.     

Effect of hypothyroidism on G protein-coupled receptor kinase 2 expression levels in rat liver, lung, and heart

GRK2 is a member of the G protein-coupled receptor kinase family that phosphorylates the activated form of beta-adrenergic and other G protein-coupled receptors and plays an important role in their desensitization and modulation. Alterations in thyroid hormone levels have been reported to lead to important changes in adrenergic receptor responsiveness and signaling in a variety of tissues. In this context, we have explored the effects of experimental hypothyroidism on GRK2 protein levels in rat heart, lung, and liver using a specific antibody. Hypothyroid animals show significant up-regulation ( approximately 50% increase compared with controls) in GRK2 levels in heart and lung at 60 days after birth, whereas a 50% reduction is detected in the liver at this stage. These alterations are selective, as beta-adrenergic receptors or other G protein-coupled receptor regulatory proteins, such as G protein-coupled receptor kinase 5 or beta-arrestin-1, display a different pattern of expression changes in the hypothyroid animals. The reported changes in GRK2 levels and in the receptor/kinase ratio predict alterations in adrenergic receptor desensitization and signal transduction efficacy consistent with those observed in thyroid disorders, thus suggesting a relevant role for the modulation of GRK2 expression in this physiopathological condition.     

Hybrid transgenic mice reveal in vivo specificity of G protein-coupled receptor kinases in the heart

G protein-coupled receptor kinases (GRKs) phosphorylate activated G protein-coupled receptors, including alpha(1B)-adrenergic receptors (ARs), resulting in desensitization. In vivo analysis of GRK substrate selectivity has been limited. Therefore, we generated hybrid transgenic mice with myocardium-targeted overexpression of 1 of 3 GRKs expressed in the heart (GRK2 [commonly known as the beta-AR kinase 1], GRK3, or GRK5) with concomitant cardiac expression of a constitutively activated mutant (CAM) or wild-type alpha(1B)AR. Transgenic mice with cardiac CAMalpha(1B)AR overexpression had enhanced myocardial alpha(1)AR signaling and elevated heart-to-body weight ratios with ventricular atrial natriuretic factor expression denoting myocardial hypertrophy. Transgenic mouse hearts overexpressing only GRK2, GRK3, or GRK5 had no hypertrophy. In hybrid transgenic mice, enhanced in vivo signaling through CAMalpha(1B)ARs, as measured by myocardial diacylglycerol content, was attenuated by concomitant overexpression of GRK3 but not GRK2 or GRK5. CAMalpha(1B)AR-induced hypertrophy and ventricular atrial natriuretic factor expression were significantly attenuated with either concurrent GRK3 or GRK5 overexpression. Similar GRK selectivity was seen in hybrid transgenic mice with wild-type alpha(1B)AR overexpression concurrently with a GRK. GRK2 overexpression was without effect on any in vivo CAM or wild-type alpha(1B)AR cardiac phenotype, which is in contrast to previously reported in vitro findings. Furthermore, endogenous myocardial alpha(1)AR mitogen-activated protein kinase signaling in single-GRK transgenic mice also exhibited selectivity, as GRK3 and GRK5 desensitized in vivo alpha(1)AR mitogen-activated protein kinase responses that were unaffected by GRK2 overexpression. Thus, these results demonstrate that GRKs differentially interact with alpha(1B)ARs in vivo such that GRK3 desensitizes all alpha(1B)AR signaling, whereas GRK5 has partial effects and, most interestingly, GRK2 has no effect on in vivo alpha(1B)AR signaling in the heart.