摘要
目的:观察胶原性关节炎(collagen-induced arthritis, CIA)大鼠炎症过程中滑膜组织G蛋白偶联受体激酶(G-protein-coupled receptor kinases,GRKs)的表达情况及白芍总苷(total glucosides of paeony, TGP)对其的影响;明确GRKs在CIA大鼠成纤维样滑膜细胞(fibroblast-like synoviocytes, FLS)中的表达情况及TGP中有效单体成分芍药苷(paeoniflorin,Pae)对其的影响。从滑膜细胞G蛋白偶联信号途径,研究GRK2在FLS的信号调节中作用及Pae治疗大鼠CIA的可能的分子机制。
方法:大鼠足趾、尾根部、背部皮内多点注射鸡Ⅱ型胶原(chicken typeⅡcollagen, CCⅡ)乳剂制备CIA模型,TGP(25、50、100 mg·kg-1·d-1)灌胃给药(intragastric administration, ig)(d14~d28),观察大鼠全身状况及足爪肿胀度,光学显微镜观察关节病理变化;Western blot检测GRKs水平。组织块法培养大鼠关节滑膜细胞,微孔生物发光法检测FLS PKA活性,放免法检测FLS cAMP水平。
结果:
1. CIA大鼠关节滑膜组织GRKs的表达及TGP对其的影响
CIA大鼠加强免疫注射后28天左右炎症反应达到高峰,光镜下可见滑膜组织中滑膜细胞增生明显,层次增多,排列紊乱,滑膜下层纤维组织增生,血管扩张、充血,炎性细胞浸润及血管翳形成,软骨中炎性细胞浸润,破坏软骨组织。Westernblot发现CIA大鼠滑膜组织中GRK2、GRK5、GRK6的表达较正常组明显升高,致炎后28天GRK2、GRK5、GRK6的表达达到高峰;46天后关节肿胀、炎症逐渐减退,GRK2、GRK5、GRK6的高表达也开始下降。TGP (25、50、100 mg﹒kg~(-1))可明显减轻CIA大鼠的足爪肿胀度,改善其多发性关节炎症状,可不同程度改善以上病理变化。TGP (25、50、100 mg·kg~(-1))可以降低CIA大鼠滑膜组织GRK5、GRK6的高表达水平;TGP(100 mg·kg-1)给药后CIA大鼠滑膜组织GRK2的表达水平明显降低。
2. GRKs在CIA大鼠滑膜细胞中的表达及Pae对其的影响
CIA大鼠滑膜细胞GRK2、GRK5、GRK6表达水平升高,Pae(10-5、10-6、10-7、10-8mol·L~(-1))可以不同程度的降低CIA大鼠滑膜细胞GRK2、GRK5、GRK6的表达。3.GRK2与G蛋白-AC-cAMP信号通路的关系及Pae对其的影响
加入GRK2抑制剂(5-甲基[2-(5-硝基-2-呋喃基)乙烯基]-2-糠酸酯,C12H9NO6)后,滑膜细胞cAMP的水平和PKA的活性升高;Pae(10-5、10-6、10-7、10-8mol·L~(-1))可以降低GRK2抑制剂引起的滑膜细胞cAMP水平的升高,Pae(10-5、10-6mol·L~(-1))可以降低GRK2抑制剂引起的滑膜细胞PKA活性的升高。
结论:
1. CIA炎症导致关节滑膜组织和滑膜细胞GRK2、GRK5、GRK6表达水平的上调; GRKs是CIA滑膜炎症、滑膜细胞异常增殖的重要分子。
2. TGP对大鼠CIA多发性关节炎、关节局部肿胀及病理形态等具有明显的改善作用;TGP、Pae可以下调CIA大鼠关节滑膜组织和滑膜细胞GRKs的高表达;而且Pae可以纠正GRK2抑制剂引起的CIA大鼠滑膜细胞脱敏障碍,恢复G蛋白-AC-cAMP信号通路的正常转导。提示TGP、Pae下调GRKs的表达,抑制GPCRs的过度脱敏,调节G蛋白-AC-cAMP信号通路的正常转导,可能是其抑制滑膜细胞增殖、维持滑膜正常功能的重要机制之一。
OBJECTIVE To investigate the expression changes of GRKs in synovium from CIA rats during the inflamation and the effects of TGP. Then we clarified the expression of GRKs in synoviocytes from CIA rats and the effects of Pae, and we attempted to investigate the role of GRK2 playing in G-Protein signal transduction pathways of synoviocyte and the effects of Pae. And further to analyze the relationship between the effects of Pae on CIA rats and its relative mechanisms by which Pae affected the process of synoviocytes through these signal transduction pathways.
METHODS The model of rat CIA was induced by injection of chicken typeⅡcollagen(CCⅡ) emulsion. TGP(25、50、100 mg·kg~(-1), ig, d14-d28) was administrated intragastric to CIA rats . Hind paw volumes of rats were measured by volume meter. Pathological changes in synovium of joint were observed by light microscope. The expression of GRKs in synoviocytes from CIA rats was measured by western blot. The expression change of cAMP was investigated by radioimmunoassay, and the activities of PKA was measured by luminescent kinase assay.
RESULTS
(1)Expression changes of GRKs in synovium from CIA rats during the inflamation and the effecrs of TGP
Inflammatory reaction occurred on d14 after immunization. There was a marked secondary inflammatory response in the models, which accompanied with paw swelling, pain, polyarthritis, decrease of body weight, and the development of inflammatory lesions. Compared with the normal rats, in CIA rats, the histological morphological examination of rats knee joints showed hyperplastic synovium, synovial lining hyperplasia, inflammatory cells infiltration, pannus formation and destruction of the cartilage, and fatty changes in the synoviocytes. At the inflammatory process (d14~d28 after immunization), we observed a profound up-regulation of GRKs’in synovium from CIA rats and during the remission phase (d46 after immunization), GRKs’expression is returning to baseline levels. TGP (25、50、100 mg·kg-1, ig, d14-28) diminished the right hind paw swelling and polyarthritic symptoms. TGP (25、50、100 mg·kg-1) the expression of GRK5、GRK6 in synovium from CIA rats. TGP(100 mg·kg-1)could decrease the expression of GRK2.
(2)Expression of GRKs in synoviocytes from CIA rats and the effecrs of Pae
Expression of GRK2、GRK5、GRK6 in synoviocytes from CIA rats was increased, Pae(10-5、10-6、10-7、10-8 mol·L~(-1))in vitro could suppress the expression of GRK2、GRK5、GRK6 in synoviocytes from CIA rats.
(3)Role of GRK2 in GPCR-AC-cAMP signaling transduction and the effects of Pae
Level of cAMP and activity of PKA increased in synoviocytes from CIA rats after GRK2 inhibito(rMethyl 5-[2-(5-nitro-2-furyl)vinyl]-2-furoate,C12H9NO6)were given. Pae(10-5、10-6、10-7、10-8 mol·L~(-1)) may decrease the level of cAMP and Pae(10-5、10-6mol·L~(-1))might suppress the activity of PKA.
CONCLUSIONS
(1)An inflammatory process in vivo induces an up-regulation of GRKs in synovium and synoviocytes from CIA rats. The up-regulation of GRKs may play an important role in proliferation of synoviocytes from CIA rats.
(2)TGP can diminish the paw swelling and polyarthritic symptoms, and decrease the expression of GRKs in synovium from CIA rats.In Synoviocytes of CIA rats,Pae may suppress the up-regulation of GRK2、GRK5 and GRK6,and at the same time recover the disorder of desensitization induced by GRK2 inhibitor. Which indicates that TGP、Pae can suppress over-desensitization of synoviocytes from CIA rats, and regulate desensitization of synoviocytes, which may be one of the important mechanisms for the effects of Pae on synoviocytes.
引文
1. Woods JM, Katschke KJ Jr, Tokuhira M, Kurata H, Arai KI, Campbell PL, Koch AE. Reduction of inflammatory cytokines and prostaglandin E2 by IL-13 gene therapy in rheumatoid arthritis synovium. J Immunol,2000 ; 165(5): 2755-63.
2. Tamai M, Kawakami A, Tanaka F, Miyashita T, Nakamura H, Iwanaga N, Izumi Y, Arima K, Aratake K, Huang M, Kamachi M, Ida H, Origuchi T, Eguchi. Significant inhibition of TRAIL-mediated fibroblast-like synovial cell apoptosis by IFN-gamma through JAK/STAT pathway by translational regulation. J Lab Clin Med,2006; 147 (4): 182-90.
3. Chen Q, Wei W. Effects and mechanisms of chaenomeles speciosa on collagen-induced arthritis in rats. Int Immunopharmacol,2003; 3(4): 593-608.
4 Brand DD, Kang AH, Rosloniec EF. Immunopathogenesis of collagen arthritis. Springer Semin Immunopathol,2003; 25(1): 3-18.
5 Lombardi MS,Kavelaars A,Heijnen CJ. Role and modulation of G protein-coupled receptor signaling in inflammatory processes. Crit Rev Immunol, 2002;22(2):141-63.
6 Lefkowitz RJ. G protein-coupled receptor kinases. Cell,1993;74(3):409-12.
7 Vroon A, Heijnen CJ, Kavelaars A.GRKs and arrestins: regulators of migration and inflammation. J Leukoc Biol,2006;80(6):1214-21.
8 Xu HM, Wei W, Jia XY, Chang Y, Zhang L Effects and mechanisms of total glucosides of paeony on adjuvant arthritis in rats. J Ethnopharmacol, 2007;109(3):442-8.
9 Wu H, Wei W, Song L, Zhang L, Chen Y, Hu X.Paeoniflorin induced immune tolerance of mesenteric lymph node lymphocytes via enhancing beta 2-adrenergic receptor desensitization in rats with adjuvant arthritis. Int Immunopharmacol,2007;7(5):662-73.
10.孙妩弋,张玲玲,吴丽,汪倪萍,王华,岳莉,张运芳,魏伟.重组人白细胞介素-1受体拮抗剂对大鼠胶原性关节炎的治疗作用.中国药理学通报,2005;21(1): 92-5.
11鄂征.组织块培养法.见:鄂征主编.组织培养技术(第二版).北京:人民卫生出版社.1988;127-30.
12王斌,陈敏珠.滑膜细胞培养上清液对成纤维细胞增殖反应的影响.安徽医科大学学报,1997; 32(5): 534-36.
13 Schurigt U,Stopfel N,Hückel M et al. Local expression of matrix metalloproteinases, cathepsins, and their inhibitors during the development of murine antigen-induced arthritis. Arthritis Res Ther, 2005;7(1):R174-88.
14 Firestein GS, Manning AM. Signal transduction and transcription factors inrheumatic disease. Arthritis Rheum,1999;42(4):609-21.
15栗占国.类风湿关节炎.见:施桂英,栗占国主编.关节炎概要(第一版).北京:中国医药科技出版社,2000:226-93.
16 Cook AD, Visvanathan K. Molecular targets in immune-mediated diseases: focus on rheumatoid arthritis. Expert Opin Ther Targets,2004;8(5): 375-90.
17 Ospelt C, Neidhart M, Gay RE, Gay S. Synovial activation in rheumatoid arthritis. Front Biosci,2004; 9: 2323-34.
18 Akaogi J, Nozaki T, Satoh M, Yamada H.Role of PGE2 and EP receptors in the pathogenesis of rheumatoid arthritis and as a novel therapeutic strategy. Endocr Metab Immune Disord Drug Targets,2006;6(4):383-94.
19 Filer A, Raza K, Salmon M, Buckley CD.The role of chemokines in leucocyte-stromal interactions in rheumatoid arthritis. Front Biosci, 2008 ; 13: 2674-85.
20 Kin NW, Sanders VM.It takes nerve to tell T and B cells what to do. J Leukoc Biol,2006;79(6):1093-104.
21 Ferguson SS.Evolving concepts in G protein-coupled receptor endocytosis: the role in receptor desensitization and signaling. Pharmacol Rev,2001;53(1):1-24.
22 Penela P, Ribas C, Mayor F Jr.Mechanisms of regulation of the expression and function of G protein-coupled receptor kinases. Cell Signal,2003;15(11):973-81.
23 Pitcher JA, Freedman NJ, Lefkowitz RJ. LinksG protein-coupled receptor kinases. Annu Rev Biochem,1998;67:653-92.
24 Kohout TA, Lefkowitz RJ. Regulation of G protein-coupled receptor kinases and arrestins during receptor desensitization. Mol Pharmacol,2003;63(1):9-18.
25 Ménard L, Ferguson SS, Zhang J, Lin FT, Lefkowitz RJ, Caron MG, Barak LS. Synergistic regulation of beta2-adrenergic receptor sequestration: intracellular complement of beta-adrenergic receptor kinase and beta-arrestin determine kinetics of internalization. Mol Pharmacol,1997;51(5):800-8.
26 Schlador ML, Nathanson NM. Synergistic regulation of m2 muscarinic acetylcholine receptor desensitization and sequestration by G protein-coupled receptor kinase-2 and beta-arrestin-1. J Biol Chem,1997;272(30):18882-90.
27 Chuang TT, Sallese M, Ambrosini G, Parruti G, De Blasi A.High expression of beta-adrenergic receptor kinase in human peripheral blood leukocytes. Isoproterenol and platelet activating factor can induce kinase translocation. J Biol Chem. 1992;267(10):6886-92.
28 Lombardi MS, Kavelaars A, Cobelens PM et al. Adjuvant arthritis induces down-regulation of G protein-coupled receptor kinases in the immune system. J Immunol,2001;166(3):1635-40.
29 Mak JC, Hisada T, Salmon M, Barnes PJ, Chung KF. Glucocorticoids reverse IL-1beta-induced impairment of beta-adrenoceptor-mediated relaxation and up-regulation of G-protein-coupled receptor kinases.Br J Pharmacol. 2002 Feb;135(4):987-96.
30 Izikson L, Klein RS, Charo IF, Weiner HL, Luster AD. Resistance to experimental autoimmune encephalomyelitis in mice lacking the CC chemokine receptor (CCR)2. J Exp Med,2000;192(7):1075-80.
31 Huang DR, Wang J, Kivisakk P, Rollins BJ, Ransohoff RM. Absence of monocyte chemoattractant protein 1 in mice leads to decreased local macrophage recruitment and antigen-specific T helper cell type 1 immune response in experimentalautoimmune encephalomyelitis. J Exp Med,2001 ;193(6):713-26.
32 Bagaeva LV, Williams LP, Segal BM. IL-12 dependent/IFN gamma independent expression of CCR5 by myelin-reactive T cells correlates with encephalitogenicity. J Neuroimmunol, 2003 ;137(1-2):109-16.
33 Oppermann M, Mack M, Proudfoot AE, Olbrich H. Differential effects of CC chemokines on CC chemokine receptor 5 (CCR5) phosphorylation and identification of phosphorylation sites on the CCR5 carboxyl terminus. J Biol Chem,1999;274(13):8875-85.
34 Olbrich H, Proudfoot AE, Oppermann M. Chemokine-induced phosphorylation of CC chemokine receptor 5 (CCR5). J Leukoc Biol,1999;65(3):281-5.
35 Aragay AM, Mellado M, Frade JM, Martin AM, Jimenez-Sainz MC, Martinez-A C, Mayor F Jr. Monocyte chemoattractant protein-1-induced CCR2B receptor desensitization mediated by the G protein-coupled receptor kinase 2. Proc Natl Acad Sci U S A,1998;95(6):2985-90.
36 Aramori I, Ferguson SS, Bieniasz PD, Zhang J, Cullen B, Cullen MG..Molecular mechanism of desensitization of the chemokine receptor CCR-5: receptor signaling and internalization are dissociable from its role as an HIV-1 co-receptor. EMBO J,1997;16(15):4606-16.
37 Vroon A, Kavelaars A, Limmroth V, Lombardi MS, Goebel MU, Van Dam AM, Caron MG, Schedlowski M, Heijnen CJ.G protein-coupled receptor kinase 2 in multiple sclerosis and experimental autoimmune encephalomyelitis. J Immunol, 2005;174(7):4400-6.
38 Vroon A, Heijnen CJ, Lombardi MS, Cobelens PM, Mayor F Jr, Caron MG, Kavelaars A.Reduced GRK2 level in T cells potentiates chemotaxis and signaling in response to CCL4. J Leukoc Biol, 2004;75(5):901-9.
39 Tager AM, Bromley SK, Medoff BD, Islam SA, Bercury SD, Friedrich EB, Carafone AD, Gerszten RE, Luster AD.Leukotriene B4 receptor BLT1 mediates early effector T cell recruitment. Nat Immunol,2003;4(10):982-90.
40 Tager AM, Bromley SK, Medoff BD. Leukotriene B4 recep tor BLT1 mediates early effector T cell recruitment. Nat Immunol,2003;4 (10):982-90.
41 Kavelaars A, Vroon A, Raatgever RP, Fong AM, Premont RT, Patel DD, Lefkowitz RJ, Heijnen CJ. Increased acute inflammation, leukotriene B4-induced chemotaxis, and signaling in mice deficient for G protein-coupled receptor kinase 6. J Immunol, 2003;171(11):6128-34.
42 Gaudreau R, Le Gouill C, Venne MH, Stankova J, Rola-Pleszczynski M. Threonine 308 within a putative casein kinase 2 site of the cytoplasmic tail of leukotriene B(4) receptor (BLT1) is crucial for ligand-induced, G-protein-coupled receptor-specific kinase 6-mediated desensitization. J Biol Chem,2002;277(35):31567-76.
43 Pronin AN, Morris AJ, Surguchov A, Benovic JL.Synucleins are a novel class of substrates for G protein-coupled receptor kinases. J Biol Chem, 2000;275(34):26515-22.
44 Ruiz-Gómez A, Humrich J, Murga C, Quitterer U, Lohse MJ, Mayor F Jr.Phosphorylation of phosducin and phosducin-like protein by G protein-coupled receptor kinase 2. J Biol Chem, 2000;275(38):29724-30.
45 Cant SH, Pitcher JA.G protein-coupled receptor kinase 2-mediated phosphorylation of ezrin is required for G protein-coupled receptor-dependent reorganization of the actin cytoskeleton. Mol Biol Cell, 2005 ;16(7):3088-99.
46 Ren XR, Reiter E, Ahn S, Kim J, Chen W, Lefkowitz RJ. Different G protein-coupled receptor kinases govern G protein and beta-arrestin-mediated signaling of V2 vasopressin receptor. Proc Natl Acad Sci U S A, 2005;102(5):1448-53.
47 Shayo C, Fernandez N, Legnazzi BL, Monczor F, Mladovan A, Baldi A, Davio C.Histamine H2 receptor desensitization: involvement of a select array of G protein-coupled receptor kinases. Mol Pharmacol,2001;60(5):1049-56.
48 Wang L, Liu S, Quarles LD, Spurney RF. Targeted overexpression of G protein-coupled receptor kinase-2 in osteoblasts promotes bone loss. Am J Physiol Endocrinol Metab,2005;288(4):E826-34.
49 Fernández N, Monczor F, Lemos B, Notcovich C, Baldi A, Davio C, Shayo C.Reduction of G protein-coupled receptor kinase 2 expression in U-937 cells attenuates H2 histamine receptor desensitization and induces cell maturation. Mol Pharmacol,2002;62(6):1506-14.
50张玲玲,魏伟,汪庆童,陈镜宇,陈尹,吴华勋.芍药苷对胶原性关节炎大鼠滑膜细胞G蛋白偶联信号的调节作用.中国药理学通报,2008;24(3):300-306
51 Thalhamer T, McGrath MA, Harnett MM. MAPKs and their relevance to arthritis and inflammation. Rheumatology (Oxford). 2008;47(4):409-14.
52 Chabaud-Riou M, Firestein GS. Expression and activation of mitogen-activated protein kinase kinases-3 and -6 in rheumatoid arthritis. Am J Pathol,2004; 164(1):177-84.
53 Han Z, Chang L,Yamanishi Y, Karin M, Firestein GS. Joint damage and inflammation in c-Jun N-terminal kinase 2 knockout mice with passive murine collagen-induced arthritis. Arthritis Rheum,2002; 46(3):818–23.
54. Zheng YQ, Wei W. Total glucosides of paeony suppresses adjuvant arthritis in rats and intervenes cytokine-signaling between different types of synoviocytes. Int Immunopharmacol,2005;5(10):1560-73.
55 Lefkowitz RJ, Whalen EJbeta-arrestins: traffic cops of cell signaling. Curr Opin Cell Biol,2004;16(2):162-8.
56 Lefkowitz RJ, Shenoy SK.Transduction of receptor signals by beta-arrestins. Science,2005;308(5721):512-7.
57 Jiménez-Sainz MC, Murga C, Kavelaars A, Jurado-Pueyo M, Krakstad BF, Heijnen CJ, Mayor F Jr, Aragay AM. G protein-coupled receptor kinase 2 negatively regulates chemokine signaling at a level downstream from G protein subunits. Mol Biol Cell,2006;17(1):25-31.
58 Ito K, Caramori G, Adcock IM. Therapeutic potential of phosphatidylinositol 3-kinase inhibitors in inflammatory respiratory disease. J Pharmacol Exp Ther, 2007;321(1):1-8.
59 Naga Prasad SV, Barak LS, Rapacciuolo A, Caron MG, Rockman HA.Agonist-dependent recruitment of phosphoinositide 3-kinase to the membrane by beta-adrenergic receptor kinase 1. A role in receptor sequestration. J Biol Chem, 2001;276(22):18953-9.
60 Liu S, Premont RT, Kontos CD, Zhu S, Rockey DC.A crucial role for GRK2 in regulation of endothelial cell nitric oxide synthase function in portal hypertension. Nat Med,2005;11(9):952-8.
61 Zhu L, Wei W, Zheng YQ, Jia XY. Effects and mechanisms of total glucosides of paeony on joint damage in rat collagen-induced arthritis. Inflamm Res. 2005; 54(5): 211-20.
62贾晓益,魏伟,郑咏秋,朱蕾.白芍总苷对胶原性关节炎滑膜组织中Bcl-2、Bax表达的影响.安徽医科大学学报,2006;41(2):143-6.
63朱蕾,魏伟,郑咏秋.白芍总苷对胶原性关节炎大鼠滑膜细胞的作用及机制.药学学报,2006;41(2):166-170.
64 Dumont JE, Jauniaux JC, Roger PP. The cyclic AMP-mediated stimulation of cell proliferation. Trends Biochem Sci,1989;14(2):67-71.
65魏伟,徐星铭,沈玉先,丁长海,张安平,徐叔云.褪黑素对类风湿关节炎外周血淋巴细胞增殖反应的影响及其G蛋白-AC-cAMP信号传导机制的研究.中国药理学通报,1998; 14(6): 530-2.
66 Ljiljana Andrijevic. The influence of 8-Cl-cAMP on tumor marker CA19.9 on experimental glioma in rats. Arch Oncol,2001; 9(1): 9-12.
67 Anderson KC, Dalton WS. Synopsis of a research roundtable presented on cell signaling in myeloma: regulation of growth and apoptosis-opportunities for new drug discovery. Mol Cancer Ther,2002; 1(14): 1361-65.
68 Tang J, Cao CP, Jian YP. Expression of PKA RI ingastric cancer cells and effects of
8-Bromo-cAMP on its expression and cells proliferation. J Gen Hos Air Force, 2001; 17(4): 203-5
69 Zheng YQ, Wei W, Zhu L, Jia XY. Relationship between G proteins-associatedtransmembrane cascades and MAPK phosphorylation induced by recombinant interleukin-1αin fibroblast-like synoviocytes from rats with adjuvant arthritis (abstract). Acta Pharmacol Sin, 2004; 25(11): 1541.
70 Zheng YQ, Wei W, Dai M, Zhu L, Jia XY, Wang Y. Interleukin-1 receptor antagonist intervenes in signaling between different types of synoviocytes in rats with adjuvant arthritis. Acta Pharmaco l Sin. 2006 Jan;27(1):111-8.
71常艳,魏伟,张磊,徐红梅.白芍总苷对IL-1α诱导胶原性关节炎大鼠成纤维样滑膜细胞功能的影响.中国药理学通报,2007;23(2):185-8
1. Lombardi MS,Kavelaars A,Heijnen CJ.Role and modulation of G protein-coupled receptor signaling in inflammatory processes. Crit Rev Immunol, 2002;22(2):141-63.
2. Ana Elorza, Susana Sarnago, Federico Mayor. Agonist-Dependent Modulation of G Protein-Coupled Receptor Kinase 2 by Mitogen-Activated Protein Kinases,2000;57(4):778-83.
3. Ana Elorza, Petronila Penela , Susana Sarnago etal.MAPK-dependent Degradation of G Protein-coupled Receptor Kinase 2. J Biol Chem,2003;278(31):29164-73.
4. Elorza A, Sarnago S and Mayor F, Jr. Agonist-dependent modulation of G protein-coupled receptor kinase 2 by mitogen-activated protein kinases. Mol Pharmacol,2000;57(4): 778-83.
5. Trudy A. Kohout and Robert J. Lefkowitz.Regulation of G Protein-Coupled Receptor Kinases and Arrestins During Receptor Desensitization . Mol Pharmacol ,2003;63(1):9-18.
6. Fraser ID, Cong M, Kim J, Rollins EN, Daaka Y, Lefkowitz RJ and Scott JD.Assembly of an A kinase-anchoring protein- 2-adrenergic receptor complex facilitates receptorphosphorylation and signaling. Curr Biol,2000;10(7):409-12.
7.Antonio Rapacciuolo,Shayela Suvarna,Liza Barki-Harrington. Protein Kinase A and G Protein-coupled Receptor Kinase Phosphorylation Mediatesβ-1 Adrenergic Receptor Endocytosis through Different Pathways. J Biol Chem,2003;278(37): 35403-11.
8. Cong M, Perry SJ, Lin FT, Fraser ID, Hu LA, Chen W, Pitcher JA, Scott JD and Lefkowitz RJ. Regulation of membrane targeting of the G protein-coupled receptor kinase 2 by protein kinase A and its anchoring protein AKAP79. J Biol Chem,2001;276:15192-9.
9. Xing-Long Yang, Ya-Li Zhang, Zhuo-Sheng et al.Pleckstrin homology domain of G protein-coupled receptor kinase-2 binds to PKC and affects the activity of PKC kinase. World J Gastroenterol, 2003;9(4):800-3.
10. Penela P, Ruiz-Gomez A, Castano JG and Mayor F, Jr. Degradation of the G protein-coupled receptor kinase 2 by the proteasome pathway. J Biol Chem,1998;273: 35238-44.
11. Penela P, Elorza A, Sarnago S and Mayor F, Jr. -arrestin and c-Src-dependent degradation of G-protein-coupled receptor kinase 2. EMBO J,2001;20(18): 5129-38.
12. Sallese M, Iacovelli L, Cumashi A, Capobianco L, Cuomo L and De Blasi A. Regulation of G protein-coupled receptor kinase subtypes by calcium sensor proteins. Biochim Biophys Acta,2000;1498(2-3): 112-21.
13. Alexey N.Pronin, Daulet K.Satpaev,Vladlen Z.Plepak et. Regulation of G protein-coupled receptor kinases by calmodulin and localization of the calmodulin binding domain. J Biol Chem,1997;272(29):18273-7.
14. Pronin AN, Carman CV and Benovic JL. Structure-function analysis of G protein-coupled receptor kinase-5. Role of the carboxyl terminus in kinase regulation. J Biol Chem,1998;273(47): 31510-8.
15. Christopher V. Carman, Michael P. Lisanti, and Jeffrey L. Benovic. Regulation of G Protein-coupled Receptor Kinases by Caveolin.J Biol Chem,1999;274(13):8858-64.
16. Schutzer WE, Reed JF, Mader SL.Decline in caveolin-1 expression and scaffolding of G protein receptor kinase-2 with age in Fischer 344 aortic vascular smooth muscle. Am J Physiol Heart Circ Physiol, 2005;288(5): 2457-64.
17. Maria Stella Lombardi, Evelyn van den Tweel, Annemieke Kavelaars et al. Hypoxia/Ischemia Modulates G Protein–Coupled Receptor Kinase 2 and ?-Arrestin-1 Levels in the Neonatal Rat Brain. Stroke,2004;35(4):981-6.
18. Maria Stella Lombardi, Annemieke Kavelaars, Petronila Penela.Oxidative Stress Decreases G Protein-coupled receptor kinase 2 in lymphocytes via a calpain-dependent mechanis .Mol Pharmacol,2002; 6(22):379–88.
19. Lombardi MS, Kavelaars A, Cobelens PM et al. Adjuvant arthritis induces down-regulation of G protein-coupled receptor kinases in the immune system. J Immunol,2001;166(3):1635-40.
20. Lombardi MS, Kavelaars A, Schedlowski M. Decreased expression and activity of G-protein-coupled receptor kinases in peripheral blood mononuclear cells of patients with rheumatoid arthritis. FASEB J,1999;13(6):715-25.
21. Hagen SA, Kondyra AL, Grocott HP et al. Cardiopulmonary bypass decreases G protein-coupled receptor kinase activity and expression in human peripheral blood mononuclear cells. Anesthesiology,2003; 98(2):343-8.
22 Ramos-Ruiz R, Penela P, Penn RB, Mayor F Jr.Analysis of the human G protein-coupled receptor kinase 2 (GRK2) gene promoter: regulation by signal transduction systems in aortic smooth muscle cells. Circulation,2000;101(17): 2083-9.
23 Kehrl JH.Heterotrimeric G protein signaling: roles in immune function and fine-tuning by RGS proteins. Immunity,1998;8(1):1-10.
24 De Vries L, Gist Farquhar MRGS proteins: more than just GAPs for heterotrimeric G proteins. Trends Cell Biol,1999;9(4):138-44.
25 Carman CV, Parent JL, Day PW, Pronin AN, Sternweis PM, Wedegaertner PB, Gilman AG, Benovic JL, Kozasa T.Selective regulation of Galpha(q/11) by an RGSdomain in the G protein-coupled receptor kinase, GRK2. J Biol Chem, 1999;274(48):34483-92.
26 Sallese M, MariggiòS, D'Urbano E, Iacovelli L, De Blasi A.Selective regulation of Gq signaling by G protein-coupled receptor kinase 2: direct interaction of kinase N terminus with activated galphaq. Mol Pharmacol,2000;57(4):826-31.
27 Usui H, Nishiyama M, Moroi K, Shibasaki T, Zhou J, Ishida J, Fukamizu A, Haga T, Sekiya S, Kimura S.RGS domain in the amino-terminus of G protein-coupled receptor kinase 2 inhibits Gq-mediated signaling. Int J Mol Med, 2000;5(4):335-40.
28 Tesmer VM, Kawano T, Shankaranarayanan A, Kozasa T, Tesmer JJ.Snapshot of activated G proteins at the membrane: the Galphaq-GRK2-Gbetagamma complex. Science,2005;310(5754):1686-90.
29. Tanja Eichmann, Kristina Lorenz, Michaela Hoffmann et al. The Amino-terminal Domain of G-protein-coupled Receptor Kinase 2 Is a Regulatory GβγBinding Site. J. Biol. Chem,2003;278(10) 8052-7
30 Chuang TT, Sallese M, Ambrosini G, Parruti G, De Blasi A.High expression of beta-adrenergic receptor kinase in human peripheral blood leukocytes. Isoproterenol and platelet activating factor can induce kinase translocation. J Biol Chem,1992;267(10):6886-92.
31 Lombardi MS, Kavelaars A, Cobelens PM, Schmidt RE, Schedlowski M, Heijnen CJ.Adjuvant arthritis induces down-regulation of G protein-coupled receptor kinases in the immune system. J Immunol,2001;166(3):1635-40.
32 Mak JC, Hisada T, Salmon M, Barnes PJ, Chung KF.Glucocorticoids reverse IL-1beta-induced impairment of beta-adrenoceptor-mediated relaxation and up-regulation of G-protein-coupled receptor kinases. Br J Pharmacol, 2002;135(4):987-96.
33 Vroon A, Lombardi MS, Kavelaars A, Heijnen CJ.Changes in the G-protein-coupled receptor desensitization machinery during relapsing-progressive experimental allergic encephalomyelitis. J Neuroimmunol, 2003;137(1-2):79-86.
34 Vroon A, Kavelaars A, Limmroth V, Lombardi MS, Goebel MU, Van Dam AM, Caron MG, Schedlowski M, Heijnen CJ.G protein-coupled receptor kinase 2 in multiple sclerosis and experimental autoimmune encephalomyelitis. J Immunol, 2005;174(7):4400-6.
35 Giorelli M, Livrea P, Trojano M.Post-receptorial mechanisms underlie functional disregulation of beta2-adrenergic receptors in lymphocytes from Multiple Sclerosis patients. J Neuroimmunol,2004;155(1-2):143-9.
36 Lombardi MS, Kavelaars A, Schedlowski M, Bijlsma JW, Okihara KL, Van de Pol M, Ochsmann S, Pawlak C, Schmidt RE, Heijnen CJ.Decreased expression and activity of G-protein-coupled receptor kinases in peripheral blood mononuclear cells of patients with rheumatoid arthritis. FASEB J,1999;13(6):715-25.
37 Ramos-Ruiz R, Penela P, Penn RB, Mayor F Jr.Analysis of the human G protein-coupled receptor kinase 2 (GRK2) gene promoter: regulation by signal transduction systems in aortic smooth muscle cells. Circulation, 2000;101(17): 2083-9.
38 Gilgun-Sherki Y, Melamed E, Offen D.The role of oxidative stress in the pathogenesis of multiple sclerosis: the need for effective antioxidant therapy. J Neurol,2004;251(3):261-8.
39 Okuda Y, Sakoda S, Yanagihara T. The pattern of cytokine gene expression in lymphoid organs and peripheral blood mononuclear cells of mice with experimental allergic encephalomyelitis. J Neuroimmunol,1998;87(1-2):147-55.
40 Huang WX, Huang P, Link H, Hillert J. Cytokine analysis in multiple sclerosis by competitive RT- PCR: A decreased expression of IL-10 and an increased expression of TNF-alpha in chronic progression. Mult Scler,1999;5(5):342-8.
41 Walsh NC, Crotti TN, Goldring SR, Gravallese EM. Rheumatic diseases: the effects of inflammation on bone. Immunol Rev,2005;208:228-51.
42 Izikson L, Klein RS, Charo IF, Weiner HL, Luster AD. Resistance to experimental autoimmune encephalomyelitis in mice lacking the CC chemokine receptor (CCR)2.J Exp Med,2000;192(7):1075-80.
43 Huang DR, Wang J, Kivisakk P, Rollins BJ, Ransohoff RM. Absence of monocyte chemoattractant protein 1 in mice leads to decreased local macrophage recruitment and antigen-specific T helper cell type 1 immune response in experimental autoimmune encephalomyelitis. J Exp Med,2001;193(6):713-26.
44 Bagaeva LV, Williams LP, Segal BM. IL-12 dependent/IFN gamma independent expression of CCR5 by myelin-reactive T cells correlates with encephalitogenicity. J Neuroimmunol,2003;137(1-2):109-16.
45 Oppermann M, Mack M, Proudfoot AE, Olbrich H. Differential effects of CC chemokines on CC chemokine receptor 5 (CCR5) phosphorylation and identification of phosphorylation sites on the CCR5 carboxyl terminus. J Biol Chem,1999;274(13):8875-85.
46 Olbrich H, Proudfoot AE, Oppermann M. Chemokine-induced phosphorylation of CC chemokine receptor 5 (CCR5). J Leukoc Biol,1999;65(3):281-5.
47 Aragay AM, Mellado M, Frade JM, Martin AM, Jimenez-Sainz MC, Martinez-A C, Mayor F Jr. Monocyte chemoattractant protein-1-induced CCR2B receptor desensitization mediated by the G protein-coupled receptor kinase 2. Proc Natl Acad Sci U S A,1998;95(6):2985-90.
48 Aramori I, Ferguson SS, Bieniasz PD, Zhang J, Cullen B, Cullen MG..Molecular mechanism of desensitization of the chemokine receptor CCR-5: receptor signaling and internalization are dissociable from its role as an HIV-1 co-receptor. EMBO J, 1997;16(15):4606-16.
49 Vroon A, Kavelaars A, Limmroth V, Lombardi MS, Goebel MU, Van Dam AM, Caron MG, Schedlowski M, Heijnen CJ.G protein-coupled receptor kinase 2 in multiple sclerosis and experimental autoimmune encephalomyelitis. J Immunol,2005;174(7):4400-6.
50 Vroon A, Heijnen CJ, Lombardi MS, Cobelens PM, Mayor F Jr, Caron MG, Kavelaars A.Reduced GRK2 level in T cells potentiates chemotaxis and signaling inresponse to CCL4. J Leukoc Biol,2004;75(5):901-9.
51 Kavelaars A, Vroon A, Raatgever RP, Fong AM, Premont RT, Patel DD, Lefkowitz RJ, Heijnen CJ. Increased acute inflammation, leukotriene B4-induced chemotaxis, and signaling in mice deficient for G protein-coupled receptor kinase 6. J Immunol,2003;171(11):6128-34.
52 Gaudreau R, Le Gouill C, Venne MH, Stankova J, Rola-Pleszczynski M. Threonine 308 within a putative casein kinase 2 site of the cytoplasmic tail of leukotriene B(4) receptor (BLT1) is crucial for ligand-induced, G-protein-coupled receptor-specific kinase 6-mediated desensitization. J Biol Chem, 2002;277(35):31567-76.
53 Pronin AN, Morris AJ, Surguchov A, Benovic JL.Synucleins are a novel class of substrates for G protein-coupled receptor kinases. J Biol Chem, 2000;275(34): 26515-22.
54 Ruiz-Gómez A, Humrich J, Murga C, Quitterer U, Lohse MJ, Mayor F Jr.Phosphorylation of phosducin and phosducin-like protein by G protein-coupled receptor kinase 2. J Biol Chem,2000;275(38):29724-30.
55 Cant SH, Pitcher JA.G protein-coupled receptor kinase 2-mediated phosphorylation of ezrin is required for G protein-coupled receptor-dependent reorganization of the actin cytoskeleton. Mol Biol Cell, 2005;16(7):3088-99.