G蛋白偶联受体对GRK功能调控的研究
|
摘要
G蛋白偶联受体（GPCR）是一类重要的细胞表面受体。GPCR的细胞内信号主要由G蛋白介导。激动剂的持续存在能引起GPCR的失敏，而激动剂诱导的GPCR磷酸化和内吞等是GPCR介导信号的负调控的主要机制。G蛋白偶联受体激酶 (GRK)是催化激动剂诱导的GPCR磷酸化以及启动GPCR脱敏的关键激酶。已发现七种GRK（GRK1-7），其中GRK2和GRK3主要分布在细胞浆，GRK的功能受GPCR和G蛋白等多种因素的严格调控。已知GRK2和GRK3只有与G蛋白((亚单位结合后才能向细胞膜转位，而且GRK只有与激活的GPCR结合以后，才能被激活并产生催化活性。现有的GRK功能调控假说认为：激动剂激活GPCR后导致游离的G((二聚体释放，随后GRK与锚定在细胞膜上的G((亚单位结合使GRK转位至细胞膜并磷酸化GPCR。但这一假说尚未得到体内实验证实，GRK向细胞膜转位和催化受体磷酸化的具体机制也不清楚。本研究在人胚胎肾细胞系（HEK293）中以(-阿片受体（DOR）为模型探讨了GPCR对GRK功能的调控机制，主要研究了激动剂诱导的GRK与G蛋白偶联受体和GRK与G((的相互作用及其对GRK细胞膜转位和GRK功能的影响。
本研究结果表明：（1）激动剂激活野生型DOR能诱导GRK2和受体的结合，使GRK2由细胞浆转位至细胞膜并催化DOR发生磷酸化。而在表达DOR羧基末端缺失突变体(31（不含有GRK磷酸化位点）的细胞中，激动剂的刺激不仅不能引起受体发生磷酸化，也不能诱导GRK2和受体的结合以及GRK2向细胞膜转位。这一结果证明，DOR的羧基末端是决定GRK2与受体结合的关键区域，并提示GPCR与GRK的结合是激动剂诱导GRK向细胞膜转位的必要条件。（2）进一步的实验结果显示，将DOR羧基末端GRK磷酸化位点邻近的酸性氨基酸突变为中性氨基酸(E355Q/D364N)后，激动剂刺激不能诱导其与GRK2的结合，并且也不能引起GRK2向细胞膜转位及使受体发生磷酸化。而所有三个GRK磷酸化位点都突变的DOR（M3）尽管不能发生磷酸化，但在激动剂刺激后能够与GRK2结合，并诱导GRK2向细胞膜转位。这些结果证明DOR的GRK磷酸化位点在GRK2与受体结合中不起重要作用，而其邻近的酸性氨基酸残基是受体结合GRK的关键位点。（3）实验结果显示，在激动剂刺激后，受体免疫复合物中不仅含有GRK，而且可检测出G(。G((亚基在受体复合体中的存在取决于受体与GRK的相互作用。共表达G((亚单位能促进激动剂诱导的GRK2和受体的结合以及GRK2向细胞膜转位。而共表达能够竞争结合G((亚单位的G(t或GRK2羧基末端能够完全抑制激动剂诱导的受体和GRK2的结合以及GRK2向细胞膜转位。这些结果证明激动剂刺激能诱导DOR-GRK2-G((复合体形成，并且这种复合体的形成是激动剂诱导的GRK2向细胞膜转位及GRK发挥功能的一个关键环节。
上述研究结果表明，G蛋白偶联受体不仅仅是GRK的底物，而且还是调控GRK功能的一个重要的因素。激动剂诱导的GPCR与GRK的结合是GRK向细胞膜转位的必要条件之一；激动剂能够诱导GPCR、GRK、G((在细胞膜上形成复合体，这种GPCR-GRK-G((复合体的形成是GRK向细胞膜转位及发挥催化功能的必要条件。
G protein-coupled receptor kinases (GRKs) catalyze agonist-induced receptor phosphorylation on the membrane and initiate receptor desensitization. Previous in vitro studies have shown that the binding of GRK to membrane-associated G(( subunits plays an important role in translocation of GRK2 from the cytoplasm to the plasma membrane. The current studies have investigated the role of the interaction of GRK2 with the activated (-opioid receptor (DOR) and G(( subunits in the membrane translocation and function of GRK2 using intact human embryonic kidney 293 cells. Our results showed that agonist treatment induced GRK2 translocation to the plasma membrane, GRK2 binding to DOR, and DOR phosphorylation in cells expressing the wild type DOR (WT), but not the mutant DOR lacking the carboxyl terminus which contains all three GRK2 phosphorylation sites. DORs with the GRK2-phosphorylation sites modified (M3) or with the acidic residues flanking phosphorylation sites mutated (E355Q/D364N) failed to be phosphorylated in response to agonist stimulation. Agonist-induced GRK2 membrane translocation and GRK-receptor association were observed in cells expressing M3, but not E355Q/D364N. Moreover, overexpression of G(( subunits promoted, whereas overexpression of transducin ??or the carboxyl terminal of GRK2 blocked GRK2 binding to DOR. Further study demonstrated that agonist stimulation induced formation of a complex containing DOR, GRK2, and G(( subunits in the cell and the agonist-stimulated formation of this complex is essential for the stable localization of GRK2 on the membrane and for its catalytic activity in vivo.
本研究结果表明：（1）激动剂激活野生型DOR能诱导GRK2和受体的结合，使GRK2由细胞浆转位至细胞膜并催化DOR发生磷酸化。而在表达DOR羧基末端缺失突变体(31（不含有GRK磷酸化位点）的细胞中，激动剂的刺激不仅不能引起受体发生磷酸化，也不能诱导GRK2和受体的结合以及GRK2向细胞膜转位。这一结果证明，DOR的羧基末端是决定GRK2与受体结合的关键区域，并提示GPCR与GRK的结合是激动剂诱导GRK向细胞膜转位的必要条件。（2）进一步的实验结果显示，将DOR羧基末端GRK磷酸化位点邻近的酸性氨基酸突变为中性氨基酸(E355Q/D364N)后，激动剂刺激不能诱导其与GRK2的结合，并且也不能引起GRK2向细胞膜转位及使受体发生磷酸化。而所有三个GRK磷酸化位点都突变的DOR（M3）尽管不能发生磷酸化，但在激动剂刺激后能够与GRK2结合，并诱导GRK2向细胞膜转位。这些结果证明DOR的GRK磷酸化位点在GRK2与受体结合中不起重要作用，而其邻近的酸性氨基酸残基是受体结合GRK的关键位点。（3）实验结果显示，在激动剂刺激后，受体免疫复合物中不仅含有GRK，而且可检测出G(。G((亚基在受体复合体中的存在取决于受体与GRK的相互作用。共表达G((亚单位能促进激动剂诱导的GRK2和受体的结合以及GRK2向细胞膜转位。而共表达能够竞争结合G((亚单位的G(t或GRK2羧基末端能够完全抑制激动剂诱导的受体和GRK2的结合以及GRK2向细胞膜转位。这些结果证明激动剂刺激能诱导DOR-GRK2-G((复合体形成，并且这种复合体的形成是激动剂诱导的GRK2向细胞膜转位及GRK发挥功能的一个关键环节。
上述研究结果表明，G蛋白偶联受体不仅仅是GRK的底物，而且还是调控GRK功能的一个重要的因素。激动剂诱导的GPCR与GRK的结合是GRK向细胞膜转位的必要条件之一；激动剂能够诱导GPCR、GRK、G((在细胞膜上形成复合体，这种GPCR-GRK-G((复合体的形成是GRK向细胞膜转位及发挥催化功能的必要条件。
G protein-coupled receptor kinases (GRKs) catalyze agonist-induced receptor phosphorylation on the membrane and initiate receptor desensitization. Previous in vitro studies have shown that the binding of GRK to membrane-associated G(( subunits plays an important role in translocation of GRK2 from the cytoplasm to the plasma membrane. The current studies have investigated the role of the interaction of GRK2 with the activated (-opioid receptor (DOR) and G(( subunits in the membrane translocation and function of GRK2 using intact human embryonic kidney 293 cells. Our results showed that agonist treatment induced GRK2 translocation to the plasma membrane, GRK2 binding to DOR, and DOR phosphorylation in cells expressing the wild type DOR (WT), but not the mutant DOR lacking the carboxyl terminus which contains all three GRK2 phosphorylation sites. DORs with the GRK2-phosphorylation sites modified (M3) or with the acidic residues flanking phosphorylation sites mutated (E355Q/D364N) failed to be phosphorylated in response to agonist stimulation. Agonist-induced GRK2 membrane translocation and GRK-receptor association were observed in cells expressing M3, but not E355Q/D364N. Moreover, overexpression of G(( subunits promoted, whereas overexpression of transducin ??or the carboxyl terminal of GRK2 blocked GRK2 binding to DOR. Further study demonstrated that agonist stimulation induced formation of a complex containing DOR, GRK2, and G(( subunits in the cell and the agonist-stimulated formation of this complex is essential for the stable localization of GRK2 on the membrane and for its catalytic activity in vivo.
引文
1. Wess J. G-protein-coupled receptors: molecular mechanisms involved in receptor activation and selectivity of G-protein recognition FASEB J. 1997, 11:346-354.
2. Ji TH, Grossmann M and Ji I. G Protein-coupled receptors. I. Diversity of receptor-ligand interactions. J Biol Chem. 1998, 273: 17299 - 17302.
3. Bargmann CI. The mechanism of anesthetic activation of TREK-1 and TASK was probed by using genetic constructs as well as by recording from excised patches. Science. 1998, 282: 2028-2033.
4. Venter JC, Adams MD, Myers EW, et al. The Sequence of the Human Genome. Science. 2001, 291: 1304-1351.
5. Carman CV, Benovic JL. G-protein-coupled receptors: turn-ons and turn-offs. Curr Opin Neurobiol. 1998, 8: 335-344.
6. Hisatomi O, Matsuda S, Satoh T, Kotaka S, Imanishi Y and Tokunaga F. A novel subtype of G-protein-coupled receptor kinase, GRK7, in teleost cone photoreceptors. FEBS Lett. 1998, 424: 159-164.
7. Inglese J, Freedman NJ, Koch W J and Lefkowitz RJ. Structure and mechanism of the G protein-coupled receptor kinases. J Biol Chem. 1993, 268: 23735-23738.
8. Premont RT, Inglese J and Lefkowitz RJ. Protein kinases that phosphorylate activated G protein-coupled receptors. FASEB J. 1995, 9: 175-182.
9. Pitcher JA, Inglese J, Higgins JB, Benovic JL, Kwatra MM, Caron MG and Lefkowitz RJ. Role of (( subunits of G proteins in targeting the (-adrenergic receptor kinase to membrane-bound receptors. Science.1992, 257: 125-128.
10. Kim CM, Dion SB and Benovic JL. Mechanism of beta-adrenergic receptor kinase activation by G proteins. J Biol Chem. 1993, 268: 15412-15418.
11. Pitcher JA, Touhara K, Parne ES and Lefkowitz RJ. Pleckstrin homology domain-mediated membrane association and activation of b-adrenergic receptor kinase requires coordinate interaction with G(( subunits and lipid. J Biol Chem. 1995, 270: 11707-11710.
12. Haga K, Kameyama K and Haga T. Synergistic activation of a G protein-coupled receptor kinase by G protein beta gamma subunits and mastoparan or related peptides. J Biol Chem. 1994, 269: 12594-12599.
13. Daaka Y, Pitcher JA, Richardson M, Stoffel RH, Robishaw JD and Lefkowitz RJ. Receptor and G(( isoform-specific interactions with G protein-coupled receptor kinases. Proc Natl Acad Sci U S A. 1997, 94: 2180-2185.
14. Benovic JL, Strasser RH, Caron MG and Lefkowitz RJ. Beta-adrenergic receptor kinase: identification of a novel protein kinase that phosphorylates the agonist-occupied form of the receptor. Proc Natl Acad Sci U S A.1986, 83: 2797-2801.
15. Penn RB, Pronin AN and Benovic JL. Regulation of G protein-coupled receptor kinases. Trends Cardiovasc Med. 2000, 10:81-89.
16. Strasser RH, Benovic JL, Caron MG and Lefkowitz RJ. Beta-agonist- and prostaglandin E1-induced translocation of the beta-adrenergic receptor kinase: evidence that the kinase may act on multiple adenylate cyclase-coupled receptors. Proc Natl Acad Sci U S A. 1986, 83: 6362-6366.
17. Pei G, Kieffer BL, Lefkowitz RJ and Freedman NJ. Agonist-dependent phosphorylation of the mouse delta-opioid receptor: involvement of G protein-coupled receptor kinases but not protein kinase C. Mol Pharmacol. 1995, 48: 173-177.
18. Zhao J, Pei G, Huang YL, Zhong FM and Ma L. Carboxyl terminus of delta opioid receptor is required for agonist-dependent receptor phosphorylation. Biochem Biophy Res Comm. 1997, 238: 71-76.
19. Guo J, Wu YL, Zhang WB, Zhao J, Devi LA, Pei G and Ma L. Identification of G Protein-Coupled Receptor Kinase 2 Phosphorylation Sites Responsible for Agonist-Stimulated (-Opioid Receptor Phosphorylation. Mol Pharmacol. 2000, 58:1050-1056.
20. Xiang B, Yu GH, Guo J, Chen L, Hu W, Pei G and Ma L. Heterologous Activation of Protein Kinase C Stimulates Phosphorylation of (-Opioid Receptor at Serine 344, Resulting in (-Arrestin- and Clathrin-mediated Receptor Internalization. J Biol Chem. 2001, 276: 4709-4716.
Cheng ZJ, Fan GH, Zhao J, Zhang Z, Wu YL, Jiang LZ, Zhu Y, Pei G and Ma L.
21. Endogenous opioid receptor-like receptor in human neuroblastoma SK-N-SH cells: activation of inhibitory G-protein and homologous desensitization. NeuroReport. 1997, 8: 1913-1918.
22. Freedman NJ, Ament AS, Oppermann M, Stoffel RH, Exum ST and Lefkowitz R J. Phosphorylation and Desensitization of Human Endothelin A and B Receptors. Evidence for G protein-coupled receptor kinase specificity. J Biol Chem. 1997, 272: 17734-17743.
23. Wang P, Gao H, NiY, Wang B, Wu Yn, Ji L, Qin L, Ma L and Pei G. (-arrestin 2 functions as a G protein-coupled receptor-activated regulator of oncoprotein Mdm2. J Biol Chem. 2003,278: 6363 - 6370.
24. Dhami GK, Anborgh PH, Dale LB, Sterne-Marr R and Ferguson SS. Phosphorylation-independent regulation of metabotropic glutamate receptor signaling by G protein-coupled receptor kinase 2. J Biol Chem. 2002, 277: 25266-25272.
25. Perry SJ and Lefkowitz RJ. Arresting developments in heptahelical receptor signaling and regulation. Trends Cell Biol. 2002, 12:130-8.
26. Kouhen OM, Wang G, Solberg J, Erickson LJ, Law PY and Loh HH. Hierarchical Phosphorylation of -Opioid Receptor Regulates Agonist-induced Receptor Desensitization and Internalization. J Biol Chem. 2000, 275: 36659-36664.
27. Pitcher JA, Freedman NJ and Lefkowitz RJ. G protein-coupled receptor kinases.
Annu Rev Biochem. 1998,67: 653-692.
28. Onorato JJ, Palczewski K, Regan JW, Caron MG, Lefkowitz RJ and Benovic JL. Role of acidic amino acids in peptide substrates of the beta-adrenergic receptor kinase and rhodopsin kinase. Biochemistry.1991, 30: 5118-5125.
29. Pak Y, O(Dowd BF and George SR. Agonist-induced desensitization of the mu opioid receptor is determined by threonine 394 preceded by acidic amino acids in the COOH-terminal tail. J Biol Chem. 1997, 272: 24961-24965.
30. Lee KB, Ptasienski JA, Bunemann M and Hosey MM. Acidic Amino Acids Flanking Phosphorylation Sites in the M2 Muscarinic Receptor Regulate Receptor Phosphorylation, Internalization, and Interaction with Arrestins. J Biol Chem. 2000, 275: 35767-35777.
31. Small KM, Brown KM, Forbes SL and Liggett SB. Polymorphic deletion of three intracellular acidic residues of the alpha2B-adrenergic receptor decreases G protein-coupled receptor kinase-mediated phosphorylation and desensitization. J Biol Chem. 2001, 276: 4917-4922.
2. Ji TH, Grossmann M and Ji I. G Protein-coupled receptors. I. Diversity of receptor-ligand interactions. J Biol Chem. 1998, 273: 17299 - 17302.
3. Bargmann CI. The mechanism of anesthetic activation of TREK-1 and TASK was probed by using genetic constructs as well as by recording from excised patches. Science. 1998, 282: 2028-2033.
4. Venter JC, Adams MD, Myers EW, et al. The Sequence of the Human Genome. Science. 2001, 291: 1304-1351.
5. Carman CV, Benovic JL. G-protein-coupled receptors: turn-ons and turn-offs. Curr Opin Neurobiol. 1998, 8: 335-344.
6. Hisatomi O, Matsuda S, Satoh T, Kotaka S, Imanishi Y and Tokunaga F. A novel subtype of G-protein-coupled receptor kinase, GRK7, in teleost cone photoreceptors. FEBS Lett. 1998, 424: 159-164.
7. Inglese J, Freedman NJ, Koch W J and Lefkowitz RJ. Structure and mechanism of the G protein-coupled receptor kinases. J Biol Chem. 1993, 268: 23735-23738.
8. Premont RT, Inglese J and Lefkowitz RJ. Protein kinases that phosphorylate activated G protein-coupled receptors. FASEB J. 1995, 9: 175-182.
9. Pitcher JA, Inglese J, Higgins JB, Benovic JL, Kwatra MM, Caron MG and Lefkowitz RJ. Role of (( subunits of G proteins in targeting the (-adrenergic receptor kinase to membrane-bound receptors. Science.1992, 257: 125-128.
10. Kim CM, Dion SB and Benovic JL. Mechanism of beta-adrenergic receptor kinase activation by G proteins. J Biol Chem. 1993, 268: 15412-15418.
11. Pitcher JA, Touhara K, Parne ES and Lefkowitz RJ. Pleckstrin homology domain-mediated membrane association and activation of b-adrenergic receptor kinase requires coordinate interaction with G(( subunits and lipid. J Biol Chem. 1995, 270: 11707-11710.
12. Haga K, Kameyama K and Haga T. Synergistic activation of a G protein-coupled receptor kinase by G protein beta gamma subunits and mastoparan or related peptides. J Biol Chem. 1994, 269: 12594-12599.
13. Daaka Y, Pitcher JA, Richardson M, Stoffel RH, Robishaw JD and Lefkowitz RJ. Receptor and G(( isoform-specific interactions with G protein-coupled receptor kinases. Proc Natl Acad Sci U S A. 1997, 94: 2180-2185.
14. Benovic JL, Strasser RH, Caron MG and Lefkowitz RJ. Beta-adrenergic receptor kinase: identification of a novel protein kinase that phosphorylates the agonist-occupied form of the receptor. Proc Natl Acad Sci U S A.1986, 83: 2797-2801.
15. Penn RB, Pronin AN and Benovic JL. Regulation of G protein-coupled receptor kinases. Trends Cardiovasc Med. 2000, 10:81-89.
16. Strasser RH, Benovic JL, Caron MG and Lefkowitz RJ. Beta-agonist- and prostaglandin E1-induced translocation of the beta-adrenergic receptor kinase: evidence that the kinase may act on multiple adenylate cyclase-coupled receptors. Proc Natl Acad Sci U S A. 1986, 83: 6362-6366.
17. Pei G, Kieffer BL, Lefkowitz RJ and Freedman NJ. Agonist-dependent phosphorylation of the mouse delta-opioid receptor: involvement of G protein-coupled receptor kinases but not protein kinase C. Mol Pharmacol. 1995, 48: 173-177.
18. Zhao J, Pei G, Huang YL, Zhong FM and Ma L. Carboxyl terminus of delta opioid receptor is required for agonist-dependent receptor phosphorylation. Biochem Biophy Res Comm. 1997, 238: 71-76.
19. Guo J, Wu YL, Zhang WB, Zhao J, Devi LA, Pei G and Ma L. Identification of G Protein-Coupled Receptor Kinase 2 Phosphorylation Sites Responsible for Agonist-Stimulated (-Opioid Receptor Phosphorylation. Mol Pharmacol. 2000, 58:1050-1056.
20. Xiang B, Yu GH, Guo J, Chen L, Hu W, Pei G and Ma L. Heterologous Activation of Protein Kinase C Stimulates Phosphorylation of (-Opioid Receptor at Serine 344, Resulting in (-Arrestin- and Clathrin-mediated Receptor Internalization. J Biol Chem. 2001, 276: 4709-4716.
Cheng ZJ, Fan GH, Zhao J, Zhang Z, Wu YL, Jiang LZ, Zhu Y, Pei G and Ma L.
21. Endogenous opioid receptor-like receptor in human neuroblastoma SK-N-SH cells: activation of inhibitory G-protein and homologous desensitization. NeuroReport. 1997, 8: 1913-1918.
22. Freedman NJ, Ament AS, Oppermann M, Stoffel RH, Exum ST and Lefkowitz R J. Phosphorylation and Desensitization of Human Endothelin A and B Receptors. Evidence for G protein-coupled receptor kinase specificity. J Biol Chem. 1997, 272: 17734-17743.
23. Wang P, Gao H, NiY, Wang B, Wu Yn, Ji L, Qin L, Ma L and Pei G. (-arrestin 2 functions as a G protein-coupled receptor-activated regulator of oncoprotein Mdm2. J Biol Chem. 2003,278: 6363 - 6370.
24. Dhami GK, Anborgh PH, Dale LB, Sterne-Marr R and Ferguson SS. Phosphorylation-independent regulation of metabotropic glutamate receptor signaling by G protein-coupled receptor kinase 2. J Biol Chem. 2002, 277: 25266-25272.
25. Perry SJ and Lefkowitz RJ. Arresting developments in heptahelical receptor signaling and regulation. Trends Cell Biol. 2002, 12:130-8.
26. Kouhen OM, Wang G, Solberg J, Erickson LJ, Law PY and Loh HH. Hierarchical Phosphorylation of -Opioid Receptor Regulates Agonist-induced Receptor Desensitization and Internalization. J Biol Chem. 2000, 275: 36659-36664.
27. Pitcher JA, Freedman NJ and Lefkowitz RJ. G protein-coupled receptor kinases.
Annu Rev Biochem. 1998,67: 653-692.
28. Onorato JJ, Palczewski K, Regan JW, Caron MG, Lefkowitz RJ and Benovic JL. Role of acidic amino acids in peptide substrates of the beta-adrenergic receptor kinase and rhodopsin kinase. Biochemistry.1991, 30: 5118-5125.
29. Pak Y, O(Dowd BF and George SR. Agonist-induced desensitization of the mu opioid receptor is determined by threonine 394 preceded by acidic amino acids in the COOH-terminal tail. J Biol Chem. 1997, 272: 24961-24965.
30. Lee KB, Ptasienski JA, Bunemann M and Hosey MM. Acidic Amino Acids Flanking Phosphorylation Sites in the M2 Muscarinic Receptor Regulate Receptor Phosphorylation, Internalization, and Interaction with Arrestins. J Biol Chem. 2000, 275: 35767-35777.
31. Small KM, Brown KM, Forbes SL and Liggett SB. Polymorphic deletion of three intracellular acidic residues of the alpha2B-adrenergic receptor decreases G protein-coupled receptor kinase-mediated phosphorylation and desensitization. J Biol Chem. 2001, 276: 4917-4922.