Abstract
A functional comparison was made between the wild-type bradykinin B2 receptor (B2wt) and the chimera B2eGFP (enhanced green-fluorescent protein fused to the C-terminus of B2wt), both stably expressed in HEK 293 cells. There was almost no difference in terms of ligand-inducible receptor phosphorylation and internalization, signal transduction (accumulation of inositol phosphates) or expression and affinity. However, stimulation for up to 8 h with 10 μM bradykinin (BK) resulted in a strong decrease in surface receptors (by 60% within 5 h) in B2wt, but not in B2eGFP. When the expression levels of both constructs where comparably reduced using a weaker promoter, long-term stimulation resulted in a reduction in surface receptors for B2wtlow to less than 20% within 1 h, whereas the chimera B2eGFPlow still displayed 50% binding activity after 2 h. A 1-h incubation in the absence of BK resulted in a recovery of 60% of the binding in B2wtlow after 1-h stimulation with BK, but of only 20% after 7-h stimulation. In contrast, B2eGFPlow levels were restored to more than 70%, even after 7-h stimulation. These data indicate that although the fusion of eGFP to B2wt does not affect its ligand-induced internalization, it strongly reduces the down-regulation, most likely by promoting receptor recycling over degradation.
References
Bachvarov, D.R., Houle, S., Bachvarova, M., Bouthillier, J., Adam, A., and Marceau, F. (2001). Bradykinin B2 receptor endocytosis, recycling, and down-regulation assessed using green fluorescent protein conjugates. J. Pharmacol. Exp. Ther.297, 19–26.Search in Google Scholar
Brink, C.B., Harvey, B.H., Bodenstein, J., Venter, D.P., and Oliver, D.W. (2004). Recent advances in drug action and therapeutics: relevance of novel concepts in G protein-coupled receptor and signal transduction pharmacology. Br. J. Clin. Pharmacol.57, 373–387.10.1111/j.1365-2125.2003.02046.xSearch in Google Scholar
Blaukat, A., Pizard. A., Breit, A., Wernstedt, C., Alhenc-Gelas, F., Müller-Esterl, W., and Dikic, I. (2001). Determination of bradykinin B2 receptor in vivo phosphorylation sites and their role in receptor function. J. Biol. Chem.276, 40431–40440.10.1074/jbc.M107024200Search in Google Scholar
Dendorfer, A., Wolfrum, S., Wellhöner, P., Korsman, K., and Dominiak, P. (1997). Intravascular and interstitial degradation of bradykinin in isolated perfused rat heart. Br. J. Pharmacol.122, 1179–1187.10.1038/sj.bjp.0701501Search in Google Scholar
Faussner, A., Proud, D., Towns, M., and Bathon, J.M. (1998). Influence of the cytosolic carboxyl termini of human B1 and B2 kinin receptors on receptor sequestration, ligand inter-nalization, and signal transduction. J. Biol. Chem.273, 2617–2623.10.1074/jbc.273.5.2617Search in Google Scholar
Faussner, A., Bauer, A., Kalatskaya, I., Jochum, M., and Fritz, H. (2003). Expression levels strongly affect ligand-induced sequestration of B2 bradykinin receptors in transfected cells. Am. J. Physiol. Heart284, 1892–1898.10.1152/ajpheart.01147.2002Search in Google Scholar
Faussner, A., Schuessler, S., Seidl, C., and Jochum, M. (2004). Inhibition of sequestration of human B2 bradykinin receptor by phenylarsine oxide or sucrose allows determination of a receptor affinity shift and ligand dissociation in intact cells. Biol. Chem.385, 835–843.10.1515/BC.2004.109Search in Google Scholar
Ferguson, S.S. (2001). Evolving concepts in G protein-coupled receptor endocytosis: the role in receptor desensitization and signalling. Pharmacol. Rev.53, 1–24.Search in Google Scholar
Fredriksson, R., Lagerström, M.C., Lundin, L., and Schiöth, H.B. (2003). The G protein-coupled receptors in the human genome form five main families. Phylogenetic analysis, paralogon groups, and fingerprints. Mol. Pharmacol.63, 1256–1272.Search in Google Scholar
Gether, U. (2000). Uncovering molecular mechanisms involved in activation of G protein-coupled receptors. Endocrine Rev.21, 90–113.10.1210/edrv.21.1.0390Search in Google Scholar
Haasemann, M., Cartaud, J., Müller-Esterl, W., and Dunia, I. (1998). Agonist-induced redistribution of bradykinin B2 receptors in caveolae. J. Cell Sci.111, 917–928.10.1242/jcs.111.7.917Search in Google Scholar
Hess, J.F., Borkowski, J.A., Young, G.S., Strader, C.D., and Ransom, R.W. (1992). Cloning and pharmacological characterization of a human bradykinin (BK-2) receptor. Biochem. Biophys. Res. Commun.184, 260–268.10.1016/0006-291X(92)91187-USearch in Google Scholar
Haglund, K., Di Fiore, P.P., and Dikic, I. (2003). Distinct monoubiquitin signals in receptor endocytosis. Trends Biochem. Sci.28, 598–603.10.1016/j.tibs.2003.09.005Search in Google Scholar PubMed
Houle, S. and Marceau, F. (2003). Wortmannin alters the intracellular trafficking of the bradykinin B2 receptor: role of phosphoinositide 3-kinase and Rab5. Biochem. J.375, 151–158.10.1042/bj20030872Search in Google Scholar
Kalatskaya, I., Schüssler, S., Blaukat, A., Müller-Esterl, W., Jochum, M., Proud, D., and Faussner, A. (2004). Mutation of tyrosine in conserved NPXXY sequence leads to constitutive phosphorylation and internalization, but not signalling of the human B2 bradykinin receptor. J. Biol. Chem.279, 31268–31276.10.1074/jbc.M401796200Search in Google Scholar
Kallal, L., Gagnon, A.W., Penn, R.B., and Benovic, J.L. (1998). Visualization of agonist-induced sequestration and down-regulation of a green fluorescent protein-tagged β2-adrenergic receptor. J. Biol. Chem.273, 322–328.10.1074/jbc.273.1.322Search in Google Scholar
Leeb-Lundberg, L.M.F., Marceau, F., Müller-Ester, W., Pettibone, D.J., and Zuraw, B.L. (2005). International union of pharmacology. XLV. Classification of the kinin receptor family: from the molecular mechanisms to pathophysiological consequences. Pharmacol. Rev.57, 27–77.Search in Google Scholar
Marchese, A., Chen, C., Kim, Y.M., and Benovic, J.L. (2003). The ins and outs of G protein-coupled receptor trafficking. Trends Biochem. Sci.28, 369–376.10.1016/S0968-0004(03)00134-8Search in Google Scholar
Maxfield, F.R. and McGraw, T.E. (2004). Endocytic recycling. Nat. Rev. Mol. Cell Biol.5, 121–132.10.1038/nrm1315Search in Google Scholar
Milligan, G. (1999). Exploring the dynamics of regulation of G protein-coupled receptors using green fluorescent protein. Br. J. Pharmacol.128, 501–510.10.1038/sj.bjp.0702824Search in Google Scholar
Mitchell, R., McCulloch, D., Lutz, E., Johnson, M., MacKenzie, C., Fennell, M., Fink, G., Zhou, W., and Sealfon, S.C. (1998). Rhodopsin-family receptors associate with small G proteins to activate phospholipase D. Nature392, 411–414.10.1038/32937Search in Google Scholar
Modrall, J.G., Nanamori, M., Sadoshima, J., Barnhart, D.C., Stanley, J.C., and Neubig, R.R., (2001). ANG II type 1 receptor downregulation does not require receptor endocytosis or G protein coupling. Am. J. Physiol. Cell Physiol.281, C801–C809.10.1152/ajpcell.2001.281.3.C801Search in Google Scholar
Mousavi, S.A., Malerod, L., Berg, T., and Kjeken R. (2004). Clathrin-dependent endocytosis. Biochem. J.377, 1–16.10.1042/bj20031000Search in Google Scholar
Perry, S.J. and Lefkowitz, R.J. (2002). Arresting developments in heptahelical receptor signalling and regulation. Trends Cell. Biol.12, 130–138.10.1016/S0962-8924(01)02239-5Search in Google Scholar
Pierce, K.L., Premont, R.T., and Lefkowitz, R.J. (2002). Seven-transmembrane receptors. Nat. Rev.3, 639–650.10.1038/nrm908Search in Google Scholar PubMed
Prado, G.N, Taylor, L., and Polgar, P. (1997). Effects of intracellular tyrosine residue mutation and carboxyl terminus truncation on signal transduction and internalization of the rat bradykinin B2 receptor. J. Biol. Chem.272, 14638–14642.10.1074/jbc.272.23.14638Search in Google Scholar PubMed
Proud, D. (1988). Kinin formation: mechanisms and role in inflammatory disorders. Annu. Rev. Immunol.6, 49–83.10.1146/annurev.iy.06.040188.000405Search in Google Scholar PubMed
Regoli, D., Rizzi, A., Perron, S.I., and Gobeil, F. Jr. (2001). Classification of kinin receptors. Biol. Chem.382, 31–35.10.1515/BC.2001.005Search in Google Scholar PubMed
Roscher, A.A., Manganiello, V.C., Jelsema, C.L., and Moss, J. (1984). Autoregulation of bradykinin receptors and bradykinin-induced prostacyclin formation in human fibroblasts. J. Clin. Invest.74, 552–558.10.1172/JCI111452Search in Google Scholar PubMed PubMed Central
Roseberry, A.G. and Hosey, M.M. (2001). Internalization of the M2 muscarinic acetylcholine receptor proceeds through an atypical pathway in HEK293 cells that is independent of clathrin and caveolae. J. Cell Science114, 739–746.10.1242/jcs.114.4.739Search in Google Scholar PubMed
Sabourin, T., Bastien, L., Bachvarov, D.R., and Marceau, F. (2002). Agonist-induced translocation of the kinin B1 receptor to caveolae-related rafts. Mol. Pharmacol.61, 546–553.10.1124/mol.61.3.546Search in Google Scholar PubMed
Shenoy, S.K. and Lefkowitz, R.J. (2005). Receptor-specific ubiquitination of β-arrestin directs assembly and targeting of seven-transmembrane receptor signalosomes. J. Biol. Chem.280, 15315–15324.10.1074/jbc.M412418200Search in Google Scholar PubMed
Simaan, M., Bédard-Goulet, S., Fessart, D., Gratton, J.-P., and Laporte, S.A. (2005). Dissociation of β-arrestin from internalized bradykinin B2 receptor is necessary for receptor recycling and resensitization. Cell. Signal.17, 1074–1083.10.1016/j.cellsig.2004.12.001Search in Google Scholar PubMed
Stewart, J.M. (2004). Bradykinin antagonist: discovery and development. Peptides25, 527–532.10.1016/j.peptides.2003.10.016Search in Google Scholar PubMed
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