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W1971800733 abstract "Proteases cleave proteinase-activated receptors (PARs) to expose N-terminal tethered ligands that bind and activate the cleaved receptors. The tethered ligand, once exposed, is always available to interact with its binding site. Thus, efficient mechanisms must prevent continuous activation, including receptor phosphorylation and uncoupling from G-proteins, receptor endocytosis, and lysosomal degradation. β-Arrestins mediate uncoupling and endocytosis of certain neurotransmitter receptors, which are activated in a reversible manner. However, the role of β-arrestins in trafficking of PARs, which are irreversibly activated, and the effects of proteases on the subcellular distribution of β-arrestins have not been examined. We studied trafficking of PAR2 and β-arrestin1 coupled to green fluorescent protein. Trypsin induced the following: (a) redistribution of β-arrestin1 from the cytosol to the plasma membrane, where it co-localized with PAR2; (b) internalization of β-arrestin1 and PAR2 into the same early endosomes; (c) redistribution of β-arrestin1 to the cytosol concurrent with PAR2 translocation to lysosomes; and (d) mobilization of PAR2 from the Golgi apparatus to the plasma membrane. Overexpression of a C-terminal fragment of β-arrestin-319–418, which interacts constitutively with clathrin but does not bind receptors, inhibited agonist-induced endocytosis of PAR2. Our results show that β-arrestins mediate endocytosis of PAR2 and support a role for β-arrestins in uncoupling of PARs. Proteases cleave proteinase-activated receptors (PARs) to expose N-terminal tethered ligands that bind and activate the cleaved receptors. The tethered ligand, once exposed, is always available to interact with its binding site. Thus, efficient mechanisms must prevent continuous activation, including receptor phosphorylation and uncoupling from G-proteins, receptor endocytosis, and lysosomal degradation. β-Arrestins mediate uncoupling and endocytosis of certain neurotransmitter receptors, which are activated in a reversible manner. However, the role of β-arrestins in trafficking of PARs, which are irreversibly activated, and the effects of proteases on the subcellular distribution of β-arrestins have not been examined. We studied trafficking of PAR2 and β-arrestin1 coupled to green fluorescent protein. Trypsin induced the following: (a) redistribution of β-arrestin1 from the cytosol to the plasma membrane, where it co-localized with PAR2; (b) internalization of β-arrestin1 and PAR2 into the same early endosomes; (c) redistribution of β-arrestin1 to the cytosol concurrent with PAR2 translocation to lysosomes; and (d) mobilization of PAR2 from the Golgi apparatus to the plasma membrane. Overexpression of a C-terminal fragment of β-arrestin-319–418, which interacts constitutively with clathrin but does not bind receptors, inhibited agonist-induced endocytosis of PAR2. Our results show that β-arrestins mediate endocytosis of PAR2 and support a role for β-arrestins in uncoupling of PARs. Proteinase-activated receptors (PARs) 1The abbreviations used are: PARs, proteinase-activated receptors; AP, activating peptide SLIGKV-NH2; ARR, arrestin; β2AR, β2-adrenergic receptor; GFP, green fluorescent protein; EGFP, enhanced green fluorescent protein; GPCR, G-protein-coupled receptor; GRK, G-protein-coupled receptor kinase; KNRK cells, Kirsten murine sarcoma virus-transformed rat kidney epithelial cells; DMEM, Dulbecco's modified Eagle's medium; BSA, bovine serum albumin; PBS, phosphate-buffered saline; LAMP-1, lysosomal acidic membrane protein-1; BAPTA-AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester).1The abbreviations used are: PARs, proteinase-activated receptors; AP, activating peptide SLIGKV-NH2; ARR, arrestin; β2AR, β2-adrenergic receptor; GFP, green fluorescent protein; EGFP, enhanced green fluorescent protein; GPCR, G-protein-coupled receptor; GRK, G-protein-coupled receptor kinase; KNRK cells, Kirsten murine sarcoma virus-transformed rat kidney epithelial cells; DMEM, Dulbecco's modified Eagle's medium; BSA, bovine serum albumin; PBS, phosphate-buffered saline; LAMP-1, lysosomal acidic membrane protein-1; BAPTA-AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester).are a new and growing family of G-protein-coupled receptors (GPCRs) for proteases (1Déry O. Corvera C.U. Steinhoff M. Bunnett N.W. Am. J. Physiol. 1998; 274: C1429-C1452Crossref PubMed Google Scholar). Thrombin activates PAR1, PAR3, and PAR4 (2Vu T.K. Hung D.T. Wheaton V.I. Coughlin S.R. Cell. 1991; 64: 1057-1068Abstract Full Text PDF PubMed Scopus (2650) Google Scholar, 3Ishihara H. Connolly A.J. Zeng D. Kahn M.L. Zheng Y.W. Timmons C. Tram T. Coughlin S.R. Nature. 1997; 386: 502-506Crossref PubMed Scopus (796) Google Scholar, 4Xu W. Andersen H. Whitmore T.E. Presnell S.R. Yee D.P. Ching A. Gilbert T. Davie E.W. Foster D.C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6642-6646Crossref PubMed Scopus (746) Google Scholar, 5Kahn M.L. Zheng Y.-W. Huang W. Bigornia V. Zeng D. Moff S. Farese R.V. Tam C. Coughlin S.R. Nature. 1998; 394: 690-694Crossref PubMed Scopus (864) Google Scholar), whereas trypsin and mast cell tryptase activate PAR2 and probably PAR4 (6Nystedt S. Emilsson K. Wahlestedt C. Sundelin J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9208-9212Crossref PubMed Scopus (826) Google Scholar, 7Molino M. Barnathan E.S. Numerof R. Clark J. Dreyer M. Cumashi A. Hoxie J. Schechter N. Woolkalis M. Brass L.F. J. Biol. Chem. 1997; 272: 4043-4049Abstract Full Text Full Text PDF PubMed Scopus (543) Google Scholar, 8Corvera C.U. Dery O. McConalogue K. Bohm S.K. Khitin L.M. Caughey G.H. Payan D.G. Bunnett N.W. J. Clin. Invest. 1997; 100: 1383-1393Crossref PubMed Scopus (266) Google Scholar). Proteases cleave PARs within the extracellular N termini to expose tethered ligand domains that bind to and activate the cleaved receptors.Despite the irreversible nature of proteolytic activation, signaling by PARs, in common with other GPCRs, is rapidly terminated. For neurotransmitter receptors, which are activated in a reversible manner, the mechanisms of signal termination have been extensively investigated (9Böhm S. Grady E.F. Bunnett N.W. Biochem. J. 1997; 322: 1-18Crossref PubMed Scopus (462) Google Scholar). These mechanisms include (a) receptor phosphorylation and uncoupling from heterotrimeric G-proteins and (b) receptor endocytosis. G-protein-coupled receptor kinases (GRKs) and β-arrestins are cytosolic proteins that translocate to the plasma membrane to mediate uncoupling and endocytosis of GPCRs for certain neurotransmitters. For example, GRK2 and -3 phosphorylate β2-adrenergic receptors (β2ARs) (10Benovic J.L. DeBlasi A. Stone W.C. Caron M.G. Lefkowitz R.J. Science. 1989; 246: 235-240Crossref PubMed Scopus (327) Google Scholar, 11Benovic J.L. Onorato J.J. Arriza J.L. Stone W.C. Lohse M. Jenkins N.A. Gilbert D.J. Copeland N.G. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1991; 266: 14939-14946Abstract Full Text PDF PubMed Google Scholar, 12Pippig S. Andexinger S. Daniel K. Puzicha M. Caron M.G. Lefkowitz R.J. Lohse M.J. J. Biol. Chem. 1993; 268: 3201-3208Abstract Full Text PDF PubMed Google Scholar). β-Arrestins interact with GRK-phosphorylated β2ARs to disrupt their association with G-proteins and terminate signaling (12Pippig S. Andexinger S. Daniel K. Puzicha M. Caron M.G. Lefkowitz R.J. Lohse M.J. J. Biol. Chem. 1993; 268: 3201-3208Abstract Full Text PDF PubMed Google Scholar, 13Lohse M.J. Benovic J.L. Codina J. Caron M.G. Lefkowitz R.J. Science. 1990; 248: 1547-1550Crossref PubMed Scopus (897) Google Scholar, 14Attramadal H. Arriza J.L. Aoki C. Dawson T.M. Codina J. Kwatra M.M. Snyder S.H. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1992; 267: 17882-17890Abstract Full Text PDF PubMed Google Scholar) and also to serve as adaptor proteins for clathrin-mediated endocytosis of β2ARs (15Ferguson S.S. Downey W.E. Colapietro A.M. Barak L.S. Menard L. Caron M.G. Science. 1996; 271: 363-366Crossref PubMed Scopus (839) Google Scholar, 16Goodman Jr., O.B. Krupnick J.G. Santini F. Gurevich V.V. Penn R.B. Gagnon A.W. Keen J.H. Benovic J.L. Nature. 1996; 383: 447-450Crossref PubMed Scopus (1153) Google Scholar). Receptor endocytosis and trafficking also contribute to resensitization and signal transduction. Resensitization requires endocytosis, dephosphorylation, and recycling of β2ARs to the plasma membrane (17Yu S.S. Lefkowitz R.J. Hausdorff W.P. J. Biol. Chem. 1993; 268: 337-341Abstract Full Text PDF PubMed Google Scholar, 18Barak L.S. Tiberi M. Freedman N.J. Kwatra M.M. Lefkowitz R.J. Caron M.G. J. Biol. Chem. 1994; 269: 2790-2795Abstract Full Text PDF PubMed Google Scholar, 19Krueger K.M. Daaka Y. Pitcher J.A. Lefkowitz R.J. J. Biol. Chem. 1997; 272: 5-8Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar, 20Zhang J. Barak L.S. Winkler K.E. Caron M.G. Ferguson S.S. J. Biol. Chem. 1997; 272: 27005-27014Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar), and endocytosis of β2ARs is required for stimulation of mitogen-activated protein kinases (21Daaka Y. Luttrell L.M. Ahn S. Della Rocca G.J. Ferguson S.S.G. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1998; 273: 685-688Abstract Full Text Full Text PDF PubMed Scopus (457) Google Scholar).Far less is known about the mechanisms that attenuate signaling by PARs. Thrombin stimulates phosphorylation of PAR1 (22Ishii K. Chen J. Ishii M. Koch W.J. Freedman N.J. Lefkowitz R.J. Coughlin S.R. J. Biol. Chem. 1994; 269: 1125-1130Abstract Full Text PDF PubMed Google Scholar), and overexpression of GRK3 inhibits thrombin signaling (22Ishii K. Chen J. Ishii M. Koch W.J. Freedman N.J. Lefkowitz R.J. Coughlin S.R. J. Biol. Chem. 1994; 269: 1125-1130Abstract Full Text PDF PubMed Google Scholar). Second messenger kinases may also participate in uncoupling because activation of protein kinase C by phorbol esters stimulates phosphorylation of PAR1 and PAR2 and inhibits signaling by PAR2 (22Ishii K. Chen J. Ishii M. Koch W.J. Freedman N.J. Lefkowitz R.J. Coughlin S.R. J. Biol. Chem. 1994; 269: 1125-1130Abstract Full Text PDF PubMed Google Scholar,23Böhm S.K. Khitin L.M. Grady E.F. Aponte G. Payan D.G. Bunnett N.W. J. Biol. Chem. 1996; 271: 22003-22016Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar). 2N. W. Bunnett, unpublished observations.2N. W. Bunnett, unpublished observations. Agonists induce endocytosis of PAR1 and PAR2 by mechanisms that have not been fully defined (23Böhm S.K. Khitin L.M. Grady E.F. Aponte G. Payan D.G. Bunnett N.W. J. Biol. Chem. 1996; 271: 22003-22016Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar, 24Hoxie J.A. Ahuja M. Belmonte E. Pizarro S. Parton R. Brass L.F. J. Biol. Chem. 1993; 268: 13756-13763Abstract Full Text PDF PubMed Google Scholar, 25Brass L.F. Pizarro S. Ahuja M. Belmonte E. Blanchard N. Stadel J.M. Hoxie J.A. J. Biol. Chem. 1994; 269: 2943-2952Abstract Full Text PDF PubMed Google Scholar, 26Hein L. Ishii K. Coughlin S.R. Kobilka B.K. J. Biol. Chem. 1994; 269: 27719-27726Abstract Full Text PDF PubMed Google Scholar, 27Molino M. Bainton D.F. Hoxie J.A. Coughlin S.R. Brass L.F. J. Biol. Chem. 1997; 272: 6011-6017Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Since internalized PAR1 and PAR2 are degraded in lysosomes, resensitization requires mobilization of intracellular stores or synthesis of PARs (23Böhm S.K. Khitin L.M. Grady E.F. Aponte G. Payan D.G. Bunnett N.W. J. Biol. Chem. 1996; 271: 22003-22016Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar, 24Hoxie J.A. Ahuja M. Belmonte E. Pizarro S. Parton R. Brass L.F. J. Biol. Chem. 1993; 268: 13756-13763Abstract Full Text PDF PubMed Google Scholar, 25Brass L.F. Pizarro S. Ahuja M. Belmonte E. Blanchard N. Stadel J.M. Hoxie J.A. J. Biol. Chem. 1994; 269: 2943-2952Abstract Full Text PDF PubMed Google Scholar, 26Hein L. Ishii K. Coughlin S.R. Kobilka B.K. J. Biol. Chem. 1994; 269: 27719-27726Abstract Full Text PDF PubMed Google Scholar).PAR2 is highly expressed in the gastrointestinal tract and pancreas (6Nystedt S. Emilsson K. Wahlestedt C. Sundelin J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9208-9212Crossref PubMed Scopus (826) Google Scholar,8Corvera C.U. Dery O. McConalogue K. Bohm S.K. Khitin L.M. Caughey G.H. Payan D.G. Bunnett N.W. J. Clin. Invest. 1997; 100: 1383-1393Crossref PubMed Scopus (266) Google Scholar, 28Böhm S.K. Kong W. Brömme D. Smeekens S.P. Anderson D.C. Connolly A. Kahn M. Nelken N.A. Coughlin S.R. Payan D.G. Bunnett N.W. Biochem. J. 1996; 314: 1009-1016Crossref PubMed Scopus (410) Google Scholar, 29Kong W. McConalogue K. Khitin L.M. Hollenberg M.D. Payan D.G. Bohm S.K. Bunnett N.W. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8884-8889Crossref PubMed Scopus (274) Google Scholar). Pancreatic trypsin activates PAR2 in enterocytes and in epithelial cells of the pancreatic duct to regulate prostanoid secretion and the activity of ion channels (29Kong W. McConalogue K. Khitin L.M. Hollenberg M.D. Payan D.G. Bohm S.K. Bunnett N.W. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8884-8889Crossref PubMed Scopus (274) Google Scholar, 30Nguyen T.D. Moody M.W. Steinhoff M. Okolo C. Koh D.-S. Bunnett N.W. J. Clin. Invest. 1999; 103: 261-269Crossref PubMed Scopus (165) Google Scholar). Tryptase, a major protease of human mast cells (31Caughey G.H. Caughey G.H. Mast Cell Proteases in Immunology and Biology. Marcel Dekker, Inc., New York1995: 305-329Google Scholar), activates PAR2 in myocytes and enteric neurons (8Corvera C.U. Dery O. McConalogue K. Bohm S.K. Khitin L.M. Caughey G.H. Payan D.G. Bunnett N.W. J. Clin. Invest. 1997; 100: 1383-1393Crossref PubMed Scopus (266) Google Scholar, 32Corvera C. Dery O. McConalogue K. Gamp P. Al-Ani B. Caughey G. Hollenberg M. Bunnett N. J. Physiol.(Lond.). 1999; (in press)Google Scholar), and PAR2 may mediate some of the mitogenic and pro-inflammatory effects of tryptase (33Mirza H. Schmidt V.A. Derian C.K. Jesty J. Bahou W.F. Blood. 1997; 90: 3914-3922Crossref PubMed Google Scholar). In view of the potential importance of PAR2 in normal regulation, inflammation, and mitogenesis, it is important to understand the mechanisms that terminate PAR2 signaling. Although we have shown that trypsin induces endocytosis and lysosomal degradation of PAR2 (23Böhm S.K. Khitin L.M. Grady E.F. Aponte G. Payan D.G. Bunnett N.W. J. Biol. Chem. 1996; 271: 22003-22016Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar), agonist-induced trafficking of PAR2 and β-arrestin1 has not been examined in real time, and the role of β-arrestins in PAR2 endocytosis is unknown.The purpose of the present study was to investigate agonist-induced trafficking of β-arrestin1 and PAR2 and to determine the role of β-arrestins in trafficking of PAR2. To examine trafficking of PAR2 and β-arrestin1 in real time, we expressed chimeras of these proteins and green fluorescent protein (GFP). To determine the role of β-arrestins in PAR2 trafficking, we expressed a C-terminal fragment, β-arrestin319–418, that serves as a dominant negative mutant for clathrin-mediated endocytosis (34Krupnick J.G. Santini F. Gagnon A.W. Keen J.H. Benovic J.L. J. Biol. Chem. 1997; 272: 32507-32512Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). Our aims were as follows: (a) generate cell lines expressing PAR2-GFP or wild type PAR2 plus β-arrestin1-GFP (ARR-GFP) or dominant negative β-arrestin-GFP (ARR319–418-GFP); (b) verify that GFP did not affect PAR2 signaling, desensitization, or trafficking or the capacity of β-arrestin to participate in these processes; (c) examine agonist-induced redistribution of PAR2 and β-arrestin1 in real time; (d) determine the fate of endocytosed PAR2 and β-arrestin1; and (e) investigate the role of β-arrestins in PAR2 endocytosis.RESULTSGeneration of Cell Lines Expressing PAR2-GFP and PAR2 Plus ARR-GFP or ARR319–418-GFPKNRK cells were used for transfection since we have extensively studied signaling and trafficking of PAR2 and other GPCRs in this cell line (23Böhm S.K. Khitin L.M. Grady E.F. Aponte G. Payan D.G. Bunnett N.W. J. Biol. Chem. 1996; 271: 22003-22016Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar, 37Grady E.F. Garland A.G. Gamp P.D. Lovett M. Payan D.G. Bunnett N.W. Mol. Biol. Cell. 1995; 6: 509-524Crossref PubMed Scopus (201) Google Scholar). The mechanism of PAR2 desensitization in KNRK cells resembles that observed in enterocytes (23Böhm S.K. Khitin L.M. Grady E.F. Aponte G. Payan D.G. Bunnett N.W. J. Biol. Chem. 1996; 271: 22003-22016Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar). Thus, KNRK cells regulate PAR2 similarly to cells that naturally express this receptor.We transfected KNRK cells with cDNA encoding PAR2-GFP in a neomycin-resistant vector or with PAR2 in a hygromycin-resistant vector plus ARR-GFP or ARR319–418-GFP in neomycin-resistant vectors. We screened cells by flow cytometry and fluorescence microscopy to obtain clonal cell lines. KNRK-PAR2-GFP clone 3C was used in all experiments since analysis by flow cytometry revealed a single population of cells that expressed PAR2-GFP at a high level (Fig.2 A). Fluorescence microscopy confirmed the high, uniform expression of PAR2-GFP, which was detected at the plasma membrane and in a prominent perinuclear pool (Fig.2 B). KNRK-PAR2 + ARR-GFP clone 16 was selected since analysis by flow cytometry identified a single population of cells that expressed PAR2 and β-arrestin1 at a high and uniform level (Fig.2 A). ARR-GFP was cytosolic with no detectable localization at the plasma membrane (Fig. 2 B). Fluorescence microscopy confirmed the co-expression of PAR2 and ARR-GFP (e.g. Fig.6). Thus, there is no co-localization of PAR2 and β-arrestin1 in the unstimulated state. KNRK-PAR2 + ARR319–418-GFP clone 17 was selected since flow cytometry indicated that a large proportion of these cells co-expressed PAR2 and ARR319–418-GFP (Fig.2 A). ARR319–418-GFP had a punctate distribution throughout the cell which resembles the distribution of clathrin in KNRK cells (37Grady E.F. Garland A.G. Gamp P.D. Lovett M. Payan D.G. Bunnett N.W. Mol. Biol. Cell. 1995; 6: 509-524Crossref PubMed Scopus (201) Google Scholar) (Fig. 2 B). Since ARR319–418-GFP constitutively interacts with clathrin (34Krupnick J.G. Santini F. Gagnon A.W. Keen J.H. Benovic J.L. J. Biol. Chem. 1997; 272: 32507-32512Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar), it is probable that this protein is co-localized with clathrin in KNRK-PAR2 + ARR319–418-GFP cells.Figure 6Localization of PAR2 and ARR-GFP in KNRK-PAR2 + ARR-GFP cells. Cells were exposed to carrier (0 min, control) or 10 nm trypsin for 2–60 min at 37 °C. PAR2 was localized with the HA.11 antibody and a Texas Red-conjugated secondary antibody (left panels). β-Arrestin1 was localized with GFP (center panels). Images in the right panels are formed by superimposition of images from the two other panels in the same row. In carrier-treated cells at 0 min, PAR2 was at the cell surface (arrow heads) and ARR-GFP was cytosolic (arrows), with no co-localization. After 2 min incubation with trypsin, PAR2 was still at the cell surface (white arrowheads) and ARR-GFP was also at the plasma membrane (yellow arrowheads) in close proximity to PAR2. After 5 min with trypsin, both PAR2 and ARR-GFP were co-localized at the plasma membrane (white arrowheads). After 10–60 min with trypsin, PAR2 and ARR-GFP were co-localized in vesicles in the superficial, then in the perinuclear location (white arrows). Scale bar = 10 μm.View Large Image Figure ViewerDownload (PPT)We analyzed cells by Western blotting with a GFP antibody to determine whether chimeric proteins of the predicted size were expressed. The antibody detected recombinant GFP with an apparent mass of ∼27 kDa (Fig. 2 C). A broad protein band of ∼60–90 kDa was detected in extracts of KNRK-PAR2-GFP cells. The predicted mass of human PAR2 is ∼44 kDa, but there is one consensus site forN-linked glycosylation. Therefore, this protein probably represents glycosylated PAR2-GFP. A prominent protein of ∼75 kDa was detected in extracts of KNRK-PAR2+ARR-GFP cells, which corresponds to the known mass of β-arrestin1 (∼48 kDa) plus GFP (∼27 kDa). A protein band of ∼40 kDa was detected in extracts of KNRK-PAR2+ARR319–418-GFP cells, which corresponds to the known mass of β-arrestin-319–418 (∼12 kDa) plus GFP (∼27 kDa). The level of expression of ARR-GFP was higher that of ARR319–418-GFP, as judged by the intensity of the immunoreactive proteins. Thus, the GFP antibody recognizes proteins of the predicted sizes in transfected cell lines. The GFP antibody is specific because it did not interact with proteins in extracts of untransfected KNRK cells, and preabsorption of the antibody with GFP-GST fusion protein markedly diminished the signal in transfected cells.Functional Characterization of Cells Expressing PAR2-GFP and PAR2 Plus ARR-GFP or ARR319–418-GFPAlthough GFP is a compact protein that has been attached to other GPCRs and β-arrestin2 without affecting signaling or trafficking (38Tarasova N.I. Stauber R.H. Choi J.K. Hudson E.A. Czerwinski G. Miller J.L. Pavlakis G.N. Michejda C.J. Wank S.A. J. Biol. Chem. 1997; 272: 14817-14824Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 39Barak L.S. Ferguson S.S. Zhang J. Martenson C. Meyer T. Caron M.G. Mol. Pharmacol. 1997; 51: 177-184Crossref PubMed Scopus (201) Google Scholar, 40Kallal L. Gagnon A.W. Penn R.B. Benovic J.L. J. Biol. Chem. 1998; 273: 322-328Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 41Barak L. Ferguson S.S.G. Zhang J. Caron M.G. J. Biol. Chem. 1997; 272: 27497-27500Abstract Full Text Full Text PDF PubMed Scopus (392) Google Scholar), it is important to verify that GFP does not affect the function of each protein to which it is coupled. To determine if GFP affected PAR2 signaling, we measured trypsin- and AP-induced Ca2+ mobilization in KNRK-PAR2-GFP cells, KNRK-PAR2 + ARR-GFP cells, and KNRK-PAR2 + ARR319–418-GFP cells. In all cell lines, trypsin and AP strongly stimulated Ca2+mobilization, although the concentration-response curve for cells expressing PAR2-GFP was right-shifted for both trypsin and AP (Fig.3 A). We did not complete full concentration-response curves for all cell lines, and thus we cannot accurately determine the efficacy and EC50 values of the responses. However, approximate EC50 values were as follows: KNRK-PAR2-GFP cells, trypsin 10 nm, AP 20 μm; KNRK-PAR2 + ARR-GFP cells, trypsin 2 nm, AP 2 μm; KNRK-PAR2-ARR319–418-GFP cells, trypsin 2 nm, AP 7 μm (mean,n = 3 experiments). In KNRK cells expressing wild type human PAR-2, we have previously reported that trypsin and AP stimulate Ca2+ mobilization with potencies of 2 nm and 18 μm, respectively (28Böhm S.K. Kong W. Brömme D. Smeekens S.P. Anderson D.C. Connolly A. Kahn M. Nelken N.A. Coughlin S.R. Payan D.G. Bunnett N.W. Biochem. J. 1996; 314: 1009-1016Crossref PubMed Scopus (410) Google Scholar). Thus, trypsin and AP are slightly less potent agonists in cells expressing PAR2-GFP. In all cell lines, 10 nm trypsin induced a prompt increase in [Ca2+]i which was maximal within a few seconds and which declined to 50% of the maximal levels within 19 ± 2 s for KNRK-PAR2-GFP cells, 21 ± 1 s for KNRK-PAR2 + ARR-GFP cells, and 99 ± 13 s for KNRK-PAR2 + ARR319–418-GFP cells (mean ± S.E., n= 3 experiments) (Fig. 3, B–D). Therefore, the increase in [Ca2+]i is more prolonged in cells expressing ARR319–418-GFP.Figure 3Trypsin and AP induced Ca2+mobilization in KNRK-PAR2-GFP cells, KNRK-PAR2 + ARR-GFP cells, and KNRK-PAR2 + ARR 319–418 -GFP cells. A, cells were exposed to graded concentrations of trypsin or AP (SLIGKV-NH2), and the peak response was subtracted from the basal value to determine the change in the fluorescence ratio (n = 3–6 observations). B and C,cells were exposed to carrier (control) or 10 nm trypsin for 2 min, washed, and then exposed to 10 nm trypsin 5 min after the first challenge. Representative results from 3 observations are shown.View Large Image Figure ViewerDownload (PPT)To examine desensitization, we exposed cells to 10 nmtrypsin, 100 μm AP, or carrier (control) for 2 min, washed cells, and challenged them again with 10 nm trypsin or 100 μm AP 5 min after the first challenge. The magnitude of responses to the second challenge was compared with the response of carrier-treated cells to determine the extent of desensitization (Fig. 3, B–D). In KNRK-PAR2-GFP cells, exposure to trypsin caused 88 ± 5% desensitization to a second trypsin exposure, and exposure to AP caused 45 ± 4% desensitization to a second AP exposure (mean ± S.E.,n = 3 experiments). In KNRK-PAR2 + ARR-GFP cells, desensitization to trypsin was 52 ± 10%, and desensitization to AP was 21 ± 18%. In KNRK-PAR2-ARR319–418GFP cells, desensitization to trypsin was 91 ± 2%, and desensitization to AP was 48 ± 5%. Thus, in all cell lines, trypsin induces stronger desensitization than AP. We have previously reported that trypsin more strongly desensitizes Ca2+ mobilization than does AP in KNRK cells expressing wild type PAR2 (23Böhm S.K. Khitin L.M. Grady E.F. Aponte G. Payan D.G. Bunnett N.W. J. Biol. Chem. 1996; 271: 22003-22016Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar). Desensitization to trypsin and AP was less in cells expressing ARR-GFP.Together, these results indicate that GFP has only minor effects on PAR2-mediated signaling, on desensitization of signal transduction, or on the ability of β-arrestin1 to participate in desensitization.Trypsin-induced Trafficking of PAR2-GFP and ARR-GFP in Real TimePAR2-GFPWe have previously examined trypsin- and AP-induced trafficking of PAR2 in populations of cells fixed at various times after stimulation (23Böhm S.K. Khitin L.M. Grady E.F. Aponte G. Payan D.G. Bunnett N.W. J. Biol. Chem. 1996; 271: 22003-22016Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar), but agonist-induced trafficking of PAR2 has not been examined in real time. We examined the effects of trypsin on the subcellular distribution of PAR2-GFP in individual KNRK-PAR2-GFP cells in real time. Before addition of trypsin, PAR2-GFP was detected at the plasma membrane and in a perinuclear compartment (Fig.4). After 2–5 min incubation with 10 nm trypsin at 37 °C, PAR2-GFP was observed at the plasma membrane and in a few superficial vesicles. After 10 min, PAR2-GFP was prominently detected in vesicles in a peripheral and a perinuclear location, and there was diminished localization at the plasma membrane. After 30–60 min, PAR2-GFP was present in vesicles in a perinuclear location.Figure 4Real time trypsin-induced trafficking of PAR2-GFP in the same KNRK-PAR2-GFP cells. Cells were observed before and after addition of trypsin (10 nm). Note the redistribution of PAR2-GFP from the plasma membrane (0 min,arrowheads) to vesicles located immediately beneath the plasma membrane (2–10 min, arrows), and finally to a cluster of vesicles in a perinuclear location (30, 60 min,arrows). Scale bar = 10 μm.View Large Image Figure ViewerDownload (PPT)ARR-GFPIn a similar manner we examined the effects of trypsin on the subcellular distribution of ARR-GFP in individual KNRK-PAR2 + ARR-GFP cells in real time. Before addition of trypsin, ARR-GFP was uniformly distributed in the cytosol and was not detected at the plasma membrane or in vesicles (Fig.5). After 2–5 min incubation with 10 nm trypsin at 37 °C, ARR-GFP was prominently detected at the plasma membrane and in a few superficial vesicles. After 10 min, ARR-GFP was present in vesicles in a peripheral and a perinuclear location and was no longer present at the plasma membrane. After 30–60 min, ARR-GFP was detected in vesicles in a perinuclear location, and the intensity of the signal in the cytoplasm was diminished.Figure 5Real time trypsin-induced trafficking of ARR-GFP in the same KNRK-PAR2 + ARR-GFP cells. Cells were observed before and after addition of trypsin (10 nm). Note the redistribution of ARR-GFP from the cytosol (0 min, arrows), to the plasma membrane (2 and 5 min, arrowheads), to vesicles located immediately beneath the plasma membrane (10 min,arrows), and finally to a cluster of vesicles in a perinuclear location (30 and 60 min, arrows). Scale bar = 10 μm.View Large Image Figure ViewerDownload (PPT)Thus, trypsin induces rapid redistribution of PAR2-GFP from the plasma membrane and into vesicles in a superficial and then a perinuclear location and stimulates translocation of ARR-GFP from the cytosol to the plasma membrane, followed by redistribution to endosomes. At 2–5 min, PAR2-GFP was detected in endosomes and at the plasma membrane, and ARR-GFP was usually present at the plasma membrane. Both proteins were clearly detected in endosomes at 10 min. If β-arrestin1 mediates endocytosis of PAR2, we would expect membrane translocation of β-arrestin1 to precede PAR2 endocytosis and that β-arrestin1 would co-localize with PAR2 in vesicles. However, this possibility could not be investigated by observation of different cell populations in real time, when only a few cells were examined in any one experiment.Simultaneous Localization of PAR2 and ARR-GFPTo examine the effect of trypsin on the subcellular distribution of PAR2 and β-arrestin1 in the same cells, we simultaneously localized these proteins in KNRK-PAR2 + ARR-GFP cells by using an antibody to the C-terminal 12CA5 epitope and a Texas Red-labeled secondary antibody to detect PA" @default.
W1971800733 created "2016-06-24" @default.
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W1971800733 date "1999-06-01" @default.
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W1971800733 title "Trafficking of Proteinase-activated Receptor-2 and β-Arrestin-1 Tagged with Green Fluorescent Protein" @default.
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W1971800733 doi "https://doi.org/10.1074/jbc.274.26.18524" @default.
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