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• W1973705262 abstract "Protease-activated receptor-1 (PAR1), a G protein-coupled receptor (GPCR) for thrombin, is irreversibly activated by a proteolytic mechanism, then internalized and degraded in lysosomes. The latter is critical for temporal fidelity of thrombin signaling. Toward understanding PAR1 down-regulation, we first investigated the pathway of PAR1 internalization. Activated PAR1 was rapidly recruited to clathrin-coated pits, where it colocalized with transferrin receptor (TfnR). Dominant-negative dynamin and clathrin hub mutants both blocked PAR1 internalization. Blockade of PAR1 internalization with dynamin K44A also inhibited activation-dependent PAR1 degradation. Thus activated PAR1 internalizes via clathrin-coated pits together with receptors that recycle and is then sorted away from such receptors and delivered to lysosomes. In the course of these studies we identified a mutant HeLa cell line, designated JT1, that was defective in PAR1 internalization. PAR1 signaled robustly in JT1 cells but was not phosphorylated or recruited to clathrin-coated pits after activation. Internalization of TfnR was intact in JT1 cells and internalization of β2-adrenergic receptor, a GPCR that internalizes and recycles, was present but perhaps reduced. Taken together, these studies suggest that PAR1 is internalized in a dynamin- and clathrin-dependent manner like TfnR and β2-adrenergic receptor but requires a distinct gene product for recruitment into this pathway. Protease-activated receptor-1 (PAR1), a G protein-coupled receptor (GPCR) for thrombin, is irreversibly activated by a proteolytic mechanism, then internalized and degraded in lysosomes. The latter is critical for temporal fidelity of thrombin signaling. Toward understanding PAR1 down-regulation, we first investigated the pathway of PAR1 internalization. Activated PAR1 was rapidly recruited to clathrin-coated pits, where it colocalized with transferrin receptor (TfnR). Dominant-negative dynamin and clathrin hub mutants both blocked PAR1 internalization. Blockade of PAR1 internalization with dynamin K44A also inhibited activation-dependent PAR1 degradation. Thus activated PAR1 internalizes via clathrin-coated pits together with receptors that recycle and is then sorted away from such receptors and delivered to lysosomes. In the course of these studies we identified a mutant HeLa cell line, designated JT1, that was defective in PAR1 internalization. PAR1 signaled robustly in JT1 cells but was not phosphorylated or recruited to clathrin-coated pits after activation. Internalization of TfnR was intact in JT1 cells and internalization of β2-adrenergic receptor, a GPCR that internalizes and recycles, was present but perhaps reduced. Taken together, these studies suggest that PAR1 is internalized in a dynamin- and clathrin-dependent manner like TfnR and β2-adrenergic receptor but requires a distinct gene product for recruitment into this pathway. protease-activated receptor(s) β2-adrenergic receptor dynamin G protein-coupled receptor transferrin receptor G protein-coupled receptor kinase hemagglutinin Dulbecco's modified Eagle's medium phosphate-buffered saline bovine serum albumin enzyme-linked immunosorbent assay polyacrylamide gel electrophoresis Thrombin, a coagulant protease generated at sites of vascular injury, elicits signaling responses in many cell types important in vascular biology and disease (1Coughlin S.R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11023-11027Crossref PubMed Scopus (526) Google Scholar). Most cellular actions of thrombin appear to be mediated by a family of protease-activated G protein-coupled receptors (PARs)1 (1Coughlin S.R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11023-11027Crossref PubMed Scopus (526) Google Scholar). PAR1, the prototype of this family, is activated by an unusual proteolytic mechanism. Thrombin binds to and cleaves the amino-terminal exodomain of PAR1 to unmask a new amino terminus that then acts as a tethered ligand, triggering transmembrane signaling (1Coughlin S.R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11023-11027Crossref PubMed Scopus (526) Google Scholar, 2Vu T.-K.H. Hung D.T. Wheaton V.I. Coughlin S.R. Cell. 1991; 64: 1057-1068Abstract Full Text PDF PubMed Scopus (2680) Google Scholar, 3Vu T.-K.H. Wheaton V.I. Hung D.T. Coughlin S.R. Nature. 1991; 353: 674-677Crossref PubMed Scopus (476) Google Scholar, 4Liu L. Vu T.-K.H. Esmon C.T. Coughlin S.R. J. Biol. Chem. 1991; 266: 16977-16980Abstract Full Text PDF PubMed Google Scholar, 5Mathews I.I. Padmanabhan K.P. Ganesh V. Tulinsky A. Ishii M. Chen J. Turck C.W. Coughlin S.R. Fenton 2nd, J.W. Biochemistry. 1994; 33: 3266-3279Crossref PubMed Scopus (166) Google Scholar, 6Chen J. Ishii M. Wang L. Ishii K. Coughlin S.R. J. Biol. Chem. 1994; 269: 16041-16045Abstract Full Text PDF PubMed Google Scholar). SFLLRN, a synthetic peptide that represents PAR1's newly formed amino terminus, can fully activate PAR1 (2Vu T.-K.H. Hung D.T. Wheaton V.I. Coughlin S.R. Cell. 1991; 64: 1057-1068Abstract Full Text PDF PubMed Scopus (2680) Google Scholar, 7Vassallo Jr., R.R. Kieber-Emmons T. Cichowski K. Brass L.F. J. Biol. Chem. 1992; 267: 6081-6085Abstract Full Text PDF PubMed Google Scholar, 8Scarborough R.M. Naughton M.A. Teng W. Hung D.T. Rose J. Vu T.K. Wheaton V.I. Turck C.W. Coughlin S.R. J. Biol. Chem. 1992; 267: 13146-13149Abstract Full Text PDF PubMed Google Scholar). The irreversibility of the mechanism by which PAR1 is activated stands in contrast to reversible ligation of most receptors. This raises the questions of how PAR1 signaling is terminated and how cells expressing PAR1 become resensitized to thrombin signaling (1Coughlin S.R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11023-11027Crossref PubMed Scopus (526) Google Scholar, 9Ishii K. Hein L. Kobilka B. Coughlin S.R. J. Biol. Chem. 1993; 268: 9780-9786Abstract Full Text PDF PubMed Google Scholar, 10Hoxie 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, 11Ishii 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, 12Trejo J. Hammes S.R. Coughlin S.R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13698-13702Crossref PubMed Scopus (120) Google Scholar). The β2-adrenergic receptor (β2-AR) has been a model system for dissecting the molecular mechanisms responsible for desensitization and resensitization of G protein-coupled receptor (GPCR) signaling (13Yu S.S. Lefkowitz R.J. Hausdorff W.P. J. Biol. Chem. 1993; 268: 337-341Abstract Full Text PDF PubMed Google Scholar, 14Krueger K.M. Daaka Y. Pitcher J.A. Lefkowitz R.J. J. Biol. Chem. 1997; 272: 5-8Abstract Full Text Full Text PDF PubMed Scopus (317) Google Scholar, 15Pippig S. Andexinger S. Lohse M.J. Mol. Pharmacol. 1995; 47: 666-676PubMed Google Scholar). β2-AR is initially uncoupled from signaling by rapid phosphorylation of the activated receptor by G protein-coupled receptor kinases (GRKs) and other kinases. Receptor phosphorylation promotes the binding of arrestin. Arrestin binding prevents receptor interaction with G proteins and thereby uncouples the receptor from signaling. Arrestin binding also facilitates recruitment of β2-AR to clathrin-coated pits and internalization from the plasma membrane (16Goodman O.J. 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 (1179) Google Scholar, 17Laporte S. Oakley R.H. Zhang J. Holt J.A. Ferguson S.S.G. Caron M.G. Barak L.S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3712-3717Crossref PubMed Scopus (529) Google Scholar). Once internalized into endosomes, ligand dissociates from β2-AR, which is then dephosphorylated and recycled to the cell surface competent to signal again. Like β2-AR, activated PAR1 is rapidly phosphorylated and uncoupled from signaling (9Ishii K. Hein L. Kobilka B. Coughlin S.R. J. Biol. Chem. 1993; 268: 9780-9786Abstract Full Text PDF PubMed Google Scholar, 11Ishii 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). Phosphorylation of PAR1 facilitates receptor internalization from the plasma membrane (18Shapiro M.J. Trejo J. Zeng D. Coughlin S.R. J. Biol. Chem. 1996; 271: 32874-32880Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 19Shapiro M.J. Coughlin S.R. J. Biol. Chem. 1998; 273: 29009-29014Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). However, instead of recycling, activated PAR1 is efficiently sorted to lysosomes (10Hoxie 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, 20Hein L. Ishii K. Coughlin S.R. Kobilka B.K. J. Biol. Chem. 1994; 269: 27719-27726Abstract Full Text PDF PubMed Google Scholar). Exchanging the cytoplasmic carboxyl tails of PAR1 and the substance P receptor, a classic GPCR that internalizes and recycles like β2-AR, switched their trafficking behaviors (12Trejo J. Hammes S.R. Coughlin S.R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13698-13702Crossref PubMed Scopus (120) Google Scholar,21Trejo J. Coughlin S.R. J. Biol. Chem. 1999; 274: 2216-2224Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). Moreover, PAR1 bearing the substance P receptor carboxyl tail displayed exaggerated and prolonged signaling responses to thrombin compared with wild-type PAR1. This was due to recycling and “resignaling” by the chimeric receptors (12Trejo J. Hammes S.R. Coughlin S.R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13698-13702Crossref PubMed Scopus (120) Google Scholar, 21Trejo J. Coughlin S.R. J. Biol. Chem. 1999; 274: 2216-2224Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). Thus sorting of activated PAR1 to lysosomes rather than recycling is critical for temporal fidelity of PAR1 signaling. The molecular mechanisms by which PAR1 is internalized and sorted to lysosomes remain largely unknown. β2-AR and other GPCRs internalize from the plasma membrane via clathrin-coated pits (16Goodman O.J. 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 (1179) Google Scholar, 17Laporte S. Oakley R.H. Zhang J. Holt J.A. Ferguson S.S.G. Caron M.G. Barak L.S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3712-3717Crossref PubMed Scopus (529) Google Scholar,22Grady E.F. Garland A.M. Gamp P.D. Lovett M. Payan D.G. Bunnett N.W. Mol. Biol. Cell. 1995; 6: 509-524Crossref PubMed Scopus (203) Google Scholar, 23Lazari M. Bertrand J.E. Nakamura K. Liu X. Krupnick J.G. Benovi J.L. Ascoli M. J. Biol. Chem. 1998; 273: 18316-18324Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). The transferrin receptor (TfnR), which internalizes and recycles, utilizes a similar if not identical pathway that is dependent upon both clathrin and dynamin, a GTPase that regulates budding of coated pits (reviewed in Refs. 24Schmid S.L. Annu. Rev. Biochem. 1997; 66: 511-548Crossref PubMed Scopus (674) Google Scholar and 25Schmid S.L. McNiven M.A. De Camilli P. Curr. Opin. Cell Biol. 1998; 10: 504-512Crossref PubMed Scopus (356) Google Scholar). However, several GPCRs appear to internalize via a distinct non-dynamin dependent pathway (26Zhang J. Ferguson S.S.G. Barak L.S. Menard L. Caron M.G. J. Biol. Chem. 1996; 271: 18302-18305Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar, 27Pals-Rylaarsdam R. Gurevich V.V. Lee K.B. Ptasienski J.A. Benovic J.L. Hosey M.M. J. Biol. Chem. 1997; 272: 23682-23689Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 28Vickery R.G. von Zastrow M. J. Cell Biol. 1999; 144: 31-43Crossref PubMed Scopus (198) Google Scholar). This raises the following questions. Is activated PAR1 first internalized via clathrin-coated pits like β2-AR and TfnR and only later sorted away from recycling receptors and delivered to lysosomes? Or does activated PAR1 internalize via a pathway distinct from that used for recycling receptors from the outset? In this study, we report that activated PAR1 colocalizes with TfnR in coated pits and is internalized via a pathway that is both dynamin- and clathrin-dependent. Blockade of PAR1 internalization with dominant-negative dynamin also inhibited PAR1 degradation. These results strongly suggest that activated PAR1 initially internalizes via the same clathrin-coated pits used by TfnR and is then sorted away from recycling receptors and delivered to lysosomes. Characterization of a mutant HeLa cell line revealed that at least one distinct gene product is required for internalization of activated PAR1 versusTfnR or β2-AR, implying that at least partially distinct mechanisms are responsible for recruiting PAR1 versusβ2-AR to clathrin-coated pits. Peptide agonist SFLLRN was synthesized as the carboxyl amide and purified by reverse phase high pressure liquid chromatography. Isoproterenol and doxycycline were from Calbiochem (La Jolla, CA). Biotinylated human holo-transferrin, biocytin, avidin, poly-l-lysine, and fibronectin were from Sigma. Monoclonal anti-FLAG M1 antibody was from Sigma. Rabbit polyclonal 1809 antibody was generated against a peptide representing the hirudin-like sequence in PAR1's amino terminus (29Hung D.T. Vu T.K. Wheaton V.I. Ishii K. Coughlin S.R. J. Clin. Invest. 1992; 89: 1350-1353Crossref PubMed Scopus (168) Google Scholar). Anti-hemagglutinin (HA) 12CA5 monoclonal antibody and human TfnR monoclonal antibody were from Roche Molecular Biochemicals. Anti-human transferrin serum was from BioPacific (Emeryville, CA). Anti-clathrin rabbit polyclonal antibody recognizing the consensus sequence in clathrin light chains was a gift from F. Brodsky, University of California, San Francisco, CA (30Acton S.L. Brodsky F.M. J. Cell Biol. 1990; 111: 1419-1426Crossref PubMed Scopus (68) Google Scholar). Anti-T7 epitope tag monoclonal antibody was from Novagen (Madison, WI). Horseradish peroxidase-conjugated streptavidin and unconjugated goat anti-mouse IgG were from Pierce (Rockford, IL). Horseradish peroxidase-conjugated goat anti-mouse secondary antibody was from Bio-Rad. The following fluorophore-conjugated secondary antibodies used in this study were from Molecular Probes (Eugene, OR): AlexaTM 488- and AlexaTM 594-conjugated goat anti-mouse antibody; AlexaTM 488- and AlexaTM594-conjugated goat anti-rabbit antibody. HeLa cells stably expressing the tetracycline-regulatable chimeric transcription factor (tetR-VP16) were generously provided by S. Schmid, Scripps Institute, La Jolla, CA. Cells were cultured in DMEM supplemented with 10% fetal bovine serum, 4.5 mg/ml glucose, 100 units/ml penicillin, 100 μg/ml streptomycin, 100 μg/ml G418 (Life Technologies, Inc., Grand Island, NY). A cDNA encoding clathrin hub fragment that contained an amino-terminal T7 epitope (MASMTGGQQMG) was also provided by F. Brodsky, University of California, San Francisco (31Liu S.H. Marks M.S. Brodsky F.M. J. Cell Biol. 1998; 140: 1023-1037Crossref PubMed Scopus (116) Google Scholar). A PAR1 cDNA containing prolactin signal sequence followed by a FLAG epitope sequence (DYKDDDD) was co-transfected with a plasmid encoding a hygromycin resistance gene; stable transfectants were selected in 250 μg/ml hygromycin and screened by surface antibody binding (32Hung D.T. Vu T.-K.H. Nelken N.A. Coughlin S.R. J. Cell Biol. 1992; 116: 827-832Crossref PubMed Scopus (127) Google Scholar). Stable transfectants expressing human β2-AR containing an amino-terminal HA epitope sequence (YPYDVPDYA) were generated similarly. A mutant HeLa cell line designated JT1 expressing PAR1 was transfected with HA-tagged β2-AR together with a plasmid encoding a puromycin resistance gene and stable transfectants were selected in 2.5 μg/ml puromycin and screened by surface antibody binding as described above. Recombinant adenoviruses encoding wild-type and mutant K44A dynamin isoforms were generated and used to infect HeLa cells as described previously (33Altschuler Y. Barbas S.M. Terlecky L.J. Tang K. Hardy S. Mostov K.E. Schmid S.L. J. Cell Biol. 1998; 143: 1871-1881Crossref PubMed Scopus (187) Google Scholar). Briefly, ∼15 plaque-forming units/cell of recombinant adenovirus was incubated with cells for 2 h at 37 °C in HEPES buffer. Cells were washed and incubated for an additional 18 h at 37 °C in DMEM containing 10% fetal bovine serum and 0.05 ng/ml doxycycline. At this concentration of doxycycline, sufficient dominant-negative dynamin was expressed to inhibit TfnR internalization by 80% but cytotoxic effects were minimal. Approximately equal amounts of wild-type and mutant dynamins were expressed as determined by immunoblot using 12CA5 antibody (both wild-type and mutant dynamins contained an amino-terminal HA epitope (34Damke H. Baba T. Warnock D.E. Schmid S.L. J. Cell Biol. 1994; 127: 915-934Crossref PubMed Scopus (1046) Google Scholar)). HeLa cells grown on fibronectin-coated glass coverslips (22 × 22 mm) were transiently transfected with 2 μg of DNA, 2 μl of LIPOFECTIN Reagent, and 25 μl of PLUS Reagent for 3 h at 37 °C according to the manufacturer's instructions (Life Technologies, Inc.). Following transfections, DMEM containing 10% fetal bovine serum was added and cells were incubated for an additional 48 h at 37 °C. To follow internalization, cells expressing FLAG-tagged PAR1 were plated in 24-well dishes (Falcon, Lincoln Park, NJ) and incubated with 1 μg/ml anti-FLAG M1 antibody for 1 h at 4 °C. Cells were washed and exposed to agonist at 37 °C for various times. Next, surface-bound antibody was removed by three washes with PBS, Ca2+- and Mg2+-free containing 0.04% EDTA for 5 min at 4 °C. In cells expressing recombinant dynamins, surface-bound antibody was removed by three sequential washes (15 min at 4 °C) in PBS containing 0.6% BSA and 0.15 m glycine, pH 2.5. Cells were lysed in 150 μl of Triton lysis buffer (50 mm Tris-HCl, pH 7.4, 100 mm NaCl, 5 mm EDTA, 3% BSA, and 1% Triton X-100) and intracellular antibody was measured by ELISA (18Shapiro M.J. Trejo J. Zeng D. Coughlin S.R. J. Biol. Chem. 1996; 271: 32874-32880Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). TfnR endocytosis was measured using a modification of a previously described procedure (34Damke H. Baba T. Warnock D.E. Schmid S.L. J. Cell Biol. 1994; 127: 915-934Crossref PubMed Scopus (1046) Google Scholar). Cells plated in 24-well dishes (Falcon) were washed and incubated in DMEM containing 1 mg/ml BSA and 10 mm HEPES, pH 7.4, for 1.5 h at 37 °C to deplete cells of transferrin. Cells were incubated with 2 μg/ml human biotinylated transferrin for 1 h at 4 °C. Unbound biotinylated transferrin was removed and medium was exchanged with warmed DMEM/BSA/HEPES, pH 7.4. Cells were incubated for various times at 37 °C. Following incubations, cells were washed and surface-bound biotinylated transferrin was masked by incubation with 100 μg/ml avidin for 1 h at 4 °C followed by 100 μg/ml biocytin for 1 h at 4 °C. Cells were washed and lysed with 200 μl of 10 mm Tris-HCl, pH 7.4, 50 mm NaCl, 1 mm EDTA, 0.1% SDS, 0.2% BSA, and 1% Triton X-100 (blocking buffer). Internalized biotinylated transferrin was measured by ELISA. Briefly, 96-well Primaria plates (Falcon) were coated with 0.5 μg of anti-human transferrin antibody overnight at 4 °C. Each well was washed, incubated for 1 h at 37 °C with blocking buffer. Lysates were added and plates incubated overnight at 4 °C. Plates were washed, incubated with blocking buffer for 5 min, and washed again. Each well was incubated with 0.5 μg/ml horseradish peroxidase-conjugated streptavidin for 1 h at room temperature, washed as described above, and incubated with 150 μl of horseradish peroxidase substrate one-step 2,2′-azino-bis-3-ethylbenz-thiazoline-6-sulfonic acid (Pierce). The OD of each well was read at 405 nm using a Molecular Devices Microplate Reader (Sunnyvale, CA). The amount of internalized biotinylated transferrin measured in cell lysates was within the linear range of the assay as assessed by direct application of biotinylated transferrin. β2-AR internalization was examined using a modification of a previously described cell surface ELISA (21Trejo J. Coughlin S.R. J. Biol. Chem. 1999; 274: 2216-2224Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). Briefly, cells stably transfected with β2-AR containing an amino-terminal HA epitope sequence were plated in 24-well dishes and incubated with 1 μg/ml 12CA5 antibody for 1 h at 4 °C. Cells were washed to remove unbound antibody, incubated for various times at 37 °C, and the amount of internalized β2-AR was determined as described. Cell lysates were prepared, processed, and immunoblotted as described previously (21Trejo J. Coughlin S.R. J. Biol. Chem. 1999; 274: 2216-2224Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). The amount of PAR1 protein was measured as above except that immunoblots were developed using Enhanced Chemiluminescence (ECL) PlusTM (Amersham Pharmacia Biotech, Arlington, IL) and quantitated using a Molecular Dynamics Storm imaging system (Sunnyvale, CA). Phosphorylation of PAR1 was determined essentially as described (21Trejo J. Coughlin S.R. J. Biol. Chem. 1999; 274: 2216-2224Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). Cells stably expressing PAR1 were grown on fibronectin-coated glass coverslips (22 × 22 mm) and incubated with anti-PAR1 1809 antiserum (1:500) for 1 h at 4 °C, washed, and exposed to agonist at 37 °C. Cells were fixed with 4% paraformaldehyde for 5 min at 4 °C, washed, and permeabilized with 100% methanol (−20 °C) for 30 s. After permeabilization, cells were washed three times with PBS (1% nonfat dry milk, 150 mm sodium acetate, pH 7) and washed again three times with PBS (1% nonfat dry milk) (blocking buffer). Cells were then incubated with either 5 μg/ml anti-HA 12CA5 antibody (dynamin expression) or 1 μg/ml anti-T7 antibody (clathrin hub expression) for 1 h at room temperature. Cells were then incubated with species-specific fluorophore-conjugated secondary antibodies for 1 h at room temperature. After secondary antibody incubations, cells were washed four times with PBS, once with Molecular ProbesSlowFade equilibration buffer, and SlowFadeanti-fade reagent was added to each coverslip before mounting. Images were collected using a Nikon Microphot-FXA fluorescence microscope (Melville, NY) fitted with a Plan Apo ×40 objective and the final composite was created using Adobe Photoshop 5.0. Cells were grown on glass coverslips (22 × 22 mm) coated with 0.1 mg/ml poly-l-lysine. Cells were equilibrated in DMEM containing 1 mg/ml BSA and 10 mmHEPES, pH 7.4, for 1 h at 37 °C and incubated with or without agonist. Cells were then incubated with 3 μg/ml anti-FLAG M1 antibody or anti-PAR1 1809 (1:500) antibody for 1 h at 4 °C to label surface PAR1. TfnR was labeled with 4 μg/ml anti-TfnR B3/25 antibody for 1 h at 4 °C. Unbound antibody was removed and plasma membrane patches were ripped from cells as described previously (34Damke H. Baba T. Warnock D.E. Schmid S.L. J. Cell Biol. 1994; 127: 915-934Crossref PubMed Scopus (1046) Google Scholar,35Sanan D.A. Anderson R.G. J. Histochem. Cytochem. 1991; 39: 1017-1024Crossref PubMed Scopus (87) Google Scholar). Isolated plasma membrane patches were fixed, permeabilized, and washed with blocking buffer as described above. For PAR1 and TfnR colocalization experiments, plasma membrane patches were incubated with species-specific fluorophore-conjugated secondary antibodies for 1 h at room temperature. For clathrin colocalization studies, plasma membrane patches were first incubated with anti-clathrin antibody (1:1000) for 1 h, washed, and incubated with species-specific fluorophore-conjugated secondary antibodies for an additional 1 h at room temperature. Cells were washed four times with PBS, once with equilibration buffer, and SlowFade anti-fade reagent was added to each coverslip before mounting. Confocal images were collected using a Bio-Rad MRC-1024 laser scanning confocal system (Cambridge, MA) configured and with a Nikon Eclipse TE300 inverted microscope and a Plan Apo ×100 oil objective. Fluorescent images, 0.5-μm X-Y sections, were collected sequentially at 512 × 512 resolution with ×2 optical zoom and processed using LaserSharp software. The final composite image was created using Adobe Photoshop 5.0. Colocalization of PAR1 with clathrin or TfnR was quantitated by counting PAR1 containing puncta specifically associated with clathrin or TfnR immunoreactive puncta indicated by the yellow color in the merged image. The data are expressed as the percent of clathrin or TfnR containing puncta that co-stained for PAR1. Cells were plated in 12-well dishes (Falcon) and labeled overnight with 2 μCi/mlmyo-[3H]inositol in DMEM containing 1 mg/ml BSA. Cells were washed and treated as described in the legend to Fig.9, and the accumulation of inositol phosphates was measured as described previously (36Nanevicz T. Wang L. Chen M. Ishii M. Coughlin S.R. J. Biol. Chem. 1996; 271: 702-706Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar).Figure 9Phosphorylation and signaling of PAR1 in wild-type and mutant HeLa cell lines. A, wild-type and mutant HeLa cells labeled with [32P]orthophosphate were incubated in the absence (Ctrl) or presence of 100 μm SFLLRN for 3 min at 37 °C. Receptor immunoprecipitates were prepared from lysates of equivalent numbers of cells and analyzed by SDS-PAGE and autoradiography. Note the failure of agonist to induce phosphorylation of PAR1 in the mutant cell line.B, PAR1 signaling in wild-type and mutant cell lines, measured as SFLLRN-triggered phosphoinositide hydrolysis. Cells were labeled with myo-[3H]inositol and incubated in the absence (Ctrl) or presence of 100 μmSFLLRN for 60 min at 37 °C in media containing 20 mmlithium chloride. Accumulated [3H]inositol phosphates were measured as described under “Materials and Methods.” The data (mean ± S.D.; n = 3) are representative of two independent experiments. Basal [3H]inositol formation was 914 cpm/well (untransfected), 515 cpm/well (wild-type), and 1024 cpm/well (mutant). The initial levels of PAR1 surface expression were 0.5 for wild-type, 0.6 for mutant, and 0.01 for untransfected cells (arbitrary units). Note both the higher basal and greater fold increased signaling produced by activated PAR1 in the mutant cell line.View Large Image Figure ViewerDownload Hi-res image Download (PPT) A recently developed HeLa cell-based system offered an opportunity for rapid, efficient and regulated expression of dominant-negative trafficking molecules using adenoviral vectors (33Altschuler Y. Barbas S.M. Terlecky L.J. Tang K. Hardy S. Mostov K.E. Schmid S.L. J. Cell Biol. 1998; 143: 1871-1881Crossref PubMed Scopus (187) Google Scholar). Accordingly, we first asked whether PAR1 undergoes activation-dependent internalization and degradation in HeLa cells like it does in fibroblasts, endothelial cells, and megakaryocyte-like cells. PAR1 internalization was assayed by measuring uptake of receptor-bound antibody. HeLa cells stably transfected with PAR1 bearing a FLAG epitope at its amino terminus were incubated with the calcium-dependent M1 FLAG antibody for 60 min at 4 °C; under these conditions only receptors residing at the cell surface were labeled with antibody. Unbound antibody was removed and cells were incubated in the presence or absence of agonist at 37 °C for various times to allow internalization of receptor-bound antibody. After these incubations, bound antibody remaining on the cell surface was removed by washing with PBS/EDTA, cells were lysed, and internalized antibody was quantitated by ELISA. In untransfected cells, virtually no antibody was bound or internalized. In PAR1-expressing cells not exposed to agonist, ∼10% of antibody initially bound to the cell surface was internalized at steady state (Fig. 1 A,Ctrl), consistent with some tonic cycling of PAR1 between the cell surface and an intracellular compartment (18Shapiro M.J. Trejo J. Zeng D. Coughlin S.R. J. Biol. Chem. 1996; 271: 32874-32880Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 20Hein L. Ishii K. Coughlin S.R. Kobilka B.K. J. Biol. Chem. 1994; 269: 27719-27726Abstract Full Text PDF PubMed Google Scholar). Exposure to PAR1 agonist peptide SFLLRN caused a rapid increase in internalized receptor-bound antibody (Fig. 1 A, SFLLRN). Studies examining agonist-induced decreases in receptor-bound antibody on the cell surface yielded similar results (data not shown). These findings strongly suggest that PAR1 undergoes agonist-triggered internalization in HeLa cells. Agonist-triggered internalization of PAR1 is phosphorylation-dependent in several fibroblast-like cell lines. HeLa cells stably expressing PAR1 labeled with [32P]orthophosphate were incubated in the presence or absence of agonist SFLLRN for 3 min at 37 °C, lysed, and then immunoprecipitated with anti-PAR1 1809 antibody (29Hung D.T. Vu T.K. Wheaton V.I. Ishii K. Coughlin S.R. J. Clin. Invest. 1992; 89: 1350-1353Crossref PubMed Scopus (168) Google Scholar). Analysis of the immunoprecipitates by SDS-PAGE and autoradiography revealed agonist-triggered PAR1 phosphorylation in transfected HeLa cells (Fig.1 B). To determine whether PAR1 underwent activation-dependent degradation in HeLa cells, PAR1-expressing cells were incubated in the presence or absence of agonist for 90 min at 37 °C. The amount of PAR1 protein remaining was then measured by immunoblot of whole cell lysates (Figs. 1 C and 2). One major transfection-" @default.
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