Protease-activated receptor-1 (PAR1) contains five the surface of ECL2 serves distinct functions critical for proper regulation of receptor trafficking and the fidelity of thrombin signaling. the carboxyl amide and purified by reverse phase-high pressure liquid chromatography at Tufts University Core Facility (Boston). PNGase F sialidase and endoglycosidase H were purchased from New England Biolabs Inc. (Ipswich MA). Tunicamycin cycloheximide carbachol epidermal growth factor (EGF) and hirudin were obtained from Sigma. Rabbit polyclonal anti-FLAG antibody and mouse monoclonal M1 and M2 anti-FLAG antibodies were purchased from Sigma. The anti-PAR1 WEDE mouse antibody was purchased from Beckman Coulter (Fullerton CA). Anti-PAR1 rabbit polyclonal antibody was described previously (21). The mouse monoclonal anti-EGF receptor LA22 antibody was from Upstate Cell Signaling Solutions/Millipore (Temecula CA). Mouse anti-early endosome antigen-1 (EEA-1) and ZD6474 (NCBI reference sequence “type”:”entrez-nucleotide” attrs :”text”:”NM_012950.2″ term_id :”82617582″ term_text :”NM_012950.2″NM_012950.2); (NCBI ZD6474 reference sequence “type”:”entrez-nucleotide” attrs :”text”:”NM_010169.3″ term_id :”133892391″ term_text :”NM_010169.3″NM_010169.3); (GenBankTM accession number “type”:”entrez-nucleotide” attrs :”text”:”X61958.1″ term_id :”940495″ term_text :”X61958.1″X61958.1); (NCBI reference sequence “type”:”entrez-nucleotide” attrs :”text”:”NM_001992.3″ term_id :”166362739″ term_text :”NM_001992.3″NM_001992.3); (GenBankTM accession number “type”:”entrez-nucleotide” attrs :”text”:”AF028727.1″ term_id :”2605876″ term_text :”AF028727.1″AF028727.1); (NCBI reference sequence “type”:”entrez-nucleotide” attrs :”text”:”XM_001503957.2″ term_id :”194220118″ term_text :”XM_001503957.2″XM_001503957.2); (NCBI reference sequence “type”:”entrez-nucleotide” attrs :”text”:”XM_546059.2″ term_id :”73952381″ term_text :”XM_546059.2″XM_546059.2) and (NCBI reference sequence “type”:”entrez-nucleotide” attrs :”text”:”NM_001085783.1″ term_id :”160420280″ term_text :”NM_001085783.1″NM_001085783.1). PAR1 Immunoprecipitation and Immunoblotting HeLa cells stably expressing FLAG-tagged PAR1 wild type or mutants were plated at 5 × 105 Mertk cells per well in 6-well culture dishes and grown overnight at 37 °C. Cells were washed and incubated with ZD6474 or without agonists diluted in DMEM made up of 1 mg/ml BSA and 10 mm HEPES pH 7.4 for various times at 37 °C. Cells were then washed with cold phosphate-buffered saline and lysed with Triton X-100 lysis buffer (50 mm Tris-HCl pH 7.4 100 mm NaCl 5 mm EDTA 50 mm NaF 10 mm NaPP 1 Triton X-100) made up of freshly added protease inhibitors. Cell lysates were solubilized for 1.5 h at 4 °C and clarified by centrifugation at 14 0 rpm for 20 min at 4 °C. The total amount of protein ZD6474 in cell lysates was quantified using a bicinchoninic acid protein assay reagent (Thermo Scientific) and equivalent amounts of lysates were used for immunoprecipitation with the M2 anti-FLAG antibody or goat anti-mouse IgG control. Immunoprecipitates were analyzed by SDS-PAGE and transferred to membranes and PAR1 was detected by immunoblotting with anti-PAR1 antibody. Immunoblots were developed with enhanced chemiluminescence (GE Healthcare) imaged by autoradiography and quantitated with ImageJ software. To assess PAR1 degradation cells were pretreated with 10 μm cycloheximide for 30 min at 37 °C and then incubated with or without agonists in serum-free media made up of 10 μm cycloheximide for various times at 37 °C. Cells were washed solubilized with Triton X-100 lysis buffer and processed and the total amount of PAR1 remaining was determined by immunoblotting as described above. Cell Surface ELISA HeLa cells stably expressing ZD6474 FLAG-tagged PAR1 wild type or mutants were plated at 1 × 105 cells per well in a fibronectin coated 24-well culture dish and grown overnight at 37 °C. Cells were washed in serum-free DMEM and then treated with agonists 100 μm SFLLRN or 10 nm thrombin diluted in DMEM/BSA/HEPES at 37 °C for various times. After treatments cells were placed on ice washed with phosphate-buffered saline and then fixed with 4% paraformaldehyde for 5 min at 4 °C. Cells were washed and then incubated with polyclonal anti-FLAG antibody diluted 1:1000 or rabbit polyclonal anti-PAR1 C5433 diluted 1:250 in DMEM/BSA/HEPES for 1 h at room temperature. Cells were washed and then incubated with secondary horseradish peroxidase-conjugated goat anti-rabbit antibody for 1 h at room temperature and washed extensively. The amount of secondary antibody bound was determined by incubation with one-step 2 2 acid (Thermo.