title: Internalisation of the protease-activated receptor 1: role of the third intracellular loop and of the cytoplasmic tail.
abstract: To analyse the mechanisms of PAR-1 internalisation, we constructed several PAR-1 mutants and stably expressed them in CHO cells. Our study shows that the Ser(306)-->Ala mutation (S306A), which eliminates a potential site of phosphorylation by PKC in the third intracellular loop of PAR-1, did not change the rate of phosphorylation but reduced the rate of thrombin-induced internalisation of the PAR-1 mutant (58 versus 78% of membrane PAR-1 in 15 min, p<0.005). Deletion of the last 43 amino acid residues of the PAR-1 cytoplasmic tail completely suppressed the thrombin phosphorylation of the mutated receptor and significantly reduced its internalisation upon activation. This deletion also inhibited the PMA-induced and the agonist-independent internalisation of the receptor. The Tyr(371)--> Ala mutation (Y371A), in a NPXXY motif of the seventh transmembrane domain of the receptor had no effect on the receptor behaviour. Our results indicate that both the C-tail and the third intracellular loop are involved in PAR-1 internalisation induced by thrombin while only the C-tail plays a role in the PMA-induced and in the agonist-independent PAR-1 internalisation.
authors.affiliation: INSERM U489, Association Claude Bernard, Hôpital Tenon, 75970 Paris cedex 20, France.
authors.forename: X
authors.initials: X
authors.lastname: Chen
authors.forename: J
authors.initials: J
authors.lastname: Berrou
authors.forename: C
authors.initials: C
authors.lastname: Vigneau
authors.forename: F
authors.initials: F
authors.lastname: Delarue
authors.forename: E
authors.initials: E
authors.lastname: Rondeau
mesh_terms: D000409:Alanine / Q000737:chemistry; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D006224:Cricetinae; D003593:Cytoplasm / Q000378:metabolism; D017353:Gene Deletion; D009154:Mutation; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011493:Protein Kinase C / Q000378:metabolism; D017434:Protein Structure, Tertiary; D044463:Receptor, PAR-1; D018179:Receptors, Thrombin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013917:Thrombin / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D014443:Tyrosine / Q000737:chemistry
chemical_list: D044463:Receptor, PAR-1; D018179:Receptors, Thrombin; D014443:Tyrosine; D011493:Protein Kinase C; D013917:Thrombin; D000409:Alanine