Internalisation of the protease-activated receptor 1: Role of the third intracellular loop and of the cytoplasmic tail

To analyse the mechanisms of PAR-I 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 ch ange the rate of phosphorylation but reduced the rate of thrombin-induced i nternalisation of the PAR-1 mutant (58 versus 78% of membrane PAR-1 in 15 m in, p <0.005). Deletion of the last 43 amino acid residues of the PAR-1 cyt oplasmic tail completely suppressed the thrombin phosphorylation of the mut ated 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 eff ect on the receptor behaviour. Our results indicate that both the C-tail an d the third intracellular loop are involved in PAR-1 internalisation induce d by thrombin while only the C-tail plays a role in the PMA-induced and in the agonist-independent PAR-1 internalisation.