Protease-activated receptor-1 down-regulation - A mutant HeLa cell line suggests novel requirements for PAR1 phosphorylation and recruitment to clathrin-coated pits

Protease-activated receptor-1 (PAR1), a G protein-coupled receptor (GPCR) f or thrombin, is irreversibly activated by a proteolytic mechanism, then int ernalized and degraded in lysosomes. The latter is critical for temporal fi delity 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 trans ferrin receptor (TfnR). Dominant-negative dynamin and clathrin hub mutants both blocked PAR1 internalization, Blockade of PARI internalization with dy namin K44A also inhibited activation-dependent PAR1 degradation. Thus activ ated PAR1 internalizes via clathrin-coated pits together with receptors tha t recycle and is then sorted away from such receptors and delivered to lyso somes. 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 cel ls and internalization of beta(2)-adrenergic receptor, a GPCR that internal izes and recycles, was present but perhaps reduced. Taken together, these s tudies suggest that PAR1 is internalized in a dynamin- and clathrin-depende nt manner like TfnR and beta(2)-adrenergic receptor but requires a distinct gene product for recruitment into this pathway.