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) 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 beta(2)-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 beta(2)-adrenergic receptor but requires a distinct gene product for recruitment into this pathway.
Trejo
J
J
Cardiovascular Research Institute, Departments of Medicine, and Cellular and Molecular Pharmacology, University of California, San Francisco, California 94143-0130, USA.
Altschuler
Y
Y
Fu
H W
HW
Mostov
K E
KE
Coughlin
S R
SR
D000256:Adenoviridae / Q000235:genetics; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002966:Clathrin / Q000819:agonists / Q000378:metabolism; D003034:Coated Pits, Cell-Membrane / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D015536:Down-Regulation; D034281:Dynamins; D004797:Enzyme-Linked Immunosorbent Assay; D020558:GTP Phosphohydrolases / Q000235:genetics / Q000378:metabolism; D005799:Genes, Dominant; D006367:HeLa Cells; D006801:Humans; D006868:Hydrolysis; D015151:Immunoblotting; D008247:Lysosomes / Q000378:metabolism; D008856:Microscopy, Fluorescence; D009154:Mutation; D010766:Phosphorylation; D011485:Protein Binding; D044463:Receptor, PAR-1; D018179:Receptors, Thrombin / Q000819:agonists / Q000235:genetics / Q000378:metabolism*; D011990:Receptors, Transferrin / Q000378:metabolism; D013997:Time Factors; D014162:Transfection
D002966:Clathrin; D018076:DNA, Complementary; D044463:Receptor, PAR-1; D018179:Receptors, Thrombin; D011990:Receptors, Transferrin; D020558:GTP Phosphohydrolases; D034281:Dynamins