Clathrin adaptor AP2 regulates thrombin receptor constitutive internalization and endothelial cell resensitization.
Protease-activated receptor 1 (PAR1), a G protein-coupled receptor for the coagulant protease thrombin, is irreversibly activated by proteolysis. Unactivated PAR1 cycles constitutively between the plasma membrane and intracellular stores, thereby providing a protected receptor pool that replenishes the cell surface after thrombin exposure and leads to rapid resensitization to thrombin signaling independent of de novo receptor synthesis. Here, we show that AP2, a clathrin adaptor, binds directly to a tyrosine-based motif in the cytoplasmic tail of PAR1 and is essential for constitutive receptor internalization and cellular recovery of thrombin signaling. Expression of a PAR1 tyrosine mutant or depletion of AP2 by RNA interference leads to significant inhibition of PAR1 constitutive internalization, loss of intracellular uncleaved PAR1, and failure of endothelial cells and other cell types to regain thrombin responsiveness. Our findings establish a novel role for AP2 in direct regulation of PAR1 trafficking, a process critically important to the temporal and spatial aspects of thrombin signaling.
Department of Pharmacology, University of North Carolina at Chapel Hill, 1106 Mary Ellen Jones Bldg., Chapel Hill, NC 27599-7365. USA.
May M
MM
Paing
Christopher A
CA
Johnston
David P
DP
Siderovski
Joann
J
Trejo
D033962:Adaptor Protein Complex 2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000378:metabolism; D002118:Calcium / Q000032:analysis; D002460:Cell Line; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D005982:Glutathione Transferase / Q000378:metabolism; D006367:HeLa Cells; D006639:Histidine / Q000737:chemistry; D006801:Humans; D015151:Immunoblotting; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D044463:Receptor, PAR-1 / Q000737:chemistry / Q000378:metabolism; D018179:Receptors, Thrombin / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020349:Surface Plasmon Resonance; D014443:Tyrosine / Q000737:chemistry; D014471:Umbilical Veins / Q000166:cytology
D033962:Adaptor Protein Complex 2; D000911:Antibodies, Monoclonal; D021122:Protein Subunits; D034741:RNA, Small Interfering; D044463:Receptor, PAR-1; D018179:Receptors, Thrombin; D011993:Recombinant Fusion Proteins; D014443:Tyrosine; D006639:Histidine; D005982:Glutathione Transferase; D002118:Calcium