Role of the thrombin receptor's cytoplasmic tail in intracellular trafficking. Distinct determinants for agonist-triggered versus tonic internalization and intracellular localization.
The G protein-coupled thrombin receptor is activated by an irreversible proteolytic mechanism and, perhaps as a result, exhibits an unusual trafficking pattern in the cell. Naive receptors tonically cycle between the cell surface and a protected intracellular pool, whereas receptors cleaved and activated at the cell surface internalize and move to lysosomes. Toward understanding how these trafficking events are regulated, we examined a series of receptor mutants. A receptor with alanine substitutions at all potential phosphorylation sites in the cytoplasmic tail failed to display agonist-triggered internalization but, like wild type receptor, displayed robust signaling, tonic cycling, and localization to both the cell surface and an intracellular pool. A truncation mutant that lacked most of the cytoplasmic tail also signaled robustly, lacked phosphorylation, and was defective in agonist-triggered internalization. However, in contrast to the specific phosphorylation site mutant, the truncation mutant did not display tonic cycling and localized exclusively to the cell surface. An analysis of a series of truncation mutants localized residues important for receptor trafficking to a 10-amino acid stretch in its cytoplasmic tail. These data suggest that phosphorylation may trigger internalization of activated thrombin receptors but that a second phosphorylation-independent signal mediates tonic internalization of naive receptors. They further suggest that maintenance of the intracellular pool of naive thrombin receptors requires tonic receptor internalization.
Shapiro
M J
MJ
Cardiovascular Research Institute, University of California, San Francisco, California 94143-0130, USA. shaun_coughlin.src@quickmail.ucsf.edu
Trejo
J
J
Zeng
D
D
Coughlin
S R
SR
D000818:Animals; D019556:COS Cells; D002451:Cell Compartmentation; D003593:Cytoplasm / Q000378:metabolism; D004705:Endocytosis; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D051381:Rats; D018179:Receptors, Thrombin / Q000737:chemistry* / Q000378:metabolism; D015398:Signal Transduction; D013329:Structure-Activity Relationship
D018179:Receptors, Thrombin