Internalization of the TXA2 Receptor α and β Isoforms ROLE OF THE DIFFERENTIALLY SPLICED COOH TERMINUS IN AGONIST-PROMOTED RECEPTOR INTERNALIZATION

Abstract Thromboxane A2(TXA2) potently stimulates platelet aggregation and smooth muscle constriction and is thought to play a role in myocardial infarction, atherosclerosis, and bronchial asthma. The TXA2receptor (TXA2R) is a member of the G protein-coupled receptor family and is found as two alternatively spliced isoforms, α (343 residues) and β (407 residues), which share the first 328 residues. In the present report, we demonstrate by enzyme-linked immunosorbent assay and immunofluorescence microscopy that the TXA2Rβ, but not the TXA2Rα, undergoes agonist-induced internalization when expressed in HEK293 cells as well as several other cell types. Various dominant negative mutants were used to demonstrate that the internalization of the TXA2Rβ is dynamin-, GRK-, and arrestin-dependent in HEK293 cells, suggesting the involvement of receptor phosphorylation and clathrin-coated pits in this process. Interestingly, the agonist-stimulated internalization of both the α and β isoforms, but not of a mutant truncated after residue 328, can be promoted by overexpression of arrestin-3, identifying the C-tails of both receptors as necessary in arrestin-3 interaction. Simultaneous mutation of two dileucine motifs in the C-tail of TXA2Rβ did not affect agonist-promoted internalization. Analysis of various C-tail deletion mutants revealed that a region between residues 355 and 366 of the TXA2Rβ is essential for agonist-promoted internalization. These data demonstrate that alternative splicing of the TXA2R plays a critical role in regulating arrestin binding and subsequent receptor internalization.