Internalization of the TXA2 receptor α and β isoforms -: Role of the differentially spliced COOH terminus in agonist-promoted receptor internalization

Thromboxane A(2) (TXA(2)) potently stimulates platelet aggregation and smooth muscle constriction and is thought to play a role in myocardial infarction, atherosclerosis, and bronchial asthma, The TXA(2) receptor (TXA(2)R) is a member of the G protein-coupled receptor family and is found as two alternatively spliced isoforms, alpha (343 residues) and beta (407 residues), which share the first 328 residues. In the present report, we demonstrate by enzyme-linked immunosorbent assay and immunofluorescence microscopy that the TXA(2)R beta, but not the TXA(2)R alpha, 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 TXA(2)R beta 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 alpha and beta 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 TXA(2)R beta did not affect agonist-promoted internalization. Analysis of various C-tail deletion mutants revealed that a region between residues 355 and 366 of the TXA(2)R beta is essential for agonist-promoted internalization. These data demonstrate that alternative splicing of the TXA(2)R plays a critical role in regulating arrestin binding and subsequent receptor internalization.