Role of the differentially spliced carboxyl terminus in thromboxane A2 receptor trafficking: identification of a distinct motif for tonic internalization

The thromboxane A(2) receptor (TP) is a G protein-coupled receptor that is expressed as two alternatively spliced isoforms, alpha (343 residues) and beta (407 residues) that share the first 328 residues. We have previously shown that TPbeta, but not TPalpha, undergoes agonist-induced internalization in a dynamin-, GRK-, and arrestin-dependent manner. In the present report, we demonstrate that TPbeta, but not TPalpha, also undergoes tonic internalization. Tonic internalization of TPbeta was temperature- and dynamin-dependent and was inhibited by sucrose and NH(4)Cl treatment but unaffected by wild-type or dominant-negative GRKs or arrestins. Truncation and site-directed mutagenesis revealed that a YX(3)phi motif (where X is any residue and phi is a bulky hydrophobic residue) found in the proximal portion of the carboxyl-terminal tail of TPbeta was critical for tonic internalization but had no role in agonist-induced internalization. Interestingly, introduction of either a YX(2)phi or YX(3)phi motif in the carboxyl-terminal tail of TPalpha induced tonic internalization of this receptor. Additional analysis revealed that tonically internalized TPbeta undergoes recycling back to the cell surface suggesting that tonic internalization may play a role in maintaining an intracellular pool of TPbeta. Our data demonstrate the presence of distinct signals for tonic and agonist-induced internalization of TPbeta and represent the first report of a YX(3)phi motif involved in tonic internalization of a cell surface receptor.