Role of the Differentially Spliced Carboxyl Terminus in Thromboxane A2 Receptor Trafficking IDENTIFICATION OF A DISTINCT MOTIF FOR TONIC INTERNALIZATION

Abstract The thromboxane A2 receptor (TP) is a G protein-coupled receptor that is expressed as two alternatively spliced isoforms, α (343 residues) and β (407 residues) that share the first 328 residues. We have previously shown that TPβ, but not TPα, undergoes agonist-induced internalization in a dynamin-, GRK-, and arrestin-dependent manner. In the present report, we demonstrate that TPβ, but not TPα, also undergoes tonic internalization. Tonic internalization of TPβ was temperature- and dynamin-dependent and was inhibited by sucrose and NH4Cl treatment but unaffected by wild-type or dominant-negative GRKs or arrestins. Truncation and site-directed mutagenesis revealed that a YX 3φ motif (whereX is any residue and φ is a bulky hydrophobic residue) found in the proximal portion of the carboxyl-terminal tail of TPβ was critical for tonic internalization but had no role in agonist-induced internalization. Interestingly, introduction of either a YX 2φ or YX 3φ motif in the carboxyl-terminal tail of TPα induced tonic internalization of this receptor. Additional analysis revealed that tonically internalized TPβ undergoes recycling back to the cell surface suggesting that tonic internalization may play a role in maintaining an intracellular pool of TPβ. Our data demonstrate the presence of distinct signals for tonic and agonist-induced internalization of TPβ and represent the first report of a YX 3φ motif involved in tonic internalization of a cell surface receptor.