Role of the Differentially Spliced Carboxyl Terminus in Thromboxane A2 Receptor Trafficking

Abstract

The thromboxane A 2 receptor (TP) is a G protein-coupled receptor that is expressed as two alternatively spliced isoforms, ␣ (343 residues) and ␤ (407 residues) that share the first 328 residues. We have previously shown that TP␤, but not TP␣, undergoes agonist-induced internalization in a dynamin-, GRK-, and arrestindependent manner. In the present report, we demonstrate that TP␤, but not TP␣, also undergoes tonic internalization. Tonic internalization of TP␤ was temperature-and dynamin-dependent and was inhibited by sucrose and NH 4 Cl treatment but unaffected by wildtype or dominant-negative GRKs or arrestins. Truncation and site-directed mutagenesis revealed that a YX 3 motif (where X is any residue and is a bulky hydrophobic residue) found in the proximal portion of the carboxyl-terminal tail of TP␤ was critical for tonic internalization but had no role in agonist-induced internalization. Interestingly, introduction of either a YX 2 or YX 3 motif in the carboxyl-terminal tail of TP␣ induced tonic internalization of this receptor. Additional analysis revealed that tonically internalized TP␤ undergoes recycling back to the cell surface suggesting that tonic internalization may play a role in maintaining an intracellular pool of TP␤. Our data demonstrate the presence of distinct signals for tonic and agonist-induced internalization of TP␤ and represent the first report of a YX 3 motif involved in tonic internalization of a cell surface receptor.Cell surface receptors provide a primary mechanism by which cells perceive their environment. Many cell surface receptors are dynamically regulated and often undergo a process of endocytic sorting (1). For some receptors (e.g. G proteincoupled and growth factor), sorting is often initiated by hormone binding, whereas for others (e.g. low density lipoprotein and transferrin), the receptors undergo continuous or tonic internalization and recycling. Recent studies have demonstrated that several GPCRs 1 including the CXCR4, thyrotropin, M 2 muscarinic, and thrombin receptors also undergo tonic internalization (2-5). Although no particular motif responsible for tonic internalization of GPCRs has been identified, tyrosine-containing (YXX and NPXY) and dileucine motifs have been shown to be determinants for a number of other receptor types (1). Various studies have demonstrated direct interaction between YXX motifs and the chain of the clathrin-associated proteins AP-1, AP-2 (Ref. 6 and references therein), and AP-3 (7, 8), allowing the efficient targeting of transmembrane proteins containing these motifs to clathrin-coated vesicles.Thromboxane has been implicated in a number of cardiovascular, bronchial, and kidney diseases (9, 10). It is produced by the sequential metabolism of arachidonic acid by cyclooxygenase and thromboxane synthase following activation of a variety of cell types including platelets, macrophages, and vascular smooth muscle cells (11). Thromboxane is a strong activator of platelet aggregation and smooth muscle cell proliferation and mediates its effects v...

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