Identification of a motif in the carboxyl terminus of CXCR2 that is involved in adaptin 2 binding and receptor internalization.

The chemokine receptor, CXCR2,1 is a member of a superfamily of G protein-coupled seven-transmembrane receptors (GPCRs) that transduce intracellular signals via heterotrimeric guanine nucleotide-binding proteins (G proteins). Upon stimulation by agonists, such as interleukin 8 (IL-8) or melanoma growth-stimulatory activity (MGSA)/growth-regulatory protein (GRO), CXCR2 activates a series of G protein-mediated events, including phosphatidylinositide hydrolysis, to generate inositol 1,4,5-trisphosphate and diacylglycerol, as well as mobilization of intracellular free Ca2+ to initiate a series of cellular responses (1). In addition, CXCR2 mediates cell chemotaxis, a distinct function of chemokine receptors (2). Like many other types of GPCRs, CXCR2 undergoes a dynamic trafficking between the cell surface and the intracellular compartments (3). Such trafficking may be involved in both transmission and termination of the receptor signals and may play an important role in mediating cell chemotaxis. For CXCR2, the most remarkable trafficking process is agonist-induced receptor internalization. Agonist-induced phosphorylation of the carboxyl terminus by G protein-coupled receptor kinases (GRKs) has been proposed to be necessary for the desensitization and internalization of many GPCRs (4–7). Clathrin-coated pits have been demonstrated to play a central role in GPCR sequestration (8, 9). β-arrestins, which interact with the phosphorylated form of agonist-stimulated GPCRs to desensitize their signaling, might act as an adaptor proteins between the receptor and clathrin to initiate clathrin-mediated internalization (10–14). In a word, for many GPCRs, receptor phosphorylation has been suggested to be required for the receptor internalization, and β-arrestins have been proposed to play an important role in this process. Although β-arrestins have been shown to bind to clathrin with high affinity (15), they are not constitutively associated with clathrin-coated vesicles (16), nor do they promote clathrin-coat assembly (17). These studies imply that some other adaptor proteins are also involved in this process. In fact, recent studies on the β2-adrenergic receptor (β2-AR), a prototypic GPCR, have shown that adaptin 2 (AP-2) functions as a clathrin adaptor for the endocytosis of β2-AR (18). Our previous studies have demonstrated that carboxyl-terminal phosphorylation is necessary for the desensitization of CXCR2 (19), and dynamin, a component of clathrin-mediated endocytosis, is essential for CXCR2 internalization and resensitization (3). However, the receptor domains that might be involved in the receptor sequestration are still poorly defined. Our preliminary data show that a carboxyl-terminal-truncated receptor (331T), which loses agonist-induced phosphorylation, still undergoes agonist-induced internalization in human embryonic kidney 293 (HEK293) cells but not in 3ASubE cells, although binding of the truncated receptor to β-arrestin 1 was severely impaired. These data suggest that additional motifs in CXCR2 might associate with other adaptor proteins to facilitate receptor endocytosis. A highly conserved carboxyl-terminal Leu-Leu motif has been demonstrated to involve the endocytosis of various membrane proteins (20–22), including G protein-coupled β2-AR (23). A Leu-Leu motif has been proposed to act as a binding site for adaptor proteins, such as AP-2, that are required for intracellular protein trafficking (24). In addition, a Leu-Ile motif has been reported to be important for the endocytosis of the leukemia inhibitory factor receptor (25). However, it has never been directly demonstrated that mutation of the dileucine residues eliminates the association of AP-2 with GPCRs. Comparable Leu-Leu, Leu-Ile, and Ile-Leu motifs are also present in the carboxyl terminus of CXCR2, suggesting a potential role of these domains in the internalization of CXCR2. In this paper, we report that the LLKIL motif in the carboxyl terminus of CXCR2 is required for association of the receptor with AP-2 and is essential for the receptor sequestration as well as for ligand-mediated chemotaxis.