Chemokine receptors (CKRs) are important physiological mediators of immune defense, inflammatory responses, and angiogenesis, and they have also been implicated in a number of viral disease processes. Here, we report that the Nef protein of human immunodeficiency virus (HIV) reduces cell surface levels of eight different members of the CC-and CXC-family of CKRs by up to 92%. This broad-range activity required specific elements in HIV SF2 Nef, including the proline-rich motif P 73 P 76 P 79 P 82 as well as the acidic cluster motif E 66 E 67 E 68 E 69 , and Nef expression induced a marked perinuclear accumulation of CKRs. Surprisingly, receptor mutagenesis demonstrated that the cytoplasmic tail of CCR5 and CXCR4, which is critical for basal and ligand-mediated endocytosis, was completely dispensable for this Nef activity. In contrast, triple-mutation of the highly conserved DRY motif in the second intracellular CKR loop abolished the Nef-mediated down-regulation of CXCR4 independently of this motif's role in CKR binding to heterotrimeric G proteins and signaling via the G␣ i subunit. Thus, we identify the lentiviral pathogenicity factor Nef as a unique and broad-range modulator of CKR cell surface levels. Nef uses a mechanism that is distinct from well-established pathways orchestrating CKR metabolism and offers an interesting tool to study the multifaceted biology of CKRs.
INTRODUCTIONChemokine receptors (CKRs) are a specialized subset of seven-transmembrane (7-TM) G protein-coupled receptors (GPCRs) that is broadly grouped into CC, CXC, CX 3 C, and C classes based on the structure of their cognate agonists (Murphy et al., 2000). All CKRs are composed of an extracellular amino-terminal domain, seven hydrophobic transmembrane domains, and a cytoplasmic carboxy-terminal tail that harbors important motifs for basal and ligand-induced signaling, desensitization, and endocytosis. CKRs transduce signals via multiple mediators, i.e., heterotrimeric G proteins, -arrestin, and GPCR kinases. Signal transduction after ligand binding is initiated by stabilizing the CKR in an active conformation that enables the binding and activation of heterotrimeric G proteins (Scheer et al., 1997;Rasmussen et al., 1999;Scheer et al., 2000;Seifert and Wenzel-Seifert, 2003). A highly conserved sequence starting with Asp-ArgTyr (DRY) in the second intracellular loop of all CKRs plays a critical role in mediating the binding and signaling via heterotrimeric G proteins. After agonist engagement, desensitization of CKRs rapidly occurs by the interaction of -arrestin with phosphorylated Ser/Thr residues in the cytoplasmic tail of CKRs. Phosphorylation abolishes the signaling via heterotrimeric G proteins, and -arrestin binding lowers the receptor cell surface expression by targeting the molecule for endocytosis (Krupnick and Benovic, 1998). In addition, some CKRs contain a dileucine-based element in their cytoplasmic tail that provides a second independent motif for receptor endocytosis. Receptor internalization, recycling, and/or degradation...