Homo- and heterodimerization have emerged as prominent features of G-protein-coupled receptors with possible impact on the regulation of their activity. Using a sensitive bioluminescence resonance energy transfer system, we investigated the formation of CXCR4 and CCR2 chemokine receptor dimers. We found that both receptors exist as constitutive homo- and heterodimers and that ligands induce conformational changes within the pre-formed dimers without promoting receptor dimer formation or disassembly. Ligands with different intrinsic efficacies yielded distinct bioluminescence resonance energy transfer modulations, indicating the stabilization of distinct receptor conformations. We also found that peptides derived from the transmembrane domains of CXCR4 inhibited activation of this receptor by blocking the ligand-induced conformational transitions of the dimer. Taken together, our data support a model in which chemokine receptor homo- and heterodimers form spontaneously and respond to ligand binding as units that undergo conformational changes involving both protomers even when only one of the two ligand binding sites is occupied.

We have shown that the chemokine and HIV receptor CCR5 is palmitoylated on a cluster of cysteine residues located at the boundary between the seventh transmembrane region and the cytoplasmic tail. Single or combined substitutions of the three cysteines (Cys-321, Cys-323, and Cys-324) or incubation of wild-type CCR5-transfected cells with the palmitic acid analog 2-bromopalmitate prevented palmitoylation of the receptor. Moreover, failure of CCR5 to be palmitoylated resulted in both accumulation in intracellular stores and a profound decrease of membrane expression of the receptor. Upon metabolic labeling, kinetic experiments showed that the half-life of palmitoylation-deficient CCR5 is profoundly decreased. Bafilomycin A1, but not a specific proteasome inhibitor, prevented early degradation of palmitoylation-deficient CCR5 and promoted its accumulation in lysosomal compartments. Although membrane expression of the CCR5 mutant was diminished, the molecules reaching the membrane were still able to interact efficiently with the chemokine ligand MIP1β and remained able to function as HIV co-receptors. Thus we conclude that palmitoylation controls CCR5 expression through regulation of the life span of this receptor.

Determining the role of lipid raft nanodomains in G protein-coupled receptor signaling remains fraught by the lack of assays directly monitoring rafts in native membranes. We thus combined extensive biochemical and pharmacological approaches to a nanoscale strategy based on bioluminescence resonance energy transfer (BRET) to assess the spatial and functional influence of cholesterol-rich liquid-ordered lipid nanodomains on β₂ adrenergic receptor (β₂AR) signaling. The data revealed that whereas β₂AR did not partition within liquid-ordered lipid phase, a pool of G protein and adenylyl cyclase (AC) were sequestered in these domains. Destabilization of the liquid-ordered phase by cholesterol depletion led to a lateral redistribution of Gα_s and AC that favored interactions between the receptor and its signaling partners as assessed by BRET. This resulted in an increased basal and agonist-promoted β₂AR-stimulated cAMP production that was partially dampened as a result of constitutive protein kinase A-dependent phosphorylation and desensitization of the receptor. This restraining influence of nanodomains on β₂AR signaling was further substantiated by showing that liquid-ordered lipid phase stabilization using caveolin overexpression or increasing membrane cholesterol amount led to an inhibition of β₂AR-associated signaling. Given the emerging concept that clustering of receptors and effectors into signaling platforms contributes to the efficacy and selectivity of signal transduction, our results support a model whereby cholesterol-promoted liquid-ordered lipid phase-embedding G_s and AC allows their lateral separation from the receptor, thus restraining the basal activity and controlling responsiveness of β₂AR signaling machinery within larger signaling platforms.

PML nuclear bodies (NBs) are dynamic intranuclear structures harboring numerous transiently or permanently localized proteins. PML, the NBs' organizer, is directly induced by interferon, and its expression is critical for antiviral host defense. We describe herein the molecular events following poliovirus infection that lead to PML-dependent p53 activation and protection against virus infection. Poliovirus infection induces PML phosphorylation through the extracellular signal-regulated kinase pathway, increases PML SUMOylation, and induces its transfer from the nucleoplasm to the nuclear matrix. These events result in the recruitment of p53 to PML NBs, p53 phosphorylation on Ser15, and activation of p53 target genes leading to the induction of apoptosis. Moreover, the knock-down of p53 by small interfering RNA results in higher poliovirus replication, suggesting that p53 participates in antiviral defense. This effect, which requires the presence of PML, is transient since poliovirus targets p53 by inducing its degradation in a proteasome- and MDM2-dependent manner. Our results provide evidence of how poliovirus counteracts p53 antiviral activity by regulating PML and NBs, thus leading to p53 degradation.

CC chemokine receptor 5 (CCR5) is a G protein-coupled receptor that governs migration of leukocytes and serves as a coreceptor for the R5 tropic strains of human immunodeficiency virus (HIV). CCR5-mediated signaling in response to CC chemokines relies on G protein activation. Desensitization, which rapidly turns off G protein-dependent signaling, involves phosphorylation of CCR5 that promotes interaction of the receptor with β-arrestins for endocytosis. Whether coupling to G proteins, desensitization, and endocytosis of CCR5 require the same structural determinants remains a matter of investigation. Here, we show that CCR5 displayed agonist-independent coupling to G proteins. This constitutive activity of the receptor was abrogated by TAK779 (N,N-dimethyl-N-[4-[[[2-(4-methylphenyl)-6,7-dihydro-5H-benzocyclohepten-8-yl]carbonyl]amino]benzyl]tetrahydro-2H-pyran-4-aminium chloride), a nonpeptidic CCR5 ligand that inhibits HIV infection and was found to depend on the integrity of the Asp-Arg-Tyr (DRY) motif. Changing Arg-126 by the neutral residue Asn (R126N-CCR5 mutant) abolished CCR5-mediated activation of G proteins, either constitutively or in response to agonists. In contrast, R126N-CCR5 not only retained agonist-promoted phosphorylation and β-arrestin-dependent endocytosis but also displayed a higher basal phosphorylation than wild-type CCR5. Expression of β-arrestin in R126N-CCR5-expressing cells resulted in receptor down-regulation, thereby suggesting that R126N-CCR5 spontaneously interacts with β-arrestins. However, although expression of β-arrestin favored wild-type CCR5-mediated chemotaxis, it failed to promote migration of cells expressing R126N-CCR5. Overall, these data indicate that structural requirements for CCR5-mediated activation of G proteins, albeit not involved in receptor desensitization and internalization, are needed for β-arrestin-mediated chemotaxis. These results have implications for how distinct biological responses of CCR5 might rely on a different set of receptor conformations.