The Composition of the ␤-2 Adrenergic Receptor Oligomer Affects Its Membrane Trafficking after Ligand-Induced Endocytosis

ABSTRACT The ␤-2 adrenergic receptor (B2AR) is well known to form oligomeric complexes in vivo, but the functional significance of this process is not fully understood. The present results identify an effect of oligomerization of the human B2AR on the membrane trafficking of receptors after agonist-induced endocytosis in stably transfected human embryonic kidney 293 cells. A sequence present in the cytoplasmic tail of the B2AR has been shown previously to be required for efficient recycling of internalized receptors. Mutation of this sequence was observed to inhibit recycling not only of the receptor containing the mutation but also of the coexpressed wild-type B2AR. Coexpression of recycling-defective mutant B2ARs also enhanced proteolytic degradation of the wild-type B2AR after agonist-induced endocytosis, consistent with trafficking of both receptors to lysosomes in an oligomeric complex. Coexpression of the ␦ opioid receptor (DOR) at similar levels produced a much smaller effect on endocytic trafficking of the B2AR, even though DOR traverses a similar membrane pathway as recycling-defective mutant B2ARs. Biochemical studies confirmed that B2AR/ B2AR-ala homomeric complexes form more readily than DOR/ B2AR heteromers in expression-matched cell clones and support the hypothesis that B2AR/B2AR-ala complexes are not disrupted by agonist. These results suggest that a significant fraction of B2ARs exists in oligomeric complexes after ligandinduced endocytosis and that the composition of the oligomeric complex influences the sorting of endocytosed receptors between functionally distinct recycling and degradative membrane pathways

CCR5 Revisited: How Mechanisms of HIV Entry Govern AIDS Pathogenesis

International audienceThe chemokine receptor CCR5 has been the focus of intensive studies since its role as a coreceptor for HIV entry was discovered in 1996. These studies lead to the development of small molecular drugs targeting CCR5, with maraviroc becoming in 2007 the first clinically approved chemokine receptor inhibitor. More recently, the apparent HIV cure in a patient transplanted with hematopoietic stem cells devoid of functional CCR5 rekindled the interest for inactivating CCR5 through gene therapy and pharmacological approaches. Fundamental research on CCR5 has also been boosted by key advances in the field of G-protein coupled receptor research, with the realization that CCR5 adopts a variety of conformations, and that only a subset of these conformations may be targeted by chemokine ligands. In addition, recent genetic and pathogenesis studies have emphasized the central role of CCR5 expression levels in determining the risk of HIV and SIV acquisition and disease progression. In this article, we propose to review the key properties of CCR5 that account for its central role in HIV pathogenesis, with a focus on mechanisms that regulate CCR5 expression, conformation, and interaction with HIV envelope glycoproteins

Effect of Mutations in the Second Extracellular Loop of CXCR4 on Its Utilization by Human and Feline Immunodeficiency Viruses

CCR5 and CXCR4 are the principal CD4-associated coreceptors used by human immunodeficiency virus type 1 (HIV-1). CXCR4 is also a receptor for the feline immunodeficiency virus (FIV). The rat CXCR4 cannot mediate infection by HIV-1(NDK) or by FIV(PET) (both cell line-adapted strains) because of sequence differences with human CXCR4 in the second extracellular loop (ECL2). Here we made similar observations for HIV-1(89.6) (a strain also using CCR5) and for a primary HIV-1 isolate. It showed the role of ECL2 in the coreceptor activity of CXCR4 for different types of HIV-1 strains. By exchanging ECL2 residues between human and rat CXCR4, we found that several amino acid differences contributed to the inactivity of the rat CXCR4 toward HIV-1(89.6). In contrast, its inactivity toward HIV-1(NDK) seemed principally due to a serine at position 193 instead of to an aspartic acid (Asp193) in human CXCR4. Likewise, a mutation of Asp187 prevented usage of CXCR4 by FIV(PET). Different mutations of Asp193, including its replacement by a glutamic acid, markedly reduced or suppressed the activity of CXCR4 for HIV-1(NDK) infection, indicating that the negative charge was not the only requirement. Mutations of Asp193 and of arginine residues (Arg183 and Arg188) of CXCR4 reduced the efficiency of HIV-1 infection for all HIV-1 strains tested. Other ECL2 mutations tested had strain-specific effects or no apparent effect on HIV-1 infection. The ECL2 mutants allowed us to identify residues contributing to the epitope of the 12G5 monoclonal antibody. Overall, residues with different charges and interspersed in ECL2 seem to participate in the coreceptor activity of CXCR4. This suggests that a conformational rather than linear epitope of ECL2 contributes to the HIV-1 binding site. However, certain HIV-1 and FIV strains seem to require the presence of a particular ECL2 residue

The composition of the beta-2 adrenergic receptor oligomer affects its membrane trafficking after ligand-induced endocytosis

ABSTRACT The beta-2 adrenergic receptor (B2AR) is well known to form oligomeric complexes in vivo but the functional significance of this process is not fully understood. The present results identify an effect of oligomerization of the human B2AR on the membrane trafficking of receptors after agonist-induced endocytosis in stably transfected HEK293 cells. A sequence present in the cytoplasmic tail of the B2AR has been shown previously to be required for efficient recycling of internalized receptors. Mutation of this sequence was observed to inhibit recycling, not only of the receptor in receptor containing the mutation, but also of the co-expressed wild type B2AR. Co-expression of recycling-defective mutant B2ARs also enhanced proteolytic degradation of the wild type B2AR after agonist-induced endocytosis, consistent with trafficking of both receptors to lysosomes in an oligomeric complex. Co-expression of the delta opioid receptor (DOR) at similar levels produced a much smaller effect on endocytic trafficking of the B2AR, even though DOR traverses a similar membrane pathway as recyclingdefective mutant B2ARs following agonist-induced endocytosis. Biochemical studies confirmed that B2AR / B2AR-ala 'homomeric' complexes form more readily than DOR / B2AR heteromers in expression-matched cell clones, and support the hypothesis that B2AR / B2AR-ala complexes are not disrupted by agonist. These results suggest that a significant fraction of B2ARs exist in oligomeric complexes after ligand-induced endocytosis, and that the composition of the oligomeric complex influences the sorting of endocytosed receptors between functionally distinct recycling and degradative membrane pathways

Structural diversity and versatility for organoaluminum complexes supported by mono- and di-anionic aminophenolate bidentate ligands

The present contribution describes the synthesis and structural characterization of structurally diverse organoaluminum species supported by variously substituted aminophenolate-type ligands: these Al complexes are all derived from the reaction of AlMe3 with aminophenols 2-CH2NH(R)-C6H3OH (1a, R = mesityl (Mes); 1b, R = 2,6-di-isopropylphenyl (Diip)) and 2-CH2NH(R)-4,6-Bu-t(2)-C6H2OH (1c, R = Mes; 1d, R = Diip). The low temperature reaction of AlMe3 with 1a-b readily affords the corresponding Al dimeric species [mu-eta(1), eta(1)-N,O-{2-CH2NH(R)-C6H4O}](2)Al2Me4 (2a-b), consisting of twelve-membered ring aluminacycles with two mu-eta(1), eta(1)-N,O-aminophenolate units, as determined by X-ray crystallographic studies. Heating a toluene solution of 2a (80 degrees C, 3 h) affords the quantitative and direct formation of the dinuclear aluminium complex Al[eta(2)-N; mu, eta(2)-O-{2-CH2N(Mes)-C6H4O}](AlMe2) (4a) while species 2b, under the aforementioned conditions, affords the formation of the Al dimeric species [eta(2)-N,O-{2-CH2N(Dipp)-C6H4O}AlMe](2) (3b), as deduced from X-ray crystallography for both 3b and 4a. In contrast, the reaction of bulky aminophenol pro-ligands 1c-d with AlMe3 afford the corresponding monomeric Al aminophenolate chelate complexes eta(2)-N,O-{2-CH2NH(R)-4,6-Bu-t(2)-C6H2O}AlMe2 (5c-d; R = Mes, Diip; Scheme 3) as confirmed by X-ray crystallographic analysis in the case of 5d. Subsequent heating of species 5c-d yields, via a methane elimination route, the corresponding Al-THF amido species eta(2)-N,O-{2-CH2N(R)-4,6-Bu-t(2)-C6H2O}Al(Me)(THF) (6c-d; R = Mes, Diip). Compounds 6c-6d, which are of the type {X-2}Al(R)(L) (L labile), may well be useful as novel well-defined Lewis acid species of potential use for various chemical transformations. Overall, the sterics of the aminophenol backbone and, to a lesser extent, the reaction conditions that are used for a given ligand/AlMe3 set essentially govern the rather diverse “structural” outcome in these reactions, with a preference toward the formation of mononuclear Al species (i.e. species 5c-d and 6c-d) as the steric demand of the chelating N,O-ligand increases. (C) 2011 Elsevier B.V. All rights reserved

HIV-1 exploits CCR5 conformational heterogeneity to escape inhibition by chemokines.

International audienceCC chemokine receptor 5 (CCR5) is a receptor for chemokines and the coreceptor for R5 HIV-1 entry into CD4(+) T lymphocytes. Chemokines exert anti-HIV-1 activity in vitro, both by displacing the viral envelope glycoprotein gp120 from binding to CCR5 and by promoting CCR5 endocytosis, suggesting that they play a protective role in HIV infection. However, we showed here that different CCR5 conformations at the cell surface are differentially engaged by chemokines and gp120, making chemokines weaker inhibitors of HIV infection than would be expected from their binding affinity constants for CCR5. These distinct CCR5 conformations rely on CCR5 coupling to nucleotide-free G proteins ((NF)G proteins). Whereas native CCR5 chemokines bind with subnanomolar affinity to (NF)G protein-coupled CCR5, gp120/HIV-1 does not discriminate between (NF)G protein-coupled and uncoupled CCR5. Interestingly, the antiviral activity of chemokines is G protein independent, suggesting that "low-chemokine affinity" (NF)G protein-uncoupled conformations of CCR5 represent a portal for viral entry. Furthermore, chemokines are weak inducers of CCR5 endocytosis, as is revealed by EC50 values for chemokine-mediated endocytosis reflecting their low-affinity constant value for (NF)G protein-uncoupled CCR5. Abolishing CCR5 interaction with (NF)G proteins eliminates high-affinity binding of CCR5 chemokines but preserves receptor endocytosis, indicating that chemokines preferentially endocytose low-affinity receptors. Finally, we evidenced that chemokine analogs achieve highly potent HIV-1 inhibition due to high-affinity interactions with internalizing and/or gp120-binding receptors. These data are consistent with HIV-1 evading chemokine inhibition by exploiting CCR5 conformational heterogeneity, shed light into the inhibitory mechanisms of anti-HIV-1 chemokine analogs, and provide insights for the development of unique anti-HIV molecules

Targeting CCR5 trafficking to inhibit HIV-1 infection

International audienceUsing a cell-based assay monitoring differential protein transport in the secretory pathway coupled to high-content screening, we have identified three molecules that specifically reduce the delivery of the major co-receptor for HIV-1, CCR5, to the plasma membrane. They have no effect on the closely related receptors CCR1 and CXCR4. These molecules are also potent in primary macrophages as they markedly decrease HIV entry. At the molecular level, two of these molecules inhibit the critical palmitoylation of CCR5 and thereby block CCR5 in the early secretory pathway. Our results open a clear therapeutics avenue based on trafficking control and demonstrate that preventing HIV infection can be performed at the level of its receptor delivery

The Second Extracellular Loop of CXCR4 Determines Its Function as a Receptor for Feline Immunodeficiency Virus

The feline homolog of the α-chemokine receptor CXCR4 has recently been shown to support cell-cell fusion mediated by CXCR4-dependent strains of human immunodeficiency virus (HIV) and strains of feline immunodeficiency virus (FIV) that have been selected for growth in the Crandell feline kidney (CrFK) cell line. In this report we demonstrate that expression of CXCR4 alone is sufficient to render cells from diverse species permissive for fusion with FIV-infected cells, suggesting that CXCR4 is the sole receptor for CrFK-tropic strains of FIV, analogous to CD4-independent strains of HIV-2. To identify the regions of CXCR4 involved in fusion mediated by FIV, we screened panels of chimeric CXCR4 molecules for the ability to support fusion with FIV-infected cells. Human CXCR4 supported fusion more efficiently than feline CXCR4 and feline/human CXCR4 chimeras, suggesting that the second and third extracellular loops of human CXCR4 contain a critical determinant for receptor function. Rat/human CXCR4 chimeras suggested that the second extracellular loop contained the principal determinant for receptor function; however, chimeras constructed between human CXCR2 and CXCR4 revealed that the first and third loops of CXCR4 contribute to the FIV Env binding site, as replacement of these domains with the corresponding domains of CXCR2 rendered the molecule nonfunctional in fusion assays. Mutation of the DRY motif and the C-terminal cytoplasmic tail of CXCR4 did not affect the ability of the molecule to support fusion, suggesting that neither signalling via G proteins nor receptor internalization was required for fusion mediated by FIV; similarly, truncation of the N terminus of CXCR4 did not affect the function of the molecule as a receptor for FIV. CXCR4-transfected feline cells were rendered permissive for infection with both the CrFK-tropic PET isolate of FIV and the CXCR4-dependent RF strain of HIV-1, and susceptibility to infection correlated well with ability to support fusion. The data suggest that the second extracellular loop of CXCR4 is the major determinant of CXCR4 usage by FIV