AP-2 α residues K297 and R340 contribute to the interaction between α-σ2 and HIV-1 Nef. The effects of single or combined mutations of AP-2 α K295, K297, K298, and/or R340 to glutamate or alanine on the interaction of AP-2 α-σ2 with HIV-1 Nef or the mouse tyrosinase cytosolic tail were investigated. The discordant α and σ1 pair was used as a negative control. Analyses were performed using the Y3H system as described in the legend to Fig. 2 and in Materials and Methods.

Identification of basic residues in the AP-2 α-σ2 hemicomplex that are not conserved in the homologous subunits of AP-1 and AP-3. (A) Sequence alignment of the trunk domains of human AP-1 γ (γ1 isoform; accession no. AAH36283), AP-2 α (αC isoform; accession no. AH06155), and AP-3 δ (accession no. AAC51761). (B) Sequence alignment of human AP-1 σ1 (σ1A isoform; accession no. AAA37243), AP-2 σ2 (accession no. AAP36470), and AP-3 63 (63A isoform; accession no. EAW48952). Alignments were performed using the ClustalW2 program (available at http://www.ebi.ac.uk/Tools/clustalw2/index.html). Amino acid numbers are indicated. Lysine and arginine residues present on AP-2 α and σ2 but not on the corresponding AP-1 and AP-3 subunits are highlighted in red. These residues were mutated to either aspartate or glutamate (Fig. 2). Black and red asterisks indicate residues that were also mutated to alanine (Fig. 3). Red asterisks denote AP-2 α residues K297 and R340, which were found to be required for the interaction with HIV-1 Nef. N/A, not applicable.

Location of AP-2 α K297 and R340 on the three-dimensional structure of the AP-2 core complex (accession no. 1GW5 and 2VGL) (15). (A and B) Surface representations with the α, β2, μ2, and σ2 subunits colored in blue, green, magenta, and gold, respectively; the binding site for polyphosphoinositides (PIPs) in the α subunit is depicted in light blue, and the α K297 and R340 residues (including their side chains) are shown in red. The image in panel B corresponds to a 60° rotation around the x axis of that shown in panel A. Residues α K297 and R340 correspond to K298 and R341 in polymer 1 of the crystal structure (15) (C) Surface representation of the image in panel A colored according to electrostatic potential (contoured as red to blue from −74 to 74 kT). (D) Magnified ribbon diagram of the region surrounding residues K297 and R340. All images were drawn with PyMOL (17).

Assembly of a tripartite complex of Nef, the cytosolic tail of CD4, and the AP-2 α-σ2 hemicomplex as demonstrated by yeast hybrid assays. (A) Plasmids used in the Y2H, Y3H, and Y4H assays. In all assays, HIV-1 Nef was expressed from pBridge as a GAL4BD fusion protein, while the cytosolic tail of human CD4 (CD4 ct) was expressed from pAD as a GAL4AD fusion protein. In the Y2H assay, no other proteins were expressed from these vectors; in the Y3H assay, either σ2-adaptin or αC-adaptin was expressed from pBridge or pAD, respectively; in the Y4H assay, both σ2 adaptin and αC adaptin were coexpressed. (B) Y2H, Y3H, and Y4H analyses of the interaction between GAL4BD-Nef and GAL4AD-CD4 in the absence or presence of one or both components of the α-σ2 hemicomplex. The plasmids used in the yeast hybrid experiments are noted to the left of the panel, with the pBridge (pBr)-based vectors in the first column and the pAD-based vectors in the second column. Row 1 corresponds to the Y2H assay, rows 2 and 3 correspond to the Y3H assays, and row 4 corresponds to the Y4H assay. Yeast from all assays were seeded onto +His and −His plates at increasing levels of OD. Yeast growth on the −His plates indicates an interaction between GAL4BD-Nef and GAL4AD-CD4. (C) Y4H analysis of the effect of several Nef and α mutants on the interaction of GAL4BD-Nef and GAL4AD-CD4 in the presence of the α-σ2 hemicomplex.

Y3H analysis of the interaction of HIV-1 Nef with AP-2 α-σ2 hemicomplexes having substitutions for nonconserved basic residues. AP-2 α and σ2 basic residues that are not conserved in the homologous subunits of AP-1 (γ and σ1) and AP-3 (δ and σ3) (Fig. 1) were mutated, individually or in combination, to glutamate or aspartate. The resulting α-σ2 constructs were tested for interaction with HIV-1 Nef or the mouse tyrosinase cytosolic tail by using a Y3H assay, as described in Materials and Methods. (A) Schematic representation of the plasmids used in the Y3H assay. Nef or the tyrosinase cytosolic tail was expressed as a GAL4BD fusion from MCS1 of pBridge, σ2 was expressed from MCS2 of pBridge, and α was expressed as a GAL4AD fusion from the MCS of pGADT7. Cotransformations of yeast cells with Nef or the tyrosinase tail and discordant combinations of AP-2 α and AP-1 σ1 constructs were used as self-activation/specificity controls. (B and C) Y3H assay results. Cotransformants were plated onto medium lacking Leu, Trp, Met, and His (−His) in the absence or presence of 1 mM 3AT (−His + 3AT) to detect interaction through HIS3 reporter gene activation. 3AT is a competitive inhibitor of the His3 protein (imidazoleglycerol-phosphate dehydratase) that increases the stringency of the assay. Cotransformants were also plated onto medium lacking Leu, Trp, and Met (+His) to control for growth and loading. Growth on −His or −His + 3AT plates is indicative of interactions. WT, wild type.

The replacement of α K297 and R340 impairs the binding of the recombinant AP-2 core to Nef in vitro. (A) Recombinant Nef and AP-2 core constructs used in the in vitro binding experiments were produced as described in Materials and Methods and subjected to SDS-PAGE on 4 to 12% acrylamide gradient gels, followed by Coomassie blue staining. Lanes corresponding to the AP-2 core complexes bearing wild-type (WT) and KR297,340EE mutant α chains show the α trunk-GST, His₆-β2 trunk, μ2-N, and full-length σ2 subunits in order of increasing mobilities (μ2-N and σ2 comigrate in this gel system). The band at ∼21 kDa (asterisk) in the lanes corresponding to wild-type and mutant AP-2 cores represents a GST degradation product. (B) Equal amounts of GST fusion proteins with wild-type (WT) or α KR297,340EE-containing AP-2 cores, or the ear domains of AP-2 αC (57) or AP-3 β3B (18) (the latter two as negative controls), were immobilized on glutathione-Sepharose beads, and the beads were incubated with recombinant His₆-Nef for 2 h at 4°C. Bound proteins were eluted and subjected to SDS-PAGE on 10% acrylamide gels, followed by immunoblotting (IB) with antibodies to the His₆ tag (upper panel) or to Nef (lower panel). The last lane on the right (labeled −) shows binding to unfused GST. The ∼60-kDa band labeled by anti-His₆ in the upper blot corresponds to the His₆-tagged β2 trunk in the recombinant AP-2 core and served as an internal loading control. Numbers to the left indicate the positions of molecular mass markers (in kilodaltons).

AP-2 α residues K297 and R340 are required for Nef-induced CD4 downregulation. HeLa cells were subjected to a 7-day siRNA-cDNA transfection protocol as described in Materials and Methods and in Table 1. Control and α siRNA-treated cells were cotransfected with three plasmids: one expressing CD4, one lacking (−) or expressing (+) Nef, and one lacking or expressing either wild-type αR (αR-WT) or αR-KR297,340EE. The cells were then prepared for FACS analysis and immunoblotting. Cells prepared for FACS analysis were either left unlabeled as a control for background fluorescence (shaded curves in all plots) or stained with PE-conjugated anti-human TfR or APC-conjugated anti-human CD4 antibodies. (A) Treatment with α siRNAs increases cell surface TfR and CD4 levels in the absence of Nef. (B) Both wild-type αR and αR-KR297,340EE prevent the increase in the levels of TfR and CD4 caused by treatment with α siRNA in the absence of Nef. (C) Nef expression downregulates CD4 in control but not α siRNA-treated HeLa cells. (D) Rescue of Nef-induced downregulation of CD4 by wild-type αR but not αR-KR297,340EE in α siRNA-treated HeLa cells. The results shown are representative of data from three independent experiments with similar results. Statistical analyses of the data from these three experiments showed that Nef-dependent CD4 downregulation (expressed as the ratio of geometric means in the absence or presence of Nef) was 4.81 ± 0.70 for control cells, 1.51 ± 0.24* for α siRNA-treated cells, 4.60 ± 0.94 for α siRNA-treated cells expressing wild-type αR, and 1.50 ± 0.04*† for α siRNA-treated cells expressing αR-KR297,340EE (mean ± standard error of the mean; n = 3), with the symbols * and † indicating values that are significantly different (P < 0.05) from those for control cells and α siRNA-treated cells expressing wild-type αR, respectively, as calculated by analysis of variance followed by two-tail Dunnett's test. (E) Aliquots of the transfected cells from all experimental groups were lysed and subjected to SDS-PAGE and immunoblotting (IB) with the antibodies indicated to the right. All cells were transfected with a plasmid encoding CD4, together with the plasmids and oligonucleotides indicated in the grid above the lanes (+, present; −, absent; KREE, KR297,340EE mutant). Notice that the anti-AP-2 α clone 100/2 recognized both endogenous isoforms of α-adaptin, αA and αC (apparent as an ∼100-kDa doublet in which the upper and lower bands represent αA and αC, respectively) (5), as well as a nonspecific band (∼85 kDa). The anti-AP-2 α clone 8/Adaptin α, however, recognized only endogenous αA-adaptin since it was raised against a fragment of this isoform. TheV5-tagged, RNAi-resistant αC rescue constructs were detected by both the 100/2 anti-α antibody and the anti-V5 antibody. Immunoblotting with anti-α-tubulin was used as a loading control. Numbers to the left indicate the positions of molecular mass markers (in kilodaltons).