FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2009205890> ?p ?o ?g. }

• W2009205890 endingPage "15778" @default.
• W2009205890 startingPage "15773" @default.
• W2009205890 abstract "The envelope glycoprotein (Env) of human immunodeficiency virus, type 1 (HIV-1) undergoes rapid internalization after its transport to the cell surface. Env internalization is dependent upon information contained within the cytosolic domain of the protein. Here, we report that the cytosolic domain of Env binds specifically to the medium chain, μ2, of the clathrin-associated protein complex AP-2, as well as to the complete AP-2 complex. The Env cytosolic domain contains two highly conserved tyrosine-based motifs (Y712SPL and Y768HRL), both of which are capable of binding to μ2 when presented as short peptides. However, only the membrane-proximal motif Y712SPL binds to μ2 and is required for internalization in the context of the whole cytosolic domain of Env. A glycine residue (Gly711) adjacent to the Y712SPL motif is also important for binding to μ2/AP-2 and internalization. These observations suggest that the accessibility of the membrane-proximal GY712SPL to μ2/AP-2 determines its function as a signal for recruitment of HIV-1 Env into clathrin-coated pits and its ensuing internalization. The envelope glycoprotein (Env) of human immunodeficiency virus, type 1 (HIV-1) undergoes rapid internalization after its transport to the cell surface. Env internalization is dependent upon information contained within the cytosolic domain of the protein. Here, we report that the cytosolic domain of Env binds specifically to the medium chain, μ2, of the clathrin-associated protein complex AP-2, as well as to the complete AP-2 complex. The Env cytosolic domain contains two highly conserved tyrosine-based motifs (Y712SPL and Y768HRL), both of which are capable of binding to μ2 when presented as short peptides. However, only the membrane-proximal motif Y712SPL binds to μ2 and is required for internalization in the context of the whole cytosolic domain of Env. A glycine residue (Gly711) adjacent to the Y712SPL motif is also important for binding to μ2/AP-2 and internalization. These observations suggest that the accessibility of the membrane-proximal GY712SPL to μ2/AP-2 determines its function as a signal for recruitment of HIV-1 Env into clathrin-coated pits and its ensuing internalization. The envelope glycoprotein (Env) of human immunodeficiency virus, type 1 (HIV-1) 1The abbreviations used are: HIV-1, human immunodeficiency virus, type 1; GST, glutathioneS-transferase; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; MESNa, 2-mercaptoethanesulfonic acid. 1The abbreviations used are: HIV-1, human immunodeficiency virus, type 1; GST, glutathioneS-transferase; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; MESNa, 2-mercaptoethanesulfonic acid. plays a critical role during the viral life cycle by mediating the attachment of virions to target cells and the fusion of viral and cellular membranes (1Freed E.O. Martin M.A. J. Biol. Chem. 1995; 270: 23883-23886Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar). Incorporation of Env, therefore, is essential for the formation of infectious viral particles. The cytosolic domain of the Env transmembrane subunit gp41 is the portion of the Env protein complex that is most likely to interact with the internal structural proteins of the virus (2Gelderblom H.R. Hausmann E.H. Ozel M. Pauli G. Koch M.A. Virology. 1987; 156: 171-176Crossref PubMed Scopus (406) Google Scholar, 3Hunter E. Semin. Virol. 1994; 5: 71-83Crossref Scopus (165) Google Scholar, 4Freed E.O. Martin M.A. J. Virol. 1996; 70: 341-351Crossref PubMed Google Scholar, 5Cosson P. EMBO J. 1996; 15: 5783-5788Crossref PubMed Scopus (189) Google Scholar). Although the cytosolic domain of Env is absolutely required for viral dissemination in vivo it seems to be dispensable for envelope incorporation into virions and, consequently, for viral replication in vitro (6Wilk T. Pfeiffer T. Bosch V. Virology. 1992; 189: 167-177Crossref PubMed Scopus (139) Google Scholar). How newly assembled virions specifically acquire Env remains therefore largely unknown. An intriguing characteristic of the Env proteins of HIV-1 and simian immunodeficiency virus is that they undergo rapid endocytosis after their transport to the cell surface (7Rowell J.F. Stanhope P.E. Siliciano R.F. J. Immunol. 1995; 155: 473-488PubMed Google Scholar, 8Sauter M.M. Pelchen-Matthews A. Bron R. Marsh M. LaBranche C.C. Vance P.J. Romano J. Haggarty B.S. Hart T.K. Lee W.M. Hoxie J.A. J. Cell Biol. 1996; 132: 795-811Crossref PubMed Scopus (126) Google Scholar). As a consequence, the internal structural proteins of these viruses need to compete with the internalization machinery of the cell in order to acquire Env (9Egan M.A. Carruth L.M. Rowell J.F. Yu X. Siliciano R.F. J. Virol. 1996; 70: 6547-6556Crossref PubMed Google Scholar). 2S. Wyss and M. Thali, unpublished observation. 2S. Wyss and M. Thali, unpublished observation. Although the functional significance of this phenomenon is not understood, it is clear that Env behaves like other plasma membrane proteins that are rapidly internalized from the cell surface. Rapid internalization involves recruitment of plasma membrane proteins to clathrin-coated pits, a process that is mediated by interaction of endocytic signals found in the cytosolic domains of the proteins with the clathrin-associated adaptor complex AP-2 (10Mellman I. Annu. Rev. Cell Dev. Biol. 1996; 12: 575-625Crossref PubMed Scopus (1325) Google Scholar, 11Robinson M.S. Trends Cell Biol. 1997; 7: 99-102Abstract Full Text PDF PubMed Scopus (123) Google Scholar, 12Marks M.S. Ohno H. Kirchhausen T. Bonifacino J.S. Trends Cell Biol. 1997; 7: 124-128Abstract Full Text PDF PubMed Scopus (277) Google Scholar). The AP-2 complex consists of two large chains (α and β2), a medium chain (μ2), and a small chain (ς2). A direct interaction between μ2 and tyrosine-based sorting signals from the cytosolic domains of several cellular integral membrane proteins has been recently demonstrated (13Ohno H. Stewart J. Fournier M.C. Bosshart H. Rhee I. Miyatake S. Saito T. Gallusser A. Kirchhausen T. Bonifacino J.S. Science. 1995; 269: 1872-1875Crossref PubMed Scopus (814) Google Scholar, 14Boll W. Ohno H. Songyang Z. Rapoport I. Cantley L.C. Bonifacino J.S. Kirchhausen T. EMBO J. 1996; 15: 5789-5795Crossref PubMed Scopus (235) Google Scholar, 15Ohno H. Fournier M.-C. Poy G. Bonifacino J.S. J. Biol. Chem. 1996; 271: 29009-29015Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar). To assess the diverse functions of the cytosolic domain of Env including its role in internalization from the plasma membrane, we are analyzing its interaction with cellular and viral proteins. Anti-Env antibodies allowed us to co-immunoprecipitate Env with the AP-2 complex from HIV-1-infected lymphocytes, demonstrating that these proteins associate in vivo. Using GST-Env tail fusion proteins, we then identified the μ2 chain of AP-2 as a protein that interacts with the cytosolic domain of Env. Binding of μ2 to the cytosolic domain of Env was dependent on the presence and the context of a tyrosine-based sorting motif that is crucial for Env internalization (7Rowell J.F. Stanhope P.E. Siliciano R.F. J. Immunol. 1995; 155: 473-488PubMed Google Scholar), but it was also influenced by a glycine residue that had not previously been identified to be important for efficient endosomal sorting. Moreover, sequence as well as context dependence of μ2 binding to the cytosolic domain of Env was mimicked by the intact AP-2 complex. The results presented here suggest that the glutathioneS-transferase (GST) gene fusion system may be useful to analyze the interaction of the cytosolic domain of Env with other cellular or viral proteins. This system may also permit further dissection of μ2 functional regions and definition of the requirements for the binding of tyrosine-based sorting signals to adaptor complexes other than AP-2. All constructs used in the in vitro binding assays were made by ligation of polymerase chain reaction (Pwo DNA polymerase; Boehringer Mannheim)-amplified DNA fragments into pGEX-3X (Amersham Pharmacia Biotech). Primer sequences were as follows: 5′-AATCCCGGGATAGTTTTTGCTGTAC-3′ and 5′-CTTAAGACCACTTGCCACCCATCTTA-3′. The polymerase chain reaction products were phosphorylated and inserted intoSmaI-linearized pGEX-3X. The expression vectors 3M9 for μ2, pcwt for HIV-1 Env, and the deletion mutants of Env have been described (13Ohno H. Stewart J. Fournier M.C. Bosshart H. Rhee I. Miyatake S. Saito T. Gallusser A. Kirchhausen T. Bonifacino J.S. Science. 1995; 269: 1872-1875Crossref PubMed Scopus (814) Google Scholar, 16Gabuzda D.H. Lever A. Terwilliger E. Sodroski J. J. Virol. 1992; 66: 3306-3315Crossref PubMed Google Scholar, 17Thali M. Charles M. Furman C. Cavacini L. Posner M. Robinson J. Sodroski J. J. Virol. 1994; 68: 674-680Crossref PubMed Google Scholar); the latter were kindly provided by D. Gabuzda and J. Sodroski. Site-directed mutagenesis was performed either in the GST fusion constructs or in the Env expression vectors using the Quick Change™ system (Stratagene). The following primers were used: R709A, 5′-GTGAATAGAGTTGCGCAGGGATATTCAC-3′; Q710A, 5′-GTGAATAGAGTTAGGGCCGGCTATTCACCATTATCG-3′; G711A, 5′-GAGTTAGGCAGGCATATTCTCCATTATCG-3′; Y712A, 5′-GAGTTAGGCAGGGAGCTTCTCCATTATCG-3′; S713A, 5′-GAGTTAGGCAGGGATATGCACCATTATCG-3′; P714A, 5′-GTTAGGCAGGGATATTCAGCATTATCGTTTC-3′; L715A, 5′-GGCAGGGATATTCGCCGGCGTCGTTTCAGACC-3′; Y768A, 5′-GTGCCTCTTCAGCGCTCACCGCTTGAGAGAC-3′; Y768C, 5′-GTGCCTCTTCAGCTGCCACCGCTTGAG-3′. Mutations were confirmed by DNA sequencing. Chronically infected Jurkat cells were established by transfecting the cells with proviral DNA (HXB2 strain; Ref. 18). Cells surviving the acute phase of virus production were propagated for 4–6 weeks in RPMI 1640 medium supplemented with 10% fetal calf serum and antibiotics (100 units/ml penicillin and 100 μg/ml streptomycin). Virus production was monitored by p24 capture (Abbott) and by SDS-PAGE analysis of purified virions. To perform the immunoblot analysis, uninfected or infected cells were washed in PBS and then lysed in 500 μl of a Triton X-100-based lysis buffer (50 mm Hepes, pH 7.4, 1% Triton X-100, 10% glycerol, 100 mm NaCl, 10 mm NaF, and 1 mm EDTA) essentially as described (19Nesterov A. Kurten R.C. Gill G.N. J. Biol. Chem. 1995; 270: 6320-6327Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). Precleared lysates were preincubated for 1.5 h with 50 μl of Protein A-Sepharose CL-4B beads (Amersham Pharmacia Biotech). Env was precipitated using the anti-Env monoclonal antibody b12 (20Roben P. Moore J. Thali M. Sodroski J. Barbas III, C.F. Burton D. J. Virol. 1994; 71: 6869-6874Google Scholar), kindly provided by Dennis Burton, immobilized on Protein A-Sepharose CL-4B overnight at 4 °C. After SDS-PAGE separation, the precipitated material was transferred onto nitrocellulose membranes. Those membranes were then probed with Sigma antibodies 100/2 (anti-α-adaptin) or 100/1 (anti-β-adaptin). Bound antibodies were visualized after binding of secondary antibodies by enhanced chemiluminescence (Pierce). GST fusion proteins were produced in Escherichia coli strain TB1 and purified on glutathione-Sepharose 4B (Amersham Pharmacia Biotech). Precultures (4 ml of LB medium supplemented with 0.5 md-sorbitol and 2.5 mm betaine) were grown overnight at 37 °C. They were transferred to 400 ml of LB medium supplemented with 1 md-sorbitol and 2.5 mm betaine and grown for 8 h at 37 °C. Cultures were shifted to 25 °C and induced overnight with 0.1 mmIsopropyl-β-d-thiogalactoside. The purification on glutathione-Sepharose 4B beads was performed according to the manufacturer's instructions including a heat shock step with 50 mm Tris-HCl (pH 7.4), 10 mm MgSO4, and 2 mm ATP for 10 min at 37 °C. [35S]Methionine-labeled μ2 was prepared using a coupled transcription-translation system (TNT®, Promega,). Precleared in vitrotranslated μ2 (2 μl) was incubated with 5 μg of GST fusion protein in 500 μl of binding buffer (0.05% (w/v) Triton X-100, 50 mm HEPES (pH 7.3), 0.1 mm CaCl2, 2 mm MgCl2, 100 mm KCl, 50 μm dithiothreitol, 10% (w/v) glycerol, 0.1% bovine serum albumin) for 2 h at room temperature. Loading buffer was added to washed beads, and the samples were run on SDS-PAGE. The amount of μ2 bound to the different GST fusion protein mutants was determined by measuring the radioactivity of the signals using the Instant ImagerTM system (Packard). T cell lysate was prepared from Jurkat cells essentially as described (21Dietrich J. Kastrup J. Nielsen B.L. Odum N. Geisler C. J. Cell Biol. 1997; 138: 227-281Crossref Scopus (155) Google Scholar). Briefly, the cells were washed three times with cytosol buffer (25 mm Hepes, pH 7.0, 125 mmCH3COOK, 2.5 mm(CH3COO)2Mg, 1.0 mm dithiothreitol, 1 mg/ml glucose), and the resulting pellet was resuspended in an equal volume of cytosol buffer containing 1 mmphenylmethylsulfonyl fluoride. The cell suspension was frozen in liquid nitrogen, thawed on ice, and drawn five times through a 21-gauge syringe. After centrifugation for 30 min at 20,000 ×g, 4 °C, the supernatant was transferred to new tubes in 100-μl aliquots and stored at −80 °C. Aliquots were precleared in 900 μl of PBS containing 1.5 mg of GST proteins on Sepharose 4B beads at 35 °C for 30 min. 300 μl of precleared lysate was incubated with 100 μg of fusion protein on Sepharose 4B beads at 35 °C for 2 h. Beads were washed three times with PBS and boiled in gel sample buffer. Afterward, SDS-PAGE proteins were transferred to nitrocellulose membranes and probed with Sigma antibody 100/2 (anti-α -adaptin) or 100/1 (anti-β -adaptin). Bound antibodies were visualized after binding of secondary antibodies by enhanced chemiluminescence (Pierce). Twenty-four hours after transfection with the Env expression plasmids, HeLa cells were metabolically labeled for 16 h with [35S]cysteine (50 μCi/ml). The labeled cells were chilled on ice and biotinylated for 30 min in 1 ml of PBS containing 1.5 mg/ml NHS-SS-biotin (Pierce). After three washes with PBS, 50 mm glycine, the cells were returned to 37 °C for different periods of time to allow for endocytosis to occur. To remove biotin from surface proteins that had not been internalized, cells were again chilled on ice and incubated twice for 15 min with 50 mm glutathione and for an additional 15 min with 60 mm 2-mercaptoethanesulfonic acid (MESNa). Free sulfhydryl groups were blocked by washing the cells three times with 50 mm glycine in PBS. The cells were then lysed in 1× radioimmune precipitation buffer, and viral proteins were immunoprecipitated with serum from an individual infected with HIV-1. Following immunoprecipitation, 90% of the material was boiled in 10% SDS, while the remaining 10% of the material was analyzed directly by SDS-PAGE to assess the amount of total Env in each sample. Biotinylated Env was recovered with streptavidin-agarose, followed by SDS-PAGE. The intensity of the Env signal was quantified using the Instant ImagerTM system. The proportion of internalized Env was determined by subtracting for each time point the activity of the glutathione/MESNa-resistant biotin-tagged Env at 0 °C from the activity of the glutathione/MESNa-resistant biotin-tagged Env at 37 °C and dividing the remaining activity by one-hundredth of the activity of Env from cells that had not undergone reduction. The overall goal of our work is to identify proteins interacting with the cytosolic domain of HIV-1 Env. Since Env is rapidly retrieved from the cell surface after its transport to the plasma membrane (7Rowell J.F. Stanhope P.E. Siliciano R.F. J. Immunol. 1995; 155: 473-488PubMed Google Scholar), we tested whether Env interacts with the clathrin-associated protein complex AP-2. As shown in Fig. 1, AP-2 was detected in anti-Env immunoprecipitates from chronically infected lymphocytes but not in anti-Env immunoprecipitates from noninfected cells. A detailed analysis of the requirements for the observed Env-AP-2 interaction cannot be performed in infected cells, because many mutations in Env will affect the efficiency of viral replication and consequently also the levels of Env expression. Env-AP-2 interactions were therefore analyzed in more detail in vitro. The cytosolic domain of Env contains two motifs that strongly resemble tyrosine-based endocytosis signals, one at position 712 (Y712SPL) and the other at position 768 (Y768HRL) (Fig. 3 A). Both motifs are well conserved among different strains of HIV-1 (18). Recently, the medium chain (μ2) of the clathrin-associated protein complex AP-2 was found to interact with tyrosine-based signals conforming to the canonical motif YXXØ, where X is any amino acid and Ø is a an amino acid with a bulky hydrophobic side chain (13Ohno H. Stewart J. Fournier M.C. Bosshart H. Rhee I. Miyatake S. Saito T. Gallusser A. Kirchhausen T. Bonifacino J.S. Science. 1995; 269: 1872-1875Crossref PubMed Scopus (814) Google Scholar, 14Boll W. Ohno H. Songyang Z. Rapoport I. Cantley L.C. Bonifacino J.S. Kirchhausen T. EMBO J. 1996; 15: 5789-5795Crossref PubMed Scopus (235) Google Scholar, 15Ohno H. Fournier M.-C. Poy G. Bonifacino J.S. J. Biol. Chem. 1996; 271: 29009-29015Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar). Therefore, we decided to test whether the cytosolic domain of Env of HIV-1 associates with μ2. The cytosolic tail was expressed as a GST fusion protein (GST-EnvCD) in E. coli. The medium chain (μ2) of the clathrin-associated protein complex AP-2 was translatedin vitro in a rabbit reticulocyte lysate. Fig. 2 shows that μ2 bound to GST-EnvCD but not to GST. For comparison, one-tenth of the in vitrotranslated μ2 is shown in the first lane. Typically, about 5–15% of the in vitro translated μ2 bound to GST-EnvCD.Figure 2Binding of the cytosolic domain of HIV-1 Env and μ2. In vitro translated [35S]Met-labeled μ2 was incubated for 2 h with bacterially expressed GST (lane 3) or a fusion protein consisting of the whole cytosolic domain of the HIV-1 envelope glycoprotein fused to GST (GST-EnvCD, lane 4). Binding of μ2 to the immobilized recombinant proteins was analyzed on SDS-PAGE. For comparison, one-tenth of the in vitro produced μ2 was applied to lane 2.View Large Image Figure ViewerDownload (PPT) To identify the binding site for μ2 in the cytosolic domain of Env, we tested the interaction of μ2 with various Env tail deletion mutants (Fig. 3 B and data not shown). Deletions that did not affect the membrane-proximal tyrosine motif Y712SPL had little or no effect on the association of μ2 with the cytosolic tail. Deletion of a region containing this membrane-proximal tyrosine-based motif, however, severely reduced the binding of μ2 to the cytosolic tail. In contrast, a deletion of region containing the motif Y768HRL had less of an effect. A more detailed analysis of the requirements for μ2 binding was then performed using mutants where single amino acids were mutated to alanine (Fig. 3 C). As expected from the results shown in Fig. 3 B, mutation of Tyr768 had very little impact on the binding of μ2 to the cytosolic domain of Env. Similarly, substitutions of amino acids Arg709, Gln710, or Ser713, respectively, by alanine influenced binding of μ2 only marginally. In contrast, binding of μ2 was decreased by mutation of Gly711 or Pro714 and even more dramatically by mutation of Tyr712 or Leu715. Thus, residues in the membrane-proximal tyrosine-based motif but not in the membrane-distal one are critical for the binding. Although the specificity of the binding of μ2 to the cytosolic domain of Env of HIV-1 was apparent from the effects on binding of the deletion mutants and single amino acid substitutions, we corroborated these results by testing the ability of different peptides to inhibit the binding of μ2 to EnvCD. If protein-protein complexes were allowed to form in the presence of the peptide RRQGYSPL, binding of μ2 was inhibited in a dose-dependent manner. No inhibition was observed if binding was performed in the presence of the peptide RRQGASPL, further confirming the specificity of this interaction (Fig. 4). Most interestingly, the peptide RRFSYHRL, the sequence of which conforms to the distal tyrosine-based signal, also interfered with the binding of μ2, while the tyrosine-mutated version of the same peptide did not. Thus, while the sequence YHRL within the context of the complete cytosolic domain of Env contributed very little to the binding of μ2 (see Fig. 3), it still was a potent μ2 binding element in the form of a small peptide. To test whether the same region of the cytosolic domain of Env important for μ2 binding represents the binding motif for intact AP-2 complexes, beads coated with some of our GST-EnvCD fusion proteins were incubated with cytosol of T cells and washed three times, and bound material was eluted. After SDS-PAGE, immunoblotting was performed using antibodies against either of the two large chains of the AP-2 complex (Fig. 5). Whereas the wild-type EnvCD, as well as the Y768C mutant, precipitated α and β-adaptin, the Y712A mutant did not. Substitution of Gly711 for alanine, however, still allowed for the precipitation of some α- and β-adaptin. These results demonstrate that the association of not only the isolated medium chain μ2 but also the intact AP-2 complex with the cytosolic domain of Env is critically dependent on the integrity of the membrane-proximal sequence Y712SPL. In addition, Gly711 also contributes to the binding of EnvCD to AP-2. The functional importance of some residues in the cytosolic domain of Env in its sorting to intracellular compartments was assessed using a biochemical internalization assay (see “Experimental Procedures”). None of the deletion mutants was tested for endocytosis, because the cytosolic domain of Env partially overlaps with the viral Rev function, which is necessary for Env expression in intact cells (22Hammarskjöld M.-L. Semin. Cell Dev. Biol. 1997; 8: 83-90Crossref PubMed Scopus (42) Google Scholar). Fig. 6shows the results of our internalization studies for three of the mutants where single amino acids had been substituted. The same mutations in the cytosolic domain of Env that led to a decrease in association with μ2 and the intact AP-2 complex (see Figs. 3 C and 5, respectively) also reduced Env internalization. On the other hand, mutation of Tyr768, which had little effect on μ2/AP-2 binding, did not affect endocytosis of Env. Using a fluorescence-activated cell sorting-based endocytosis assay, Siliciano and colleagues (7Rowell J.F. Stanhope P.E. Siliciano R.F. J. Immunol. 1995; 155: 473-488PubMed Google Scholar) had already demonstrated that the Tyr712in the membrane-proximal motif but not Tyr768 in the membrane-distal motif was important for internalization of Env from the cell surface. Our data thus confirm those results and also show that glycine at position −1 with respect to the membrane-proximal Y712SPL motif (Gly711) influences the recruitment of Env into clathrin-coated pits. Most importantly, these data establish a correlation between the binding of the cytosolic domain of Env to μ2, as well as to the intact AP2 complex, in vitro and the efficiency of Env internalization in vivo. The intracellular domain of HIV-1 Env is absolutely required for viral dissemination, but very little is known about its functions. This prompted us to look for proteins interacting with the cytosolic domain of Env. Here we report that the medium chain μ2 of the clathrin-associated protein complex AP-2 as well as the complete AP-2 complex binds the cytosolic domain of Env fused to GST. Furthermore, Env and AP-2 form a complex in vivo, i.e. in infected lymphocytes. Binding of μ2/AP-2 to the cytosolic domain of Env correlates well with Env internalization from the cell surface. Such fusion proteins may therefore be useful not only as probes to identify other proteins interacting with the cytosolic domain of Env but also to analyze the requirements of functional interactions of the adaptor protein complexes or its subunits with the cytosolic domain of Env. Determinants in the cytosolic domain of Env have been implicated in the directed release of HIV-1 observed in polarized epithelial cells as well as in the retrieval of Env from the cell surface (7Rowell J.F. Stanhope P.E. Siliciano R.F. J. Immunol. 1995; 155: 473-488PubMed Google Scholar, 8Sauter M.M. Pelchen-Matthews A. Bron R. Marsh M. LaBranche C.C. Vance P.J. Romano J. Haggarty B.S. Hart T.K. Lee W.M. Hoxie J.A. J. Cell Biol. 1996; 132: 795-811Crossref PubMed Scopus (126) Google Scholar, 23Lodge R. Gottlinger H. Gabuzda D. Cohen E.A. Lemay G. J. Virol. 1994; 68: 4857-4861Crossref PubMed Google Scholar, 24Lodge R. Lalonde J.P. Lemay G. Cohen E.A. EMBO J. 1997; 16: 695-705Crossref PubMed Scopus (154) Google Scholar). In both instances, a membrane-proximal tyrosine (Tyr712) was demonstrated to be part of the signal(s) responsible for the respective sorting process. Tyrosines have been identified as critical components of sorting signals in the cytosolic domains of different cellular membrane proteins destined for various cellular compartments (10Mellman I. Annu. Rev. Cell Dev. Biol. 1996; 12: 575-625Crossref PubMed Scopus (1325) Google Scholar, 12Marks M.S. Ohno H. Kirchhausen T. Bonifacino J.S. Trends Cell Biol. 1997; 7: 124-128Abstract Full Text PDF PubMed Scopus (277) Google Scholar). Together, these data led us to investigate whether the cytosolic domain of Env interacts with elements of the cellular sorting machinery. As a probe, we used a protein where GST was fused to the cytosolic domain of Env. It was demonstrated previously that GST fusion proteins containing tyrosine-based sorting signals can bind proteins involved in the sorting of cellular membrane proteins (13Ohno H. Stewart J. Fournier M.C. Bosshart H. Rhee I. Miyatake S. Saito T. Gallusser A. Kirchhausen T. Bonifacino J.S. Science. 1995; 269: 1872-1875Crossref PubMed Scopus (814) Google Scholar). Here, we demonstrated that the cytosolic domain of Env in the context of a GST fusion protein binds to μ2. Such an interaction between the whole cytosolic domain of Env and μ2 was not observed in the yeast two-hybrid system, 3H. Ohno and J. Bonifacino, unpublished observations. thus demonstrating the usefulness of the in vitro binding approach in order to study these interactions. Having established that the cytosolic domain of Env in the context of a GST fusion protein can bind to μ2, we sought to define the requirements for this interaction. The cytosolic domain of Env contains two YXXØ motifs. Both motifs are highly conserved among almost all primary viral isolates analyzed so far (18), indicating that they belong to functionally important regions of this domain. The results of the present study demonstrate that only the membrane-proximal tyrosine-based motif Y712SPL, but not the more C-terminally located motif Y768HRL, is important for μ2 binding. The lack of a contribution of the YHRL motif to μ2 binding was not due to a lower intrinsic affinity of this motif for μ2. Using the yeast two-hybrid system, Ohno et al. (25Ohno H. Aguilar R.C. Fournier M.C. Hennecke S. Cosson P. Bonifacino J.S. Virology. 1997; 238: 305-315Crossref PubMed Scopus (152) Google Scholar) recently showed that the membrane-distal motif as such can bind to μ2. Our peptide competition experiments shown in Fig. 4 indicated that the YHRL motif may have even a slightly higher affinity for μ2. The results of the in vitro binding assays presented here thus suggest that μ2 cannot access the YHRL motif in the context of the whole cytosolic domain. Support for this hypothesis is provided by the analysis of Env internalization (Fig. 6). Our analysis as well as a previous study (7Rowell J.F. Stanhope P.E. Siliciano R.F. J. Immunol. 1995; 155: 473-488PubMed Google Scholar) found that tyrosine at position 712 but not tyrosine at position 768 is critical for Env internalization. Remarkably, the correlation between binding of μ2 to the cytosolic domain of Env and endocytosis of Env holds even for the Gly711 to Ala mutant. Glycine at position −1 relative to the critical Y712SPL motif has not been previously recognized to influence the internalization of membrane receptors. Mutant G711A showed an intermediate phenotype in the μ2 binding assay as well as in the endocytosis assay. Together, our data suggest that binding of μ2 to the cytosolic domain of Env defines the specificity for the recruitment of the protein by the AP-2 complex. Therefore, we tested whether the cytosolic domain of Env also associates with the intact AP-2 complexin vitro and, if so, whether the specificity of binding to EnvCD was the same for μ2 and the intact complex. Our results showed, first, that EnvCD-AP-2 association was likewise dependent on the intactness of the membrane-proximal but not of the membrane-distal tyrosine-based motif. Second, we could reproducibly precipitate the large chains α- and β-adaptin of AP-2 with the Gly711to alanine mutant, although its interaction with the intact AP-2 complex may be weaker than its association with μ2. Therefore, the strength of interaction of μ2 with cognate signals in the cytosolic domains of membrane proteins may be modulated by an interaction of μ2 with the other subunits of AP-2. Appropriate spacing of the tyrosine signal was shown to be crucial for the sorting of a cellular protein to lysosomes (26Rohrer J. Schweizer A. Russell D. Kornfeld S. J. Cell Biol. 1996; 132: 565-576Crossref PubMed Scopus (215) Google Scholar), presumably mediated by the AP-1 complex. A possible explanation for the different recognition by μ2/AP-2 of the Y712SPL motif and the Y768HRL motif may thus be due to different placement relative to the plasma membrane. Alternatively, folding of the cytosolic domain of Env may be such that Y768HRL is not accessible to μ2/AP-2. The Y768HRL motif is positioned within one of two amphipathic regions of the cytosolic domain of Env that have been proposed to associate with the inner face of the plasma membrane (27Haffar O.K. Dowbenko D.J. Berman P.W. Virology. 1991; 180: 439-441Crossref PubMed Scopus (35) Google Scholar, 28Srinivas S.K. Srinivas R.V. Anantharamaiah G.M. Segrest J.P. Compans R.W. J. Biol. Chem. 1992; 267: 7121-7127Abstract Full Text PDF PubMed Google Scholar). It could thus be that the conformational context of the Y768HRL motif in vivo prevents its recognition by the AP-2 complex in cells. The situation may be different for the region close to the membrane-spanning domain of Env. This region is predicted to form neither α-helices nor β-sheets (29Rost B. Methods Enzymol. 1996; 266: 525-539Crossref PubMed Google Scholar). The Y712SPL motif within that context may thus adopt the “tight turn” structure, which was predicted to be the conformational determinant shared by tyrosine-based internalization signals (30Trowbridge I.S. Collawn J.F. Hopkins C.R. Annu. Rev. Cell Biol. 1993; 9: 129-161Crossref PubMed Scopus (700) Google Scholar). Our results do not allow us to exclude the possibility that the membrane-distal motif contributes to the EnvCD-AP-2 binding but that the interaction with Tyr768 is dependent on the interaction of AP-2 with the membrane-proximal motif. However, since the Tyr768 to Cys mutation does not affect Env internalization (Fig. 6), we do not consider this to be a very likely scenario. Preliminary results suggest that the described Env fusion proteins not only bind with remarkable specificity to μ2/AP-2 but that they can also selectively interact with other cellular sorting complexes. Work is in progress to test whether the association with those complexes also correlates with the in vivo sorting of Env. Such an analysis is particularly desirable with regard to the membrane-proximal region of the Env tail, which is involved not only in Env endocytosis (Ref. 7Rowell J.F. Stanhope P.E. Siliciano R.F. J. Immunol. 1995; 155: 473-488PubMed Google Scholar and this study) but also in the polarized sorting of Env (24Lodge R. Lalonde J.P. Lemay G. Cohen E.A. EMBO J. 1997; 16: 695-705Crossref PubMed Scopus (154) Google Scholar). This situation is reminiscent of the co-linearity of endocytosis signals and basolateral sorting signals that is observed in a number of cellular proteins (31Rodriguez-Boulan E. Zurzolo C. J. Cell. Sci. 1993; 17: 9-12Crossref Google Scholar). In summary, these studies show that the μ2 subunit of AP-2 as well as the intact AP-2 complex bind to a tyrosine-based signal within cytosolic domain of Env involved in endocytosis. A second tyrosine-based motif in the cytosolic tail of Env that is not relevant for Env internalization seems to be largely shielded from recognition by μ2/AP-2. In all, our data describe a case where components of the cellular sorting machinery discriminate between two potential binding sites based on their position in the cytosolic domain of the protein. Thus, our data demonstrate that it is not possible to predict if a putative μ2 binding sequence will function in vivo, based on in vitro studies alone, underscoring the importance of corroborating data from binding experiments with functional studiesin vivo. We thank Walter Hunziker for critical reading of the manuscript and discussions, Kenneth Raj for useful technical hints, D. Gabuzda and J. Sodroski for HIV-1 Env deletion mutants, and Dennis Burton for the gift of antibody b12." @default.
• W2009205890 created "2016-06-24" @default.
• W2009205890 creator A5002533467 @default.
• W2009205890 creator A5008968738 @default.
• W2009205890 creator A5072633844 @default.
• W2009205890 creator A5081989475 @default.
• W2009205890 date "1998-06-01" @default.
• W2009205890 modified "2023-10-12" @default.
• W2009205890 title "A Membrane-proximal Tyrosine-based Signal Mediates Internalization of the HIV-1 Envelope Glycoprotein via Interaction with the AP-2 Clathrin Adaptor" @default.
• W2009205890 cites W1499450468 @default.
• W2009205890 cites W1542639585 @default.
• W2009205890 cites W1545718647 @default.
• W2009205890 cites W1547371126 @default.
• W2009205890 cites W1632486555 @default.
• W2009205890 cites W170446279 @default.
• W2009205890 cites W1978689611 @default.
• W2009205890 cites W1979815209 @default.
• W2009205890 cites W1980219322 @default.
• W2009205890 cites W1983561321 @default.
• W2009205890 cites W2014876277 @default.
• W2009205890 cites W2019469658 @default.
• W2009205890 cites W2024764884 @default.
• W2009205890 cites W2032232188 @default.
• W2009205890 cites W2058032164 @default.
• W2009205890 cites W2071846646 @default.
• W2009205890 cites W2074333462 @default.
• W2009205890 cites W2092072942 @default.
• W2009205890 cites W2108623121 @default.
• W2009205890 cites W2116795517 @default.
• W2009205890 cites W2131134035 @default.
• W2009205890 cites W2134344512 @default.
• W2009205890 cites W2151057922 @default.
• W2009205890 cites W2152517932 @default.
• W2009205890 cites W2163519490 @default.
• W2009205890 cites W2178812731 @default.
• W2009205890 cites W2248336029 @default.
• W2009205890 cites W2320549884 @default.
• W2009205890 doi "https://doi.org/10.1074/jbc.273.25.15773" @default.
• W2009205890 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/9624176" @default.
• W2009205890 hasPublicationYear "1998" @default.
• W2009205890 type Work @default.
• W2009205890 sameAs 2009205890 @default.
• W2009205890 citedByCount "185" @default.
• W2009205890 countsByYear W20092058902012 @default.
• W2009205890 countsByYear W20092058902013 @default.
• W2009205890 countsByYear W20092058902014 @default.
• W2009205890 countsByYear W20092058902015 @default.
• W2009205890 countsByYear W20092058902016 @default.
• W2009205890 countsByYear W20092058902017 @default.
• W2009205890 countsByYear W20092058902018 @default.
• W2009205890 countsByYear W20092058902019 @default.
• W2009205890 countsByYear W20092058902020 @default.
• W2009205890 countsByYear W20092058902021 @default.
• W2009205890 countsByYear W20092058902022 @default.
• W2009205890 countsByYear W20092058902023 @default.
• W2009205890 crossrefType "journal-article" @default.
• W2009205890 hasAuthorship W2009205890A5002533467 @default.
• W2009205890 hasAuthorship W2009205890A5008968738 @default.
• W2009205890 hasAuthorship W2009205890A5072633844 @default.
• W2009205890 hasAuthorship W2009205890A5081989475 @default.
• W2009205890 hasBestOaLocation W20092058901 @default.
• W2009205890 hasConcept C108625454 @default.
• W2009205890 hasConcept C12554922 @default.
• W2009205890 hasConcept C139770010 @default.
• W2009205890 hasConcept C1491633281 @default.
• W2009205890 hasConcept C181199279 @default.
• W2009205890 hasConcept C183786373 @default.
• W2009205890 hasConcept C185592680 @default.
• W2009205890 hasConcept C2776165026 @default.
• W2009205890 hasConcept C28005876 @default.
• W2009205890 hasConcept C55493867 @default.
• W2009205890 hasConcept C62478195 @default.
• W2009205890 hasConcept C63162447 @default.
• W2009205890 hasConcept C86803240 @default.
• W2009205890 hasConcept C95444343 @default.
• W2009205890 hasConcept C98539663 @default.
• W2009205890 hasConceptScore W2009205890C108625454 @default.
• W2009205890 hasConceptScore W2009205890C12554922 @default.
• W2009205890 hasConceptScore W2009205890C139770010 @default.
• W2009205890 hasConceptScore W2009205890C1491633281 @default.
• W2009205890 hasConceptScore W2009205890C181199279 @default.
• W2009205890 hasConceptScore W2009205890C183786373 @default.
• W2009205890 hasConceptScore W2009205890C185592680 @default.
• W2009205890 hasConceptScore W2009205890C2776165026 @default.
• W2009205890 hasConceptScore W2009205890C28005876 @default.
• W2009205890 hasConceptScore W2009205890C55493867 @default.
• W2009205890 hasConceptScore W2009205890C62478195 @default.
• W2009205890 hasConceptScore W2009205890C63162447 @default.
• W2009205890 hasConceptScore W2009205890C86803240 @default.
• W2009205890 hasConceptScore W2009205890C95444343 @default.
• W2009205890 hasConceptScore W2009205890C98539663 @default.
• W2009205890 hasIssue "25" @default.
• W2009205890 hasLocation W20092058901 @default.
• W2009205890 hasOpenAccess W2009205890 @default.
• W2009205890 hasPrimaryLocation W20092058901 @default.
• W2009205890 hasRelatedWork W1934518428 @default.
• W2009205890 hasRelatedWork W1970268623 @default.
• W2009205890 hasRelatedWork W2009043160 @default.

• next