Developmentally regulated expression of P-glycoprotein (multidrug resistance) activity in mouse thymocytes.

P-glycoprotein (P-gly) is the transmembrane efflux pump responsible for multidrug resistance in tumor cells. The activity of P-gly in mature peripheral lymphocytes is lineage specific, with CD8+ T cells and natural killer (NK) cells expressing high levels as compared to CD4+ T cells and B cells. We have now investigated P-gly activity in immature and mature subsets of mouse thymocytes. Our data indicate that P-gly activity is undetectable in immature CD4-8- and CD4+8+ thymocyte subsets. Among mature thymocytes, P-gly activity is absent in the CD4+ subset but present in the more mature (HSAlow) fraction of CD8+ cells. Furthermore, while thymic CD4-8- T cell receptor (TCR) gamma delta cells have little P-gly activity, a minor subset of CD4-8- or CD4+ TCR alpha beta + thymocytes bearing the NK1.1 surface marker expresses high levels of P-gly activity. Collectively, our results indicate that P-gly activity arises late during thymus development and is expressed in a lineage-specific fashion.

The role of proteolytic processing and the stable signal peptide in expression of the Old World arenavirus envelope glycoprotein ectodomain.

Maturation of the arenavirus GP precursor (GPC) involves proteolytic processing by cellular signal peptidase and the proprotein convertase subtilisin kexin isozyme 1 (SKI-1)/site 1 protease (S1P), yielding a tripartite complex comprised of a stable signal peptide (SSP), the receptor-binding GP1, and the fusion-active transmembrane GP2. Here we investigated the roles of SKI-1/S1P processing and SSP in the biosynthesis of the recombinant GP ectodomains of lymphocytic choriomeningitis virus (LCMV) and Lassa virus (LASV). When expressed in mammalian cells, the LCMV and LASV GP ectodomains underwent processing by SKI-1/S1P, followed by dissociation of GP1 from GP2. The GP2 ectodomain spontaneously formed trimers as revealed by chemical cross-linking. The endogenous SSP, known to be crucial for maturation and transport of full-length arenavirus GPC was dispensable for processing and secretion of the soluble GP ectodomain, suggesting a specific role of SSP in the stable prefusion conformation and transport of full-length GPC.

Regulation of P-selectin glycoprotein ligand 1 (PSGL-1) interactions with selectins : role of the decameric repeats and of the cytoplasmic domain

SUMMARY BACKGROUND: P-selectin glycoprotein ligand 1 (PSGL-1) is a major selectin ligand, mediating leukocyte rolling along inflamed vascular wall. It is a mucin-like homodimer composed of a N-terminal domain which binds selectins, followed by 14-16 decameric repeats (DR), a transmembrane domain and a cytoplasmic tail, which may be involved in regulating leukocyte rolling and in generating intracellular signals, through its binding to moesin and Syk. P- and L-selectin binding is dependent on core-2 O-glycosylation and tyrosine sulfation of PSGL-1 N-terminus. However, a minor part of E-selectin-mediated rolling is dependent on N-terminal O-glycans; additional binding sites may thus be involved. In this project, we studied whether (1) PSGL-1 DR and (2) PSGL-1 cytoplasmic residues which bind moesin, were also involved in the regulation of selectin-dependent rolling. METHODS: Several mutated cDNAs were obtained: (1) PSGL-1 DR were either deleted, or substituted by platelet GPlba macroglycopeptide, (2) Ser-336, -348, Lys-337 and Arg-338 were mutated to alanine; moreover, truncation mutants retaining only 6 or 2 cytoplasmic residues were also generated. Transfected CHO expressing mutant PSGL-1 were tested for their ability to bind soluble selectin chimeras and to support selectin-dependent rolling under flow conditions. RESULTS: (1) Deletion of the DR had a dramatic effect on P- and L-selectin-dependent cell recruitment and rolling stability, which could only partially be compensated for, by GPlba substitution. In addition, we observed that DR create a binding site for E-selectin and thus support PSGL-1-dependent rolling. (2) Flow assays revealed that the moesin-binding site, in particular Ser-336, plays a crucial role in regulating the recruitment, velocity and rolling stability of PSGL-1-expressing cells on P- and L-selectin. CONCLUSIONS: Data presented here highlight the structure -function relationship of PSGL-1 DR. Moreover, they reveal a crucial role for the moesin-binding residues in regulating P-and L-selectin-dependent rolling. RÉSUMÉ CONTEXTE: PSGL-1 (P-selectin glycoprotein ligand 1) est un ligand majeur des sélectines permettant le roulement des leucocytes le long de la paroi vasculaire enflammée. C'est un homodimère de type mucine, composé d'un domaine N-terminal liant les sélectines, suivi de 14-16 répétitions décamèriques (RD), d'un domaine transmembranaire et d'une queue cytoplasmique qui pourrait être impliquée dans la régulation du roulement leucocytaire et la génération de signaux intracellulaires, via sa liaison à la moésine et à Syk. La liaison à la Pet à la L-sélectine dépend de la présentation par le N-terminus de PSGL-1 de O-glycans sur des structures core-2 et de tyrosines sulfatées. Cependant, une fraction mineure du roulement médié par la E-sélectine dépend des O-glycans N-terminaux; des sites de liaisons supplémentaires pourraient donc être impliqués. Dans ce projet, nous avons étudié si (1) les RD de PSGL-1 ainsi que (2) les résidus cytoplasmiques liant la moésine, étaient impliqués dans la régulation du roulement dépendant des sélectines. MÉTHODES: Plusieurs ADN codant des formes mutées de PSGL-1 ont été obtenus: (1) Les RD de PSGL-1 ont été soit ôtées, soit remplacées par le macroglycopeptide de la GPlba plaquettaire, (2) les Ser-336, -348, la Lys-337 et l'Arg-338 ont été mutées en alanine; par ailleurs, des mutants tronqués ne retenant plus que 6 ou 2 résidus cytoplasmiques ont également été générés. Des CHO transfectées exprimant PSGL-1 muté ont été testées pour leur capacité à lier des sélectines chimériques solubles et à soutenir un roulement dépendant des sélectines dans des conditions de flux. RÉSULTATS: (1) La perte des RD a eu un effet dramatique sur le recrutement cellulaire et la stabilité de roulement dépendant des P- et L-sélectine, qui n'a pu être que partiellement compensé par la substitution par la GPlba. De plus, nous avons observé que les RD forment un site de liaison pour la E-sélectine et soutiennent ainsi le roulement dépendant de PSGL-1. (2) Les tests de flux ont révélé que le site de liaison à la moésine, notamment la Ser-336, joue un rôle crucial dans la régulation du recrutement, de la vitesse et de la stabilité du roulement des cellules exprimant PSGL-1 sur les P- et L-sélectine. CONCLUSIONS; Les données présentées ici ont permis d'éclaircir la relation structure -fonction des RD de PSGL-1. Par ailleurs, elles révèlent un rôle crucial pour les résidus liant la moésine dans le roulement dépendant des P- et L-sélectine. RÉSUMÉ DESTINÉ À UN LARGE PUBLIC Pour accomplir ses fonctions, le sang circule sur un réseau de 96'000 kilomètres; ainsi, il approvisionne les cellules de l'organisme en énergie, il transporte diverses substances, il assure la défense contre les pathogènes et il participe à la régulation de la température corporelle. Le sang contient plusieurs types de cellules: la grande majorité sont les globules rouges, auxquels il faut ajouter les plaquettes (dont le rôle est de colmater les lésions vasculaires) et les globules blancs (leucocytes) qui, bien que présents en très faible quantité (moins de 0.01 %), jouent un rôle crucial en cas d'infection ou d'inflammation. Une attaque par un pathogène provoque plusieurs changements (rougeur, chaleur, gonflement, douleur), qui sont des manifestations de l'inflammation. Pour atteindre l'agent infectieux, des globules blancs spécialisés (les granulocytes) doivent quitter la circulation sanguine. Afin de faciliter leur capture, les vaisseaux sanguins vont exprimer des protéines telles que les sélectines, qui sont reconnues par une protéine leucocytaire appelée PSGL-1 (P-selectin glycoprotein ligand 7). L'interaction des sélectines avec PSGL-1 soutient le roulement du globule blanc le long de la paroi vasculaire, à une vitesse très inférieure à celle du flux sanguin. Ce roulement conduit à l'activation du globule blanc par des molécules de l'inflammation, permettant son adhésion ferme, puis son arrêt. Finalement, le granulocyte va migrer à travers la paroi du vaisseau pour atteindre et éliminer les causes de l'inflammation. L'adhésion est un processus intéressant à caractériser, car outre l'inflammation, il est également impliqué dans l'artériosclérose, l'infarctus, la métastatisation et la thrombose. Dans ce travail, nous nous sommes intéressés à définir les rôles des différents domaines de PSGL-1 dans la régulation de son interaction avec les sélectines. En effet, en plus de son extrémité extracellulaire de haute affinité pour les sélectines, PSGL-1 est composé de plusieurs séquences répétées hautement glycosylées et d'une courte région intracellulaire, dont les fonctions n'avaient pas été étudiées auparavant. En créant des formes mutées de PSGL-1, nous avons pu montrer qu'un roulement efficace des leucocytes nécessite la présence des régions répétitives et du domaine intracellulaire au complet.

Molecular characterization of the processing of arenavirus envelope glycoprotein precursors by subtilisin kexin isozyme-1/site-1 protease.

A crucial step in the life cycle of arenaviruses is the biosynthesis of the mature fusion-active viral envelope glycoprotein (GP) that is essential for virus-host cell attachment and entry. The maturation of the arenavirus GP precursor (GPC) critically depends on proteolytic processing by the cellular proprotein convertase (PC) subtilisin kexin isozyme-1 (SKI-1)/site-1 protease (S1P). Here we undertook a molecular characterization of the SKI-1/S1P processing of the GPCs of the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) and the pathogenic Lassa virus (LASV). Previous studies showed that the GPC of LASV undergoes processing in the endoplasmic reticulum (ER)/cis-Golgi compartment, whereas the LCMV GPC is cleaved in a late Golgi compartment. Herein we confirm these findings and provide evidence that the SKI-1/S1P recognition site RRLL, present in the SKI-1/S1P prodomain and LASV GPC, but not in the LCMV GPC, is crucial for the processing of the LASV GPC in the ER/cis-Golgi compartment. Our structure-function analysis revealed that the cleavage of arenavirus GPCs, but not cellular substrates, critically depends on the autoprocessing of SKI-1/S1P, suggesting differences in the processing of cellular and viral substrates. Deletion mutagenesis showed that the transmembrane and intracellular domains of SKI-1/S1P are dispensable for arenavirus GPC processing. The expression of a soluble form of the protease in SKI-I/S1P-deficient cells resulted in the efficient processing of arenavirus GPCs and rescued productive virus infection. However, exogenous soluble SKI-1/S1P was unable to process LCMV and LASV GPCs displayed at the surface of SKI-I/S1P-deficient cells, indicating that GPC processing occurs in an intracellular compartment. In sum, our study reveals important differences in the SKI-1/S1P processing of viral and cellular substrates.

P-glycoprotein-mediated resistance to chemotherapy in cancer cells: using recombinant cytosolic domains to establish structure-function relationships

Resistance to chemotherapy in cancer cells is mainly mediated by overexpression of P-glycoprotein (Pgp), a plasma membrane ATP-binding cassette (ABC) transporter which extrudes cytotoxic drugs at the expense of ATP hydrolysis. Pgp consists of two homologous halves each containing a transmembrane domain and a cytosolic nucleotide-binding domain (NBD) which contains two consensus Walker motifs, A and B, involved in ATP binding and hydrolysis. The protein also contains an S signature characteristic of ABC transporters. The molecular mechanism of Pgp-mediated drug transport is not known. Since the transporter has an extraordinarily broad substrate specificity, its cellular function has been described as a "hydrophobic vacuum cleaner". The limited knowledge about the mechanism of Pgp, partly due to the lack of a high-resolution structure, is well reflected in the failure to efficiently inhibit its activity in cancer cells and thus to reverse multidrug resistance (MDR). In contrast to the difficulties encountered when studying the full-length Pgp, the recombinant NBDs can be obtained in large amounts as soluble proteins. The biochemical and biophysical characterization of recombinant NBDs is shown here to provide a suitable alternative route to establish structure-function relationships. NBDs were shown to bind ATP and analogues as well as potent modulators of MDR, such as hydrophobic steroids, at a region close to the ATP site. Interestingly, flavonoids also bind to NBDs with high affinity. Their binding site partly overlaps both the ATP-binding site and the steroid-interacting region. Therefore flavonoids constitute a new promising class of bifunctional modulators of Pgp.

Age-related changes of the multidrug resistance P-glycoprotein function in normal human peripheral blood T lymphocytes

The multidrug resistance P-glycoprotein is a transmembrane efflux pump expressed by lymphocytes and is involved in their cytolytic activity. In the present study, we investigated the age-related changes of P-glycoprotein function in normal peripheral blood lymphocytes. Blood samples from 90 normal volunteers (age range, 0 to 86 years) were analyzed. P-glycoprotein function was assessed by the flow cytometric rhodamine 123 assay. P-glycoprotein function was highest in cord blood and progressively declined with age in peripheral blood T CD4+ and CD8+ cells. In contrast, P-glycoprotein function did not vary with age in CD19+ B or CD16+CD56+ natural killer cells. These data suggest that the decline in P-glycoprotein function in T CD4+ and CD8+ lymphocytes as a function of age may contribute to the decrease in T cell cytolytic activity with aging.

Synaptic vesicle glycoprotein 2A modulates vesicular release and calcium channel function at peripheral sympathetic synapses

Synaptic vesicle glycoprotein (SV)2A is a transmembrane protein found in secretory vesicles and is critical for Ca2+-dependent exocytosis in central neurons, although its mechanism of action remains uncertain. Previous studies have proposed, variously, a role of SV2 in the maintenance and formation of the readily releasable pool (RRP) or in the regulation of Ca2+ responsiveness of primed vesicles. Such previous studies have typically used genetic approaches to ablate SV2 levels; here, we used a strategy involving small interference RNA (siRNA) injection to knockdown solely presynaptic SV2A levels in rat superior cervical ganglion (SCG) neuron synapses. Moreover, we investigated the effects of SV2A knockdown on voltage-dependent Ca2+ channel (VDCC) function in SCG neurons. Thus, we extended the studies of SV2A mechanisms by investigating the effects on vesicular transmitter release and VDCC function in peripheral sympathetic neurons. We first demonstrated an siRNA-mediated SV2A knockdown. We showed that this SV2A knockdown markedly affected presynaptic function, causing an attenuated RRP size, increased paired-pulse depression and delayed RRP recovery after stimulus-dependent depletion. We further demonstrated that the SV2A–siRNA-mediated effects on vesicular release were accompanied by a reduction in VDCC current density in isolated SCG neurons. Together, our data showed that SV2A is required for correct transmitter release at sympathetic neurons. Mechanistically, we demonstrated that presynaptic SV2A: (i) acted to direct normal synaptic transmission by maintaining RRP size, (ii) had a facilitatory role in recovery from synaptic depression, and that (iii) SV2A deficits were associated with aberrant Ca2+ current density, which may contribute to the secretory phenotype in sympathetic peripheral neurons.

P-glycoprotein expression in lamina propria lymphocytes of duodenal biopsy samples in dogs with chronic idiopathic enteropathies

P-glycoprotein (p-gp) is a transmembrane protein functioning as a drug-efflux pump in the intestinal epithelium. Human patients with inflammatory bowel disease (IBD) who fail to respond to treatment with steroids express high levels of p-gp in lamina propria lymphocytes. The purpose of this study was to investigate p-gp expression in duodenal biopsy samples of dogs with chronic enteropathies and to evaluate the expression of p-gp after treatment with a known inducer of p-gp (prednisolone). Duodenal biopsy samples from 48 dogs were evaluated immunohistochemically with the mouse monoclonal antibody C219 for expression of p-gp in lamina propria lymphocytes. Biopsy samples were available from 15 dogs after treatment with prednisolone and 16 dogs after dietary therapy alone ("elimination diet"). Treatment with prednisolone resulted in an increase in p-gp expression (P=0.005). In contrast, dietary treatment alone produced no significant change in p-gp expression (P=0.59). A low p-gp score before initiation of steroid treatment was significantly associated with a positive response to treatment (P=0.01). These results indicate that lamina propria lymphocyte expression of p-gp is upregulated after prednisolone treatment in dogs with IBD, and that mucosal expression of p-gp may be of value in predicting the response to therapy.

The platelet collagen receptor glycoprotein VI is a member of the immunoglobulin superfamily closely related to FcalphaR and the natural killer receptors

We have cloned the platelet collagen receptor glycoprotein (GP) VI from a human bone marrow cDNA library using rapid amplification of cDNA ends with platelet mRNA to complete the 5' end sequence. GPVI was isolated from platelets using affinity chromatography on the snake C-type lectin, convulxin, as a critical step. Internal peptide sequences were obtained, and degenerate primers were designed to amplify a fragment of the GPVI cDNA, which was then used as a probe to screen the library. Purified GPVI, as well as Fab fragments of polyclonal antibodies made against the receptor, inhibited collagen-induced platelet aggregation. The GPVI receptor cDNA has an open reading frame of 1017 base pairs coding for a protein of 339 amino acids including a putative 23-amino acid signal sequence and a 19-amino acid transmembrane domain between residues 247 and 265. GPVI belongs to the immunoglobulin superfamily, and its sequence is closely related to FcalphaR and to the natural killer receptors. Its extracellular chain has two Ig-C2-like domains formed by disulfide bridges. An arginine residue is found in position 3 of the transmembrane portion, which should permit association with Fcgamma and its immunoreceptor tyrosine-based activation motif via a salt bridge. With 51 amino acids, the cytoplasmic tail is relatively long and shows little homology to the C-terminal part of the other family members. The ability of the cloned GPVI cDNA to code for a functional platelet collagen receptor was demonstrated in the megakaryocytic cell line Dami. Dami cells transfected with GPVI cDNA mobilized intracellular Ca(2+) in response to collagen, unlike the nontransfected or mock transfected Dami cells, which do not respond to collagen.

Organization and function of platelet glycoprotein Ib-IX complex

Glycoprotein (GP) Ib-IX complex, the second most abundant receptor expressed on the platelet surface, plays critical roles in haemostasis and thrombosis by binding to its ligand, von Willebrand factor (vWF). Defect or malfunction of the complex leads to severe bleeding disorders, heart attack or stroke. Comprised of three type I transmembrane subunits—GPIbα, GPIbβ and GPIX, efficient expression of the GPIb-IX complex requires all three subunits, as evident from genetic mutations identified in the patients and reproduced in transfected Chinese hamster ovary (CHO) cells. However, how the subunits are assembled together and how the complex function is regulated is not fully clear. By probing the interactions among the three subunits in transfected cells, we have demonstrated that the transmembrane domains of the three subunits interact with one another, facilitating formation of the two membrane-proximal disulfide bonds between GPIbα and GPIbβ. We have also identified the interface between extracellular domains of GPIbβ and GPIX, and provided evidence suggesting a direct interaction between extracellular domains of GPIbα and GPIX. All of these interactions are not only critical for correct assembly and consequently efficient expression of the GPIb-IX complex on the cell surface, but also for its function, such as the proper ligand binding, since removing the two inter-subunit disulfide bonds significantly hampers vWF binding to the complex under both static and physiological flow conditions. The two inter-subunit disulfide bonds are also critical for regulating the ectodomain shedding of GPIbα by the GPIbβ cytoplasmic domain. Mutations in the juxtamembrane region of the GPIbβ cytoplasmic domain deregulate GPIbα shedding, and such deregulation is further enhanced when the two inter-subunit disulfide bonds are removed. In summary, we have established the overall organization of the GPIb-IX complex, and the importance of proper organization on its function. ^

Evidence for two nonidentical drug-interaction sites in the human P-glycoprotein

Human P-glycoprotein (Pgp) confers multidrug resistance to cancer cells by ATP-dependent extrusion of a great many structurally dissimilar hydrophobic compounds. The manner in which Pgp recognizes these different substrates is unknown. The protein shows internal homology between its N- and C-terminal halves, each comprised of six putative transmembrane helices and a consensus ATP binding/utilization site. Photoactive derivatives of certain Pgp substrates specifically label two regions, one on each half of the protein. In this study, using [125I]iodoarylazidoprazosin ([125I]IAAP), a photoactive analog of prazosin, we have demonstrated the presence of two nonidentical drug-interaction sites within Pgp. Taking advantage of a highly susceptible trypsin cleavage site in the linker region of Pgp, we characterized the [125I]IAAP binding to the N- and C-terminal halves. cis(Z)-Flupentixol, a modulator of Pgp function, preferentially increased the affinity of [125I]IAAP for the C-terminal half of the protein (C-site) by reducing the Kd from 20 to 6 nM without changing the labeling or affinity (Kd = 42–46 nM) of the N-terminal half (N-site). Also, the concentration of vinblastine (Pgp substrate) and cyclosporin A (Pgp modulator) required for 50% inhibition of [125I]IAAP binding to the C-site was increased 5- to 6-fold by cis(Z)-flupentixol without any effect on the N-site. In addition, [125I]IAAP binding to the N-site was less susceptible than to C-site to inhibition by vanadate which blocks ATP hydrolysis and drug transport. These data demonstrate the presence of at least two nonidentical substrate interaction sites in Pgp.

P-glycoprotein function involves conformational transitions detectable by differential immunoreactivity

The MDR1 P-glycoprotein (Pgp), a member of the ATP-binding cassette family of transporters, is a transmembrane ATPase efflux pump for various lipophilic compounds, including many anti-cancer drugs. mAb UIC2, reactive with the extracellular moiety of Pgp, inhibits Pgp-mediated efflux. UIC2 reactivity with Pgp was increased by the addition of several Pgp-transported compounds or ATP-depleting agents, and by mutational inactivation of both nucleotide-binding domains (NBDs) of Pgp. UIC2 binding to Pgp mutated in both NBDs was unaffected in the presence of Pgp transport substrates or in ATP-depleted cells, whereas the reactivities of the wild-type Pgp and Pgps mutated in a single NBD were increased by these treatments to the level of the double mutant. These results indicate the existence of different Pgp conformations associated with different stages of transport-associated ATP hydrolysis and suggest trapping in a transient conformation as a mechanism for antibody-mediated inhibition of Pgp.

Dissection of De Novo Membrane Insertion Activities of Internal Transmembrane Segments of ATP-Binding-Cassette Transporters: Toward Understanding Topological Rules for Membrane Assembly of Polytopic Membrane Proteins

The membrane assembly of polytopic membrane proteins is a complicated process. Using Chinese hamster P-glycoprotein (Pgp) as a model protein, we investigated this process previously and found that Pgp expresses more than one topology. One of the variations occurs at the transmembrane (TM) domain including TM3 and TM4: TM4 inserts into membranes in an Nin-Cout rather than the predicted Nout-Cin orientation, and TM3 is in cytoplasm rather than the predicted Nin-Cout orientation in the membrane. It is possible that TM4 has a strong activity to initiate the Nin-Cout membrane insertion, leaving TM3 out of the membrane. Here, we tested this hypothesis by expressing TM3 and TM4 in isolated conditions. Our results show that TM3 of Pgp does not have de novo Nin-Cout membrane insertion activity whereas TM4 initiates the Nin-Cout membrane insertion regardless of the presence of TM3. In contrast, TM3 and TM4 of another polytopic membrane protein, cystic fibrosis transmembrane conductance regulator (CFTR), have a similar level of de novo Nin-Cout membrane insertion activity and TM4 of CFTR functions only as a stop-transfer sequence in the presence of TM3. Based on these findings, we propose that 1) the membrane insertion of TM3 and TM4 of Pgp does not follow the sequential model, which predicts that TM3 initiates Nin-Cout membrane insertion whereas TM4 stops the insertion event; and 2) “leaving one TM segment out of the membrane” may be an important folding mechanism for polytopic membrane proteins, and it is regulated by the Nin-Cout membrane insertion activities of the TM segments.

Lumenal and Transmembrane Domains Play a Role in Sorting Type I Membrane Proteins on Endocytic Pathways

Previous studies have shown that when the cytosolic domains of the type I membrane proteins TGN38 and lysosomal glycoprotein 120 (lgp120) are added to a variety of reporter molecules, the resultant chimeric molecules are localized to the trans-Golgi network (TGN) and to lysosomes, respectively. In the present study we expressed chimeric constructs of rat TGN38 and rat lgp120 in HeLa cells. We found that targeting information in the cytosolic domain of TGN38 could be overridden by the presence of the lumenal and transmembrane domains of lgp120. In contrast, the presence of the transmembrane and cytosolic domains of TGN38 was sufficient to deliver the lumenal domain of lgp120 to the trans-Golgi network. On the basis of steady-state localization of the various chimeras and antibody uptake experiments, we propose that there is a hierarchy of targeting information in each molecule contributing to sorting within the endocytic pathway. The lumenal and cytosolic domains of lgp120 contribute to sorting and delivery to lysosomes, whereas the transmembrane and cytosolic domains of TGN38 contribute to sorting and delivery to the trans-Golgi network.

A binding pocket for a small molecule inhibitor of HIV-1 entry within the transmembrane helices of CCR5

HIV-1 entry into CD4+ cells requires the sequential interactions of the viral envelope glycoproteins with CD4 and a coreceptor such as the chemokine receptors CCR5 and CXCR4. A plausible approach to blocking this process is to use small molecule antagonists of coreceptor function. One such inhibitor has been described for CCR5: the TAK-779 molecule. To facilitate the further development of entry inhibitors as antiviral drugs, we have explored how TAK-779 acts to prevent HIV-1 infection, and we have mapped its site of interaction with CCR5. We find that TAK-779 inhibits HIV-1 replication at the membrane fusion stage by blocking the interaction of the viral surface glycoprotein gp120 with CCR5. We could identify no amino acid substitutions within the extracellular domain of CCR5 that affected the antiviral action of TAK-779. However, alanine scanning mutagenesis of the transmembrane domains revealed that the binding site for TAK-779 on CCR5 is located near the extracellular surface of the receptor, within a cavity formed between transmembrane helices 1, 2, 3, and 7.