Nef-Induced CD4 and Major Histocompatibility Complex Class I (MHC-I) Down-Regulation Are Governed by Distinct Determinants: N-Terminal Alpha Helix and Proline Repeat of Nef Selectively Regulate MHC-I Trafficking

The Nef protein of primate lentiviruses triggers the accelerated endocytosis of CD4 and of class I major histocompatibility complex (MHC-I), thereby down-modulating the cell surface expression of these receptors. Nef acts as a connector between the CD4 cytoplasmic tail and intracellular sorting pathways both in the Golgi and at the plasma membrane, triggering the de novo formation of CD4-specific clathrin-coated pits (CCP). The downstream partners of Nef in this event are the adapter protein complex (AP) of CCP and possibly a subunit of the vacuolar ATPase. Whether Nef-induced MHC-I down-regulation stems from a similar mechanism is unknown. By comparing human immunodeficiency virus type 1 (HIV-1) Nef mutants for their ability to affect either CD4 or MHC-I expression, both in transient-transfection assays and in the context of HIV-1 infection, it was determined that Nef-induced CD4 and MHC-I down-regulation constitute genetically and functionally separate properties. Mutations affecting only CD4 regulation mapped to residues previously shown to mediate the binding of Nef to this receptor, such as W57 and L58, as well as to an AP-recruiting dileucine motif and to an acidic dipeptide in the C-terminal region of the protein. In contrast, mutation of residues in an alpha-helical region in the proximal portion of Nef and amino acid substitutions in a proline-based SH3 domain-binding motif selectively affected MHC-I down-modulation. Although both the N-terminal alpha-helix and the proline-rich region of Nef have been implicated in recruiting Src family protein kinases, the inhibitor herbimycin A did not block MHC-I down-regulation, suggesting that the latter process is not mediated through an activation of this family of tyrosine kinases.The Nef protein of primate lentiviruses plays a multifaceted role in the life cycle of these pathogens (reviewed in reference 17). Produced in abundance from the earliest stage of viral gene expression, Nef associates with the membranes of infected cells by virtue of its N-terminal myristoylation (21, 36, 46), and it accomplishes several distinct functions. First, it down-regulates the cell surface expression of class I major histocompatibility complex (MHC-I), preventing the recognition and lysis of infected cells by cytotoxic lymphocytes (14, 48, 50, 66). Second, it decreases the surface expression of CD4, the primary viral receptor (1, 25, 36). Third, it stimulates virion infectivity by as yet ill-defined influences exerted during viral particle formation (3, 13, 54, 72, 73). Finally, it alters T-cell activation pathways, an effect that can be observed both in tissue culture and in transgenic mice (7, 9, 37, 51, 71).Several lines of evidence indicate that Nef down-modulates CD4 by acting as a receptor-specific sorting adapter. The Nef effect is exerted at a posttranslational level and, unlike phorbol myristate acetate-induced down-regulation, does not require phosphorylation of the CD4 cytoplasmic tail (25). The membrane-proximal 20 amino acids of CD4, including an essential dileucine motif, are necessary for Nef-mediated down-modulation and can transfer Nef sensitivity to another integral membrane protein (1). Although difficult to detect in mammalian cells, an interaction between Nef and CD4 could be demonstrated in insect cells infected with baculovirus vectors, in the yeast two-hybrid system, and in vitro with recombinant Nef protein and CD4-derived peptide (35, 39, 64). In these last two settings, the importance of the CD4 dileucine motif for association with Nef was confirmed (35, 64). Nuclear magnetic resonance (NMR) analyses further defined the CD4 binding site of Nef (33, 35). A pocket formed of the noncontiguous amino acids WLE₅₉, GGL₉₇, R₁₀₆, and L₁₁₀ bound a peptide corresponding to the CD4 tail, albeit with a low affinity. Supporting the functional relevance of these data, a mutation targeting WL₅₈ abrogated Nef-induced CD4 down-regulation (42). Additionally, human immunodeficiency virus type 1 (HIV-1), HIV-2, and simian immunodeficiency virus (SIV) Nef proteins require slightly different sequences in the CD4 cytoplasmic tail for efficient down-modulation, arguing against the existence of a cellular intermediate bridging Nef with CD4 (43).While it now appears well established that Nef binds CD4, overwhelming evidence also indicates that the viral protein interacts with the endocytic machinery. HIV-1 Nef can trigger the de novo formation of clathrin-coated pits (CCP) that preferentially incorporate CD4 (20). Furthermore, a chimeric integral membrane protein composed of the extracellular and transmembrane domains of CD4 or CD8 with Nef as its cytoplasmic tail undergoes rapid internalization and causes an increase in the clathrin lattice on the inner side of the cell membrane (20, 53). Not strictly a cell surface phenomenon, Nef-induced CD4 down-regulation additionally reflects some degree of intracellular retention and rerouting from the Golgi to the endosomal compartment (53).The model in which Nef acts as a connector between CD4 and CCP implies that the viral protein recognizes some component of the internalization machinery. Two such downstream partners have been recently proposed: the μ chain of the so-called adapter protein complexes (AP) (48, 60), and a subunit of the vacuolar ATPase, NBP1 (52). APs are heterotetrameric complexes which normally associate with receptor cytoplasmic tails containing tyrosine-based (8, 27, 56) and perhaps dileucine-based (40) signals and which recruit clathrin to induce the formation of CCP (24, 28, 69). AP-1 is present in the Golgi, and AP-2 is found at the plasma membrane (62). A third class of AP, AP-3, was recently identified and might be involved in lysosomal targeting (15, 18, 70). Nef proteins from HIV-1, HIV-2, and SIV were found to associate with the μ chain of both the Golgi (μ1) and plasma membrane (μ2) APs (48, 60). Mutating tyrosine residues at the N terminus of SIV Nef abrogated the Nef-μ interaction and prevented Nef-mediated CD4 down-regulation (60). In HIV-1 Nef, where these tyrosine-based motifs are absent, mutating a dileucine motif in a C-terminal disordered loop of the protein abrogated CD4 down-modulation (16). Furthermore, a 10- to 11-amino-acid sequence including this Nef-derived dileucine motif induced the accelerated internalization of a chimeric integral membrane protein (10, 16). Finally, the dileucine-dependent binding of HIV-1 Nef to APs could be demonstrated both in vitro and in tissue culture (16, 30). In another study, direct interactions between HIV-1 Nef and NBP1, the catalytic subunit of the vacuolar ATPase (V-ATPase), correlated with CD4 down-regulation (52). However, loss of interaction with NBP1 led to only a partial loss of the effect of Nef on CD4.Although less information is available about the mechanisms of Nef-induced MHC-I down-regulation, this receptor also exhibits increased rates of internalization and lysosomal degradation in the presence of the viral protein (66). Furthermore, HLA-A and HLA-B accumulate in the Golgi and colocalize with clathrin-coated vesicles in this setting (31, 48). Whether the parallel between CD4 and MHC-I down-modulation can be extended further is, however, unknown.To address this question, we analyzed the ability of a series of HIV-1 Nef mutants to down-regulate CD4 and MHC-I and to trigger in cis the accelerated endocytosis of a chimeric integral membrane protein. The results of our experiments support a model in which Nef uses distinct domains for connecting CD4 with cellular mediators of protein sorting and for down-modulating MHC-I. Additionally, we identify an N-terminal domain of Nef, shown by NMR to be an alpha-helix (5), as being crucial for MHC-I down-regulation.     

The Human Scavenger Receptor CD36: GLYCOSYLATION STATUS AND ITS ROLE IN TRAFFICKING AND FUNCTION*

Human CD36 is a class B scavenger receptor expressed in a variety of cell types such as macrophage and adipocytes. This plasma membrane glycoprotein has a wide range of ligands including oxidized low density lipoprotein and long chain fatty acids which involves the receptor in diseases such as atherosclerosis and insulin resistance. CD36 is heavily modified post-translationally by N-linked glycosylation, and 10 putative glycosylation sites situated in the large extracellular loop of the protein have been identified; however, their utilization and role in the folding and function of the protein have not been characterized. Using mass spectrometry on purified and peptide N-glycosidase F-deglycosylated CD36 and also by comparing the electrophoretic mobility of different glycosylation site mutants, we have determined that 9 of the 10 sites can be modified by glycosylation. Flow cytometric analysis of the different glycosylation mutants expressed in mammalian cells established that glycosylation is necessary for trafficking to the plasma membrane. Minimally glycosylated mutants that supported trafficking were identified and indicated the importance of carboxyl-terminal sites Asn-247, Asn-321, and Asn-417. However, unlike SRBI, no individual site was found to be essential for proper trafficking of CD36. Surprisingly, these minimally glycosylated mutants appear to be predominantly core-glycosylated, indicating that mature glycosylation is not necessary for surface expression in mammalian cells. The data also show that neither the nature nor the pattern of glycosylation is relevant to binding of modified low density lipoprotein.Human CD36, originally identified in platelets as glycoprotein IV (1), is a class B scavenger receptor localized to the plasma membrane. It is not expressed ubiquitously but is present in a variety of different cells and tissue types including epithelial cells (2), macrophages (3), endothelial cells of the microvasculature (4), and smooth muscle (5). Its function is complex, and its involvement in different disease scenarios, such as cancer (6), atherosclerosis (3, 7, 8), malaria (9), and insulin resistance (10), most likely reflects the interaction of the receptor with a particular ligand in a specific cell type. For example, CD36 expressed in monocytic macrophages functions as a scavenger receptor for the uptake of oxidized LDL² (3, 11). Under certain physiological conditions, this results in the lipid loading of macrophages at the site of tissue damage in the arterial wall, leading to foam cell formation and plaque development, a key early stage in the pathogenesis of atherosclerosis (8, 12). In fat and muscle cells, CD36 plays an essential role in lipid homeostasis by uptake of long chain fatty acids (13). In this case CD36 deficiency has been linked to disorders in lipid metabolism, giving rise to increased incidences of insulin resistance and cardiomyopathies (11, 14, 15).Although much is known about the function of CD36, less is known about its structure. CD36 has no bacterial homologues but is a member of a protein family that also includes the mammalian proteins LIMPII (16), CLA-1 (17), SRBI (18), and the Drosophila proteins Croquemort (19) and emp (20). The sequence of 471 amino acids has two short hydrophobic regions at the carboxyl and amino termini separated by a large hydrophilic region (21); however, the topology of the protein is unclear with both ditopic (22) and type I (23) topological models proposed. Both are consistent in predicting that the large hydrophilic region is extracellular, which is clearly supported by epitope mapping studies (24). The protein is heavily modified post-translationally. The six extracellular cysteines, which are highly conserved within the orthologous CD36 subfamily, have been shown to be linked by disulfide bonds in bovine Cd36 (25), and the remaining four cysteines, two at each terminus, are palmitoylated (26), lending credence to the ditopic topological model.CD36 is also modified by N-linked glycosylation, which accounts for the observation that the protein migrates with an apparent molecular mass of 78–94 kDa on SDS-PAGE (4, 27) despite a theoretical mass for the polypeptide of 53 kDa. N-Linked glycosylation is a common modification of extracellular and secreted proteins, and defects in the glycosylation pathways lead to a wide range of serious diseases known collectively as congenital disorders of glycosylation (28). Glycosylation can be important for correct folding of proteins (29, 30) either by directly inducing and/or stabilizing the tertiary fold of the polypeptide (31) or via an affinity for lectin chaperones such as calnexin or calreticulin (32). Glycosylation has also been shown to be important for the trafficking of certain glycoproteins through affinity for lectin transport machinery (33). The glycosylation status of bovine Cd36 has already been determined with all eight putative sites shown to be glycosylated (34). Human and bovine CD36 are 83% identical (93% when similar residues are included) and share 7 glycosylation sites (human has 10 putative glycosylation sites). In the related mouse SRBI, which is 33% identical (54% similar) to human CD36, there are 11 putative N-linked glycosylation sites, only 3 of which are shared with the human protein. Site-directed mutagenesis of each of the 11 sites independently in SRBI in an otherwise wild type protein indicates that all are glycosylated, with two (Asn-108 and Asn-173) important for either trafficking or folding. Mutagenesis of either of these two residues resulted in very little cell surface expression of the protein (35); however, neither site is conserved in human CD36.To gain further understanding of the role of glycosylation of CD36, we used mutagenesis and biophysical analysis (mass spectrometry and gel electrophoresis) to identify unequivocally which glycosylation sites are occupied in human CD36. Antibody and ligand binding studies with these mutant proteins also provided insights into the role of glycosylation and site occupancy in the trafficking and function of the protein.     

CD4 Glycoprotein Degradation Induced by Human Immunodeficiency Virus Type 1 Vpu Protein Requires the Function of Proteasomes and the Ubiquitin-Conjugating Pathway

The human immunodeficiency virus type 1 (HIV-1) vpu gene encodes a type I anchored integral membrane phosphoprotein with two independent functions. First, it regulates virus release from a post-endoplasmic reticulum (ER) compartment by an ion channel activity mediated by its transmembrane anchor. Second, it induces the selective down regulation of host cell receptor proteins (CD4 and major histocompatibility complex class I molecules) in a process involving its phosphorylated cytoplasmic tail. In the present work, we show that the Vpu-induced proteolysis of nascent CD4 can be completely blocked by peptide aldehydes that act as competitive inhibitors of proteasome function and also by lactacystin, which blocks proteasome activity by covalently binding to the catalytic β subunits of proteasomes. The sensitivity of Vpu-induced CD4 degradation to proteasome inhibitors paralleled the inhibition of proteasome degradation of a model ubiquitinated substrate. Characterization of CD4-associated oligosaccharides indicated that CD4 rescued from Vpu-induced degradation by proteasome inhibitors is exported from the ER to the Golgi complex. This finding suggests that retranslocation of CD4 from the ER to the cytosol may be coupled to its proteasomal degradation. CD4 degradation mediated by Vpu does not require the ER chaperone calnexin and is dependent on an intact ubiquitin-conjugating system. This was demonstrated by inhibition of CD4 degradation (i) in cells expressing a thermally inactivated form of the ubiquitin-activating enzyme E₁ or (ii) following expression of a mutant form of ubiquitin (Lys₄₈ mutated to Arg₄₈) known to compromise ubiquitin targeting by interfering with the formation of polyubiquitin complexes. CD4 degradation was also prevented by altering the four Lys residues in its cytosolic domain to Arg, suggesting a role for ubiquitination of one or more of these residues in the process of degradation. The results clearly demonstrate a role for the cytosolic ubiquitin-proteasome pathway in the process of Vpu-induced CD4 degradation. In contrast to other viral proteins (human cytomegalovirus US2 and US11), however, whose translocation of host ER molecules into the cytosol occurs in the presence of proteasome inhibitors, Vpu-targeted CD4 remains in the ER in a transport-competent form when proteasome activity is blocked.

The human immunodeficiency virus type 1 (HIV-1)-specific accessory protein Vpu performs two distinct functions in the viral life cycle (11, 12, 29, 34, 46, 47, 50–52; reviewed in references 31 and 55): enhancement of virus particle release from the cell surface, and the selective induction of proteolysis of newly synthesized membrane proteins. Known targets for Vpu include the primary virus receptor CD4 (63, 64) and major histocompatibility complex (MHC) class I molecules (28). Vpu is an oligomeric class I integral membrane phosphoprotein (35, 48, 49) with a structurally and functionally defined domain architecture: an N-terminal transmembrane anchor and C-terminal cytoplasmic tail (20, 34, 45, 47, 50, 65). Vpu-induced degradation of endoplasmic reticulum (ER) membrane proteins involves the phosphorylated cytoplasmic tail of the protein (50), whereas the virion release function is mediated by a cation-selective ion channel activity associated with the membrane anchor (19, 31, 45, 47).CD4 is a 55-kDa class I integral membrane glycoprotein that serves as the primary coreceptor for HIV entry into cells. CD4 consists of a large lumenal domain, a transmembrane peptide, and a 38-residue cytoplasmic tail. It is expressed on the surface of a subset of T lymphocytes that recognize MHC class II-associated peptides, and it plays a pivotal role in the development and maintenance of the immune system (reviewed in reference 30). Down regulation of CD4 in HIV-1-infected cells is mediated through several independent mechanisms (reviewed in references 5 and 55): intracellular complex formation of CD4 with the HIV envelope protein gp160 (8, 14), endocytosis of cell surface CD4 induced by the HIV-1 nef gene product (1, 2), and ER degradation induced by the HIV-1 vpu gene product (63, 64).Vpu-induced degradation of CD4 is an example of ER-associated protein degradation (ERAD). ERAD is a common outcome when proteins in the secretory pathway are unable to acquire their native structure (4). Although it was thought that ERAD occurs exclusively inside membrane vesicles of the ER or other related secretory compartments, this has gained little direct experimental support. Indeed, there are several recent reports that ERAD may actually represent export of the target protein to the cytosol, where it is degraded by cytosolic proteases. It was found that in yeast, a secreted protein, prepro-α-factor (pαF), is exported from microsomes and degraded in the cytosol in a proteasome-dependent manner (36). This process was dependent on the presence of calnexin, an ER-resident molecular chaperone that interacts with N-linked oligosaccharides containing terminal glucose residues (3). In mammalian cells, two human cytomegalovirus (HCMV) proteins, US2 and US11, were found to cause the retranslocation of MHC class I molecules from the ER to the cytosol, where they are destroyed by proteasomes (61, 62). In the case of US2, class I molecules were found to associate with a protein (Sec61) present in the channel normally used to translocate newly synthesized proteins into the ER (termed the translocon), leading to the suggestion that the ERAD substrates are delivered to the cytosol by retrograde transport through the Sec61-containing pore (61). Fujita et al. (24) reported that, similar to these findings, the proteasome-specific inhibitor lactacystin (LC) partially blocked CD4 degradation in transfected HeLa cells coexpressing CD4, Vpu, and HIV-1 Env glycoproteins. In the present study, we show that Vpu-induced CD4 degradation can be completely blocked by proteasome inhibitors, does not require the ER chaperone calnexin, but requires the function of the cytosolic polyubiquitination machinery which apparently targets potential ubiquitination sites within the CD4 cytoplasmic tail. Our findings point to differences between the mechanism of Vpu-mediated CD4 degradation and ERAD processes induced by the HCMV proteins US2 and US11 (61, 62).     

Quantitative Proteome Analysis of Temporally Resolved Phagosomes Following Uptake Via Key Phagocytic Receptors

Macrophages operate at the forefront of innate immunity and their discrimination of foreign versus “self” particles is critical for a number of responses including efficient pathogen killing, antigen presentation, and cytokine induction. In order to efficiently destroy the particles and detect potential threats, macrophages express an array of receptors to sense and phagocytose prey particles. In this study, we accurately quantified a proteomic time-course of isolated phagosomes from murine bone marrow-derived macrophages induced by particles conjugated to seven different ligands representing pathogen-associated molecular patterns, immune opsonins or apoptotic cell markers. We identified a clear functional differentiation over the three timepoints and detected subtle differences between certain ligand-phagosomes, indicating that triggering of receptors through a single ligand type has mild, but distinct, effects on phagosome proteome and function. Moreover, our data shows that uptake of phosphatidylserine-coated beads induces an active repression of NF-κB immune responses upon Toll-like receptor (TLR)-activation by recruitment of anti-inflammatory regulators to the phagosome. This data shows for the first time a systematic time-course analysis of bone marrow-derived macrophages phagosomes and how phagosome fate is regulated by the receptors triggered for phagocytosis.Macrophages exist in many different tissue subsets, are extremely plastic in response to cytokines and pathogen-associated molecular patterns and perform a wide range of biological functions (1, 2). One of the most important functions of macrophages is phagocytosis, defined as the active uptake of large particles (>0.5 μm) by cells (3). Phagocytosis is an important cellular mechanism for almost all eukaryotes, highly conserved in evolution (4), and, in mammals, is a key part of the innate immune response to invading microorganisms. Moreover, during homeostasis and development, macrophages phagocytose apoptotic cells and cell debris to recycle cellular building blocks (5, 6). Phagocytosis is induced through the binding of particles as diverse as microbes, apoptotic cells, or even inert beads to cell surface receptors. After internalization, newly formed phagosomes engage in a maturation process that involves fusion with various organelles, including endosomes and ultimately lysosomes (7, 8). This leads to the formation of phagolysosomes that degrade the foreign matter. Antigens from the particle are presented via MHC class I and II molecules, bridging innate and adaptive immunity.In order to effectively phagocytose the diverse types of particulates they can encounter, macrophages express a vast array of receptors to sense and respond to the different ligands; however, only a small subset are solely sufficient to trigger phagocytosis (9). The classic phagocytic receptors are the Fc receptor, which internalizes immunoglobulin-bound particles (10), and the complement receptors, which binds to complement-opsonized particles (11). Other well characterized ligands for phagocytic receptors include mannan, a polysaccharide common in bacterial membranes and fungal cell walls (12), that activates mannose receptors (13, 14); lipopolysaccharide (LPS)¹, a glycolipid that constitutes the major portion of the outermost membrane of Gram-negative bacteria, that triggers CD14 as well as scavenger receptors and toll-like receptors (15–20); and phosphatidylserine (PS), a lipid normally restricted to the inner leaflet of eukaryotic plasma membranes, but exposed in the outer leaflet during apoptosis. PS provides an “eat me” signal for macrophage clearance (21, 22) and triggers a range of receptors including TIM-4, BAI1, and Stabilin-2 (23–27). Similarly, calreticulin, an endoplasmic reticulum protein that is also transported to the plasma membrane serves as a apoptotic signal has been proposed as a phagocytic ligand triggering the phagocytic receptor low-density lipoprotein receptor-related protein (LRP) (28–30).Although it is established that phagosome function is affected by various activation states, including rate of maturation, degradative capacity, and antigen cross-presentation capabilities (31–33), controversy exists around whether phagosome activity can be controlled directly, without prior activation, by receptor engagement at the phagosome level during biogenesis (34–38). Here, we dissect the role that individual ligands play in controlling downstream phagosome maturation using a reductionist strategy of ligating single ligands to microparticles and analyzing resulting phagosomes by quantitative proteomics and fluorescent phagosome functional assays.     

Inhibition and Activation by CD244 Depends on CD2 and Phospholipase C-��1

Regulation by the NK and T cell surface receptor CD244 in mice and humans depends both on engagement at the cell surface by CD48 and intracellular interactions with SAP and EAT-2. Relevance to human disease by manipulating CD244 in mouse models is complicated by rodent CD2 also binding CD48. We distinguish between contributions of mouse CD244 and CD2 on engagement of CD48 in a mouse T cell hybridoma. CD2 and CD244 both contribute positively to the immune response as mutation of proline-rich motifs or tyrosine motifs in the tails of CD2 and CD244, respectively, result in a decrease in antigen-specific interleukin-2 production. Inhibitory effects of mouse CD244 are accounted for by competition with CD2 at the cell surface for CD48. In humans CD2 and CD244 are engaged separately at the cell surface but biochemical data suggest a potential conserved intracellular link between the two receptors through FYN kinase. We identify a novel signaling mechanism for CD244 through its potential to recruit phospholipase C-γ1 via the conserved phosphorylated tyrosine motif in the tail of the adaptor protein EAT-2, which we show is important for function.The CD2 family of cell surface receptors is differentially expressed on immune cells (1, 2) and is involved in regulating both innate and adaptive immunity (3). These receptors have related extracellular immunoglobulin superfamily domains and interact either homophilically or heterophilically within the CD2 family (1, 2). The CD2 family contains a subgroup of receptors termed the SLAM family that have a conserved tyrosine signaling motif in their cytoplasmic region TXYXX(I/V) referred to as an immunoreceptor tyrosine-based switch motif (ITSM).² The SLAM family of receptors include CD244 (2B4), NTB-A (Ly-108), CD319 (CRACC, CS-1), CD150 (SLAM), CD84, and CD229 (Ly-9). Defects in signaling and aberrant expression of these receptors have been implicated in several immunodeficiency and autoimmune disorders in humans and mice (4–8). Within the SLAM family, CD244 is unusual in that it shares its ligand CD48 with the receptor CD2 in rodents, whereas in humans CD2 has evolved to interact with CD58 (9). The affinity of CD244 for CD48 in rodents is 6–9-fold higher than the still functionally relevant CD2/CD48 interaction (10). CD244 and CD2 have different cytoplasmic regions comprised of tyrosine motifs or proline-rich motifs, respectively.CD244 is predominantly found on NK cells and cytotoxic T cells and primarily characterized as an activating receptor (11–15). CD2 is found on the same cells as CD244 but is also expressed on all T cells, both activated and resting, and has an activating or costimulatory function upon engagement of ligand (9). The tyrosine motifs found in the cytoplasmic tail of CD244 have been shown to bind the SH2 domains of cytoplasmic adaptor proteins SAP and EAT-2 and FYN kinase (16–18) and are important to its function (5, 19–21). In contrast to SH2 interactions of CD244, several SH3 domain-mediated interactions have been reported for the cytoplasmic region of CD2 including CD2AP/CMS, CIN85, FYN, and LCK (22–26).The activating function of CD244 was called into question when a study using cells from a CD244 knock-out mouse showed that CD244 had an inhibitory effect as loss of CD244 resulted in enhanced NK killing of target cells (27). This suggested that previous results in mice where positive effects were seen may have been due to blocking CD244 ligand engagement as opposed to cross-linking with antibodies against CD244 (27). This has led to proposals that there are differences in function between mouse and human CD244 as there is more evidence to suggest that human CD244 is a positive regulator enhancing cytotoxicity and cytokine production (13, 15, 28). However, other more recent studies have shown the mouse CD244/CD48 interaction to be important for cytokine production and effector functions such as cytotoxicity against tumor targets in CD244-deficient mice (29). Long and short forms of CD244 have been cloned from mice with the short form being described as activating and the long form inhibitory (27, 30). Only the long form of CD244 is present in humans and is regarded as activating (14).Positive signaling by CD244 has been attributed to the recruitment of SAP (18), which is a signaling adaptor molecule comprised of a single SH2 domain encoded by the SH2D1A gene and has the ability to recruit the kinase FYN by binding its SH3 domain (31, 32). Loss of the SAP/FYN interaction can lead to X-linked lymphoproliferative disease in humans (17). The molecular basis of in vitro inhibitory effects observed with CD244 in mice on ligation with mAb or ligand remains elusive (33). Protein tyrosine and inositol phosphatases have been reported to associate with CD244 (18, 19, 34) but our studies using surface plasmon resonance found them to be very weak and unlikely to bind competitively compared with the SAP family of adaptors or FYN (16). The SAP-related adaptor EAT-2 has been reported to have an active inhibitory effect that is dependent on tyrosine motifs in the tail of EAT-2 (35) but its mechanism is not understood. The only interaction reported for the tail of EAT-2 is with FYN kinase and studies overexpressing EAT-2 in a T cell hybridoma resulted in increased IL-2 production upon antigen stimulation (16).The conservation between mouse and human CD244 cytoplasmic regions and associated adaptors suggests that both function in a similar way. We have explored the main difference between mouse and human CD244, which is the extracellular interaction through CD48 ligation in the mouse. This has revealed that inhibitory effects of CD244 ligation in mice can be due to competition between CD244 and CD2 for CD48. We have also found that the adaptor protein EAT-2 binds PLCγ1 providing a molecular basis for the important role CD244 plays in regulating cellular cytotoxicity (13, 36). We demonstrate that there is a potentially shared signaling mechanism through the FYN kinase that links CD2 and CD244 intracellularly even though in humans CD2 and CD244 no longer share a cell surface ligand.     

Caveolae-mediated Internalization of Occludin and Claudin-5 during CCL2-induced Tight Junction Remodeling in Brain Endothelial Cells

Disturbance of the tight junction (TJ) complexes between brain endothelial cells leads to increased paracellular permeability, allowing leukocyte entry into inflamed brain tissue and also contributing to edema formation. The current study dissects the mechanisms by which a chemokine, CCL2, induces TJ disassembly. It investigates the potential role of selective internalization of TJ transmembrane proteins (occludin and claudin-5) in increased permeability of the brain endothelial barrier in vitro. To map the internalization and intracellular fate of occludin and claudin-5, green fluorescent protein fusion proteins of these TJ proteins were generated and imaged by fluorescent microscopy with simultaneous measurement of transendothelial electrical resistance. During CCL2-induced reductions in transendothelial electrical resistance, claudin-5 and occludin became internalized via caveolae and further processed to early (EEA1+) and recycling (Rab4+) endosomes but not to late endosomes. Western blot analysis of fractions collected from a sucrose gradient showed the presence of claudin-5 and occludin in the same fractions that contained caveolin-1. For the first time, these results suggest an underlying molecular mechanism by which the pro-inflammatory chemokine CCL2 mediates brain endothelial barrier disruption during CNS inflammation.The blood-brain barrier is situated at the cerebral endothelial cells and their linking tight junctions. Increased brain endothelial barrier permeability is associated with remodeling of inter-endothelial tight junction (TJ)² complex and gap formation between brain endothelial cells (paracellular pathway) and/or intensive pinocytotic vesicular transport between the apical and basal side of brain endothelial cells (transcellular pathway) (1, 2). The transcellular pathway can be either passive or active and is characterized by low conductance and high selectivity. In contrast, the paracellular pathway is exclusively passive, being driven by electrochemical and osmotic gradients, and has a higher conductance and lower selectivity (3).Brain endothelial barrier paracellular permeability is maintained by an equilibrium between contractile forces generated at the endothelial cytoskeleton and adhesive forces produced at endothelial cell-cell junctions and cell-matrix contacts (1–3). A dynamic interaction among these structural elements controls opening and closing of the paracellular pathway and serves as a fundamental mechanism regulating blood-brain exchange. How this process occurs is under intense investigation. Two possible mechanisms may potentially increase paracellular permeability: phosphorylation of TJ proteins and/or endocytosis of transmembrane TJ proteins.Changes in TJ protein phosphorylation seem to be required to initiate increased brain endothelial permeability and a redistribution of most TJ proteins away from the cell border (4–8). Endocytosis may also be involved in remodeling TJ complexes between endothelial cells. Several types of endocytosis may be involved in TJ protein uptake, including clathrin- and caveolae-mediated endocytosis and macropinocytosis (for reviews, see Refs. 8 and 9–12). After first forming cell membrane-derived endocytotic vesicles, these vesicles fuse with early endosomes whose contents are further sorted for transport to lysosomes for degradation or recycling back to the plasma membrane for reuse (11).Although there is a lack of definitive knowledge regarding endocytotic internalization of brain endothelial cell TJ proteins, several studies on epithelial cells have indicated that occludin may be internalized via caveolae-mediated endocytosis whereas ZO-1, claudin-1, and junctional adhesion molecules-A may undergo macropinocytosis in response to stimuli such as TNF-α and INF-γ (13, 14). In contrast, there is evidence that Ca²⁺ may induce internalization of claudin-1 and occludin via clathrin-coated vesicles (8, 14–16). All of these studies pinpoint endocytosis as an underlying process in TJ complex remodeling and redistribution, and thus regulation of paracellular permeability in epithelial cells.The present study examines whether internalization of transmembrane TJ proteins could be one process by which adhesion between brain endothelial cells is changed during increased paracellular permeability. Our results show that a pro-inflammatory mediator, the chemokine CCL2, induces disassembly of the TJ complex by triggering caveolae-dependent internalization of transmembrane TJ proteins (occludin and claudin-5). Once internalized, occludin and claudin-5 are further processed to recycling endosomes awaiting return to the plasma membrane.     

The Cell Tropism of Human Immunodeficiency Virus Type 1 Determines the Kinetics of Plasma Viremia in SCID Mice Reconstituted with Human Peripheral Blood Leukocytes

Most individuals infected with human immunodeficiency virus type 1 (HIV-1) initially harbor macrophage-tropic, non-syncytium-inducing (M-tropic, NSI) viruses that may evolve into T-cell-tropic, syncytium-inducing viruses (T-tropic, SI) after several years. The reasons for the more efficient transmission of M-tropic, NSI viruses and the slow evolution of T-tropic, SI viruses remain unclear, although they may be linked to expression of appropriate chemokine coreceptors for virus entry. We have examined plasma viral RNA levels and the extent of CD4⁺ T-cell depletion in SCID mice reconstituted with human peripheral blood leukocytes following infection with M-tropic, dual-tropic, or T-tropic HIV-1 isolates. The cell tropism was found to determine the course of viremia, with M-tropic viruses producing sustained high viral RNA levels and sparing some CD4⁺ T cells, dual-tropic viruses producing a transient and lower viral RNA spike and extremely rapid depletion of CD4⁺ T cells, and T-tropic viruses causing similarly lower viral RNA levels and rapid-intermediate rates of CD4⁺ T-cell depletion. A single amino acid change in the V3 region of gp120 was sufficient to cause one isolate to switch from M-tropic to dual-tropic and acquire the ability to rapidly deplete all CD4⁺ T cells.The envelope gene of human immunodeficiency virus type 1 (HIV-1) determines the cell tropism of the virus (11, 32, 47, 62), the use of chemokine receptors as cofactors for viral entry (4, 17), and the ability of the virus to induce syncytia in infected cells (55, 60). Cell tropism is closely linked to but probably not exclusively determined by the ability of different HIV-1 envelopes to bind CD4 and the CC or the CXC chemokine receptors and initiate viral fusion with the target cell. Macrophage-tropic (M-tropic) viruses infect primary cultures of macrophages and CD4⁺ T cells and use CCR5 as the preferred coreceptor (2, 5, 15, 23, 26, 31). T-cell-tropic (T-tropic) viruses can infect primary cultures of CD4⁺ T cells and established T-cell lines, but not primary macrophages. T-tropic viruses use CXCR4 as a coreceptor for viral entry (27). Dual-tropic viruses have both of these properties and can use either CCR5 or CXCR4 (and infrequently other chemokine receptors [25]) for viral entry (24, 37, 57). M-tropic viruses are most frequently transmitted during primary infection of humans and persist throughout the duration of the infection (63). Many, but not all, infected individuals show an evolution of virus cell tropism from M-tropic to dual-tropic and finally to T-tropic with increasing time after infection (21, 38, 57). Increases in replicative capacity of viruses from patients with long-term infection have also been noted (22), and the switch to the syncytium-inducing (SI) phenotype in T-tropic or dual-tropic isolates is associated with more rapid disease progression (10, 20, 60). Primary infection with dual-tropic or T-tropic HIV, although infrequent, often leads to rapid disease progression (16, 51). The viral and host factors that determine the higher transmission rate of M-tropic HIV-1 and the slow evolution of dual- or T-tropic variants remain to be elucidated (4).These observations suggest that infection with T-tropic, SI virus isolates in animal model systems with SCID mice grafted with human lymphoid cells or tissue should lead to a rapid course of disease (1, 8, 44–46). While some studies in SCID mice grafted with fetal thymus and liver are in agreement with this concept (33, 34), our previous studies with the human peripheral blood leukocyte-SCID (hu-PBL-SCID) mouse model have shown that infection with M-tropic isolates (e.g., SF162) causes more rapid CD4⁺ T-cell depletion than infection with T-tropic, SI isolates (e.g., SF33), despite similar proviral copy numbers, and that this property mapped to envelope (28, 41, 43). However, the dual-tropic 89.6 isolate (19) caused extremely rapid CD4⁺ T-cell depletion in infected hu-PBL-SCID mice that was associated with an early and transient increase in HIV-1 plasma viral RNA (29). The relationship between cell tropism of the virus isolate and the pattern of disease in hu-PBL-SCID mice is thus uncertain. We have extended these studies by determining the kinetics of HIV-1 RNA levels in serial plasma samples of hu-PBL-SCID mice infected with primary patient isolates or laboratory stocks that differ in cell tropism and SI properties. The results showed significant differences in the kinetics of HIV-1 replication and CD4⁺ T-cell depletion that are determined by the cell tropism of the virus isolate.     

Greater Transport Efficiencies of the Membrane Fatty Acid Transporters FAT/CD36 and FATP4 Compared with FABPpm and FATP1 and Differential Effects on Fatty Acid Esterification and Oxidation in Rat Skeletal Muscle

In selected mammalian tissues, long chain fatty acid transporters (FABPpm, FAT/CD36, FATP1, and FATP4) are co-expressed. There is controversy as to whether they all function as membrane-bound transporters and whether they channel fatty acids to oxidation and/or esterification. Among skeletal muscles, the protein expression of FABPpm, FAT/CD36, and FATP4, but not FATP1, correlated highly with the capacities for oxidative metabolism (r ≥ 0.94), fatty acid oxidation (r ≥ 0.88), and triacylglycerol esterification (r ≥ 0.87). We overexpressed independently FABPpm, FAT/CD36, FATP1, and FATP4, within a normal physiologic range, in rat skeletal muscle, to determine the effects on fatty acid transport and metabolism. Independent overexpression of each fatty acid transporter occurred without altering either the expression or plasmalemmal content of other fatty acid transporters. All transporters increased fatty acid transport, but FAT/CD36 and FATP4 were 2.3- and 1.7-fold more effective than FABPpm and FATP1, respectively. Fatty acid transporters failed to alter the rates of fatty acid esterification into triacylglycerols. In contrast, all transporters increased the rates of long chain fatty acid oxidation, but the effects of FABPpm and FAT/CD36 were 3-fold greater than for FATP1 and FATP4. Thus, fatty acid transporters exhibit different capacities for fatty acid transport and metabolism. In vivo, FAT/CD36 and FATP4 are the most effective fatty acid transporters, whereas FABPpm and FAT/CD36 are key for stimulating fatty acid oxidation.Uptake of long chain fatty acids across the plasma membrane had long been considered to occur via passive diffusion. However, in recent years, there has been a fundamental shift in our understanding, and it is now widely recognized that long chain fatty acids cross the plasma membrane via a protein-mediated mechanism (for reviews, see Refs. 1–3). A number of fatty acid transporters have been identified, including fatty acid translocase/CD36 (FAT/CD36), plasma membrane-associated fatty acid binding proteins (FABPpm), and a family of fatty acid transport proteins (FATP1–6)⁵ (for reviews, see Refs. 1 and 4). Selected stimuli (muscle contraction, insulin, and AICAR) induce the translocation of selected fatty acid transporters (FABPpm, FAT/CD36, and FATP1) from an intracellular depot to the plasma membrane, in both heart and skeletal muscle, resulting in concurrently increased rates of fatty acid transport (for a review, see Ref. 1). Some fatty acid transporters have now also been implicated in the dysregulation of fatty acid metabolism in heart and skeletal muscle in models of insulin resistance and type 1 and 2 diabetes, including FAT/CD36 (5–9), FATP1 (10, 11), and possibly FATP4 (11, 12) but not FABPpm (5–7). Thus, in recent years, it has become widely accepted that (a) long chain fatty acids traverse the plasma membrane via a protein-mediated mechanism and (b) some of the fatty acid transporters are central to the dysregulation in skeletal muscle fatty acid metabolism in obesity and type 2 diabetes.In vivo, many of the fatty acid transporters are frequently co-expressed in different tissues. FAT/CD36 and FABPpm are ubiquitously expressed (1), whereas FATP1–6 exhibit a somewhat tissue-specific distribution pattern (13, 14). The reason for the co-expression of different fatty acid transporters within the same tissue remains unclear. It has been speculated that selected fatty acid transporters may need to interact with each other (15, 16). Alternatively, it is also possible that (a) different fatty acid transporters have discrepant transport capacities, and (b) selected transporters may channel fatty acids differentially to fatty acid oxidation and esterification into triacylglycerols in mammalian tissue.Recent evidence has shown that the transport capacities among FATPs can differ substantially, as revealed by overexpression (14, 17, 18) or knockdown studies (19), but there is little agreement as to which FATP is most effective. Extensive studies by DiRusso et al. (17) in yeast revealed that when FATP1–6 were overexpressed to similar levels (qualitative assessment), FATP4 exhibited 1.7- and 3-fold greater fatty acid transport effectiveness compared with FATP1 and FATP2, respectively, whereas no fatty acid transport capacities were attributable to FATP3, -5, and -6 (17). In contrast, in HEK293 cells, the FATP6 transport capacity was 3- and 6.5-fold greater than FATP1 and FATP4, respectively (14), whereas in 3T3-L1 adipocytes, a fatty acid transport role was evident only for FATP1 and not FATP4 (19). Others have also questioned the transport role of FATP4 (20). These discrepant findings with respect to the transport effectiveness of FATPs may reflect, in part, the use of diverse cell types with ill defined metabolic needs and/or machinery for fatty acid uptake and metabolism. Indeed, several recent reports indicate that fatty acid transport cannot be adequately examined in some cells, because these appear to lack accessory proteins that may be involved in fatty acid transport (21, 22). In addition, extrapolation of results from cultured cells to metabolically important tissue in vivo may also be problematic, since cells and mammalian tissues probably have different requirements for fatty acid utilization, and their regulation of fatty acid uptake may also differ. For example, the mechanisms regulating the acute contraction-induced up-regulation of fatty acid transport and oxidation, such as occurs in heart and skeletal muscle, is probably absent in selected cell cultures.Assessment of fatty acid transporter effectiveness, in vivo, cannot be determined in knock-out animals, since compensatory responses in some fatty acid transporters (FATP1 and -4) occur when another fatty acid transporter (FAT/CD36) has been ablated (23, 24). Thus, the relative effectiveness of selected fatty acid transporters on fatty acid transport in vivo remains unknown. In addition, whether fatty acid transporters channel fatty acids to a particular metabolic fate, as has been suggested based on studies in cultured cells (18, 19, 25), may depend on the cell type being examined.It is desirable to discern the effectiveness of selected fatty acid transporters in mammalian tissues that have a well known system for transporting and utilizing fatty acids and in which fatty acid transporters can be independently up-regulated without disturbing the expression of other fatty acid transporters. These criteria can be satisfied in rat skeletal muscle in which genes can be up-regulated under controlled conditions within a physiologically meaningful range (26–28). Therefore, in the present study, we have compared the independent transport effectiveness of fatty acid transporters (FABPpm, FAT/CD36, FATP1, and FATP4) in skeletal muscle, without disturbing the expression and plasmalemmal content of other fatty acid transporters. In addition, we also examined the contributions of these transporters to fatty acid oxidation and esterification into triacylglycerols. These are the first studies to reveal that in vivo (a) the fatty acid transport effectiveness of fatty acid transporters differs considerably, and (b) in skeletal muscle, these transporters serve to channel fatty acids to oxidation, not esterification into triacylglycerols.