Jean-Louis Carpentier

Nef-Induced CD4 Degradation: A Diacidic-Based Motif in Nef Functions as a Lysosomal Targeting Signal through the Binding of β-COP in Endosomes

The Nef protein of primate lentiviruses downregulates the cell surface expression of CD4 through a two-step process. First, Nef connects the cytoplasmic tail of CD4 with adaptor protein complexes (AP), thereby inducing the formation of CD4-specific clathrin-coated pits that rapidly endocytose the viral receptor. Second, Nef targets internalized CD4 molecules for degradation. Here we show that Nef accomplishes this second task by acting as a connector between CD4 and the beta subunit of COPI coatomers in endosomes. A sequence encompassing a critical acidic dipeptide, located nearby but distinct from the AP-binding determinant of HIV-1 Nef, is responsible for beta-COP recruitment and for routing to lysosomes. A novel class of endosomal sorting motif, based on acidic residues, is thus revealed, and beta-COP is identified as its downstream partner.

Mechanism of Nef-induced CD4 endocytosis: Nef connects CD4 with the mu chain of adaptor complexes

The Nef protein of primate lentiviruses down‐regulates the cell surface expression of CD4 and probably MHC I by connecting these receptors with the endocytic machinery. Here, we reveal that Nef interacts with the μ chains of adaptor complexes, key components of clathrin‐coated pits. For human immunodeficiency virus type 2 (HIV‐2) and simian immunodeficiency virus (SIV) Nef, this interaction occurs via tyrosine‐based motifs reminiscent of endocytosis signals. Mutating these motifs prevents the binding of SIV Nef to the μ chain of plasma membrane adaptor complexes, abrogates its ability to induce CD4 internalization, suppresses the accelerated endocytosis of a chimeric integral membrane protein harboring Nef as its cytoplasmic domain and confers a dominant‐negative phenotype to the viral protein. Taken together, these data identify μ adaptins as downstream mediators of the down‐modulation of CD4, and possibly MHC I, by Nef.

The HIV-1 Nef protein acts as a connector with sorting pathways in the Golgi and at the plasma membrane

The HIV Nef protein down-regulates the cell surface expression of CD4 and of MHC I at least in part through accelerated endocytosis. To investigate further the mechanism of this effect, we created chimeric integral membrane proteins comprising the extracellular and transmembrane regions of CD4 or CD8 and Nef as the cytoplasmic domain. These fusion molecules could down-modulate CD4 in trans in a dileucine-dependent manner. Furthermore, in spite of lacking receptor-derived internalization signals, the Nef-containing chimeras underwent both Golgi retention and rapid endocytosis via clathrin-coated pits. Taken together, these data suggest that Nef down-regulates CD4 and probably MHC I by physically connecting these receptors with sorting pathways in the Golgi and at the plasma membrane.

CD14-dependent endotoxin internalization via a macropinocytic pathway.

Gram-negative bacterial endotoxin (a lipopolysaccharide (LPS)) specifically binds to CD14, a glycosylphosphatidyl inositol (GPI)-anchored surface myeloid glycoprotein. This interaction leads to cell activation, but it also promotes LPS internalization and detoxification. In this work, we investigated the route of LPS and CD14 internalization and the relevance of CD14 GPI anchor in the endocytic pathway. In promonocytic THP-1 cells transfected with a GPI or a chimeric integral form of CD14, we showed by differential buoyancy in sucrose density gradients that these two forms of CD14 were sorted to different plasma membrane subdomains. However, both forms of CD14 associated preferentially with the same surface microfilament-enriched microvilli or ruffles. Electron microscopic studies indicated that CD14 internalized via macropinocytosis, a process resembling that of phagocytosis, different from “classical” receptor-mediated endocytic pathways, such as clathrin-coated pits or caveolae. With cell warming, the CD14-enriched ruffles fused and formed large vesicles. Later, these vacuoles made stacks and condensed into phago-lysosomes. CD14 was specifically associated with all of these structures. Radiolabeled LPS internalization paralleled CD14 internalization. Confocal microscopic studies confirmed the co-localization of LPS and CD14 both at the cell surface and in endosomal compartments. The microfilament-disrupting, macropinocytosis blocking agent cytochalasin D inhibited LPS and CD14 internalization but did not prevent LPS-dependent activation, indicating that these two processes are dissociated.

The neck of caveolae is a distinct plasma membrane subdomain that concentrates insulin receptors in 3T3-L1 adipocytes.

Insulin receptors (IRs) segregate on plasma membrane microvilli, but in cells devoid of microvilli, such as adipocytes, the localization of IRs is a matter of controversy. In the present study, we examined the distribution of IRs in the plasma membrane of 3T3-L1 adipocytes. Quantitative electron microscopy indicates that IRs are predominantly associated with the neck, but not the bulb, of caveolae. Caveola necks represent distinct microdomains of the plasma membrane. Indeed, as shown by freeze–fracture analysis, intramembrane particles are concentrated as necklaces around the craters of caveolae. In addition, subcellular fractionation suggests that the neck and the bulb of caveolae present a different resistance to detergent solubility. Finally, cytoskeletal components, including actin, are highly enriched in the membrane area underlying the neck part of caveolae. IRs coimmunoprecipitate with cytoskeletal components, and disruption of the actin cytoskeleton alters IRs expression, localization, and signaling, thus supporting the notion that caveola necks are involved in intracellular signaling by IRs. Together, these results suggest that cytoskeletal proteins anchor IRs to microdomains in the caveola necks of 3T3-L1 adipocytes. By homology with IR localization in other cell types, we suggest that the necks of caveolae may represent the counterpart of microvillar domains in cells poor in microvilli such as adipocytes and that they play an important role as signaling platforms.

INSULIN RECEPTOR INTERNALIZATION : MOLECULAR MECHANISMS AND PHYSIOPATHOLOGICAL IMPLICATIONS

SummaryThe initial interaction between insulin and its receptor on target cell surface is followed by a series of surface and intracellular steps which participate in the control of insulin action. Abnormalities of any of these steps could result in mishandling of the receptor leading to defective modulation of receptor number on the cell surface and to inappropriate cell sensitivity to the hormone. Thus, the identification of each of these steps as well as understanding the mechanisms governing them is obligatory to unravel some aspects of the pathogenesis of insulin resistance states. This was the goal of the studies we have carried out during recent years using combined molecular and cellular biology as well as biochemical techniques. These studies allowed us to propose the following ordered sequence of events: 1) insulin binds to receptors preferentially associated with microvilli on the cell surface; 2) insulin triggers receptor kinase activation and autophosphorylation which not only results in initiation of the various biological signals leading to insulin action but also in redistribution of the hormone-receptor complex in the plane of the membrane; 3) on the non-villous domain of the cell surface, insulin receptors anchor to clathrin-coated pits through specific “internalization sequences” present in their cytoplasmic juxtamembrane domain; 4) insulin-receptor complexes are internalized together with other receptors present in the same clathrincoated pits through the formation of clathrin-coated vesicles; 5) the complexes are delivered to endosomes, the acidic pH of which induces the dissociation of insulin molecules from insulin receptors and their sorting in different directions; 6) insulin molecules are targetted to late endosomes and lysosomes where they are degraded; 7) receptors are recycled back to the cell surface in order to be reused.

p56Lck anchors CD4 to distinct microdomains on microvilli

Cell-surface microvilli play a central role in adhesion, fusion, and signaling processes. Some adhesion and signaling receptors segregate on microvilli but the determinants of this localization remain mostly unknown. In this study, we considered CD4, a receptor involved in immune response and HIV infection, and p56Lck, a CD4-associated tyrosine kinase. Analysis of CD4 trafficking reveals that p56Lck binds tightly to CD4 independently of its activation state and inhibits CD4 internalization. Electron microscopy analysis established that p56Lck mediates CD4 association with microvilli whereas biochemical data indicate that p56Lck expression renders CD4 insoluble by the nonionic detergent Triton X-100. In addition, cytoskeleton-disrupting agent increased CD4 solubility, suggesting the involvement of cytoskeletal elements in CD4 anchoring to microvilli. This concept was supported further by the observation that the lateral mobility of CD4 within the plasma membrane was decreased in cells expressing p56Lck. Finally, isolation of detergent-resistant membranes revealed that the complex CD4-p56Lck is enriched within these domains as opposed to conditions in which CD4 does not interact with p56Lck. In conclusion, our results show that p56Lck targets CD4 to specialized lipid microdomains preferentially localized on microvilli. This localization, which prevents CD4 internalization, might facilitate CD4-mediated adhesion processes and could correspond to the signaling site of the receptor.

The cell biology of the insulin receptor

SummaryFollowing initial binding to specific cell surface receptors insulin is internalized in target cells. The fate of the internalized insulin-receptor complexes and how the processes involved are regulated is reviewed. The implications of these events in the effects of insulin on its target cells and in the physiopathology of diabetes and insulin resistance states are also considered.

An arginine to cysteine252 mutation in insulin receptors from a patient with severe insulin resistance inhibits receptor internalisation but preserves signalling events

AbstractAims/hypothesis. We examined the properties of a mutant insulin receptor (IR) with an Arg252 to Cys (IRR252C) substitution in the α-subunit originally identified in a patient with extreme insulin resistance and acanthosis nigricans. Methods. We studied IR cell biology and signalling pathways in Chinese Hamster Ovary cells overexpressing this IRR252C. Results. Our investigation showed an impairment in insulin binding to IRR252C related mostly to a reduced affinity of the receptor for insulin and to a reduced rate of IRR252C maturation; an inhibition of IRR252C-mediated endocytosis resulting in a decreased insulin degradation and insulin-induced receptor down-regulation; a maintenance of IRR252C on microvilli even in the presence of insulin; a similar autophosphorylation of mutant IRR252C followed by IRS 1/IRS 2 phosphorylation, p85 association with IRS 1 and IRS 2 and Akt phosphorylation similar to those observed in cells expressing wild type IR (IRwt); and finally, a reduced insulin-induced Shc phosphorylation accompanied by decreased ERK1/2 phosphorylation and activity and of thymidine incorporation into DNA in cells expressing IRR252C as compared to cells expressing IRwt. Conclusion/interpretation. These observations suggest that: parameters other than tyrosine kinase activation participate in or control the first steps of IR internalisation or both; IR-mediated IRS 1/2 phosphorylation can be achieved from the cell surface and microvilli in particular; Shc phosphorylation and its subsequent signalling pathway might require IR internalisation; defective IR endocytosis correlates with an enhancement of some biological responses to insulin and attenuation of others.

Mechanisms of HIV Receptor and Co-Receptor Down-Regulation by Prostratin: Role of Conventional and Novel PKC Isoforms:

Prostratin is an unusual non-tumour promoting phorbol ester with potential as an inductive adjuvant therapy for highly active antiretroviral therapy (HAART) due to its ability to up-regulate viral expression from latent provirus. In addition, prostratin is also able to inhibit de novo HIV infection most probably because it induces down-regulation of HIV receptors from the surface of target cells. In this study, we investigate the mechanisms by which prostratin down-regulates HIV receptor and co-receptor surface expression in lymphocytic and monocytic cell lines. Our results indicate that prostratin induces down-regulation of surface expression of CD4 and CXCR4, but not CCR5, in various cell lines. Down-regulation of CD4 and CXCR4 by prostratin is achieved by internalization through receptor-mediated endocytosis and/or macropinocytosis, which is then followed by degradation of these molecules. Because prostratin is a protein kinase C (PKC) activator, we next examined the potential contribution of distinct PKC isoforms to down-regulate CD4 and CXCR4 in response to prostratin stimulation. Although exposure of cells to prostratin or phorbol-myristate-acetate (PMA) induces the translocation of several PKC isoforms to the plasma membrane, the use of specific PKC inhibitors revealed that novel PKCs are the main mediators of the prostratin-induced CD4 down-regulation, whereas both conventional and novel PKCs contribute to CXCR4 down-regulation. Altogether these results showed that prostratin, through the activation of conventional and/or novel PKC isoforms, rapidly reduces cell surface expression of CD4 and CXCR4, but not CCR5, by inducing their internalization and degradation.