Ligand-induced rapid redistribution of lysosomes is temporally distinct from endosome translocation

When many ligands bind to cell-surface receptors, ligand-receptor complexes are internalized via clathrin coated pits by a process called receptor-mediated endocytosis. The cytoplasmic fate of ligands internalized within endocytic vesicles or endosomes is variable. For example, maternal immunoglobulins are transported through the cytoplasm of neonatal intestinal epithelial cells and are exocytosed at the basolateral surface. However, other ligands are degraded as a result of their delivery to the lysosomal compartment of cells. Although the translocation of endosomes to the Golgi region in the cell centre seems to be a general phenomenon presumably coupled to ligand degradation by lysosomes and endosomes and lysosomes undergo saltatory movements within the cytoplasm, the spatial control of interaction between the two structures is not understood. To address this problem we have begun to examine the spatial and temporal intracellular distribution of endosomes and lysosomes. Utilizing a new fluorescent microscopic approach, we have now been able simultaneously to visualize endosome and lysosome populations in living cells. Our results suggest that a specific relocation of lysosomes is rapidly induced upon binding of different types of ligands to the cell surface; this migration of lysosomes to the Golgi region of the cell precedes the translocation of endosomes into the same area.     

Impairment of dynamin's GAP domain stimulates receptor-mediated endocytosis

Dynamin is a GTP-hydrolysing protein that is an essential participant in clathrin-mediated endocytosis by cells. It self-assembles into 'collars' in vitro which also formin vivo at the necks of invaginated coated pits. This self-assembly stimulates dynamin's GTPase activity and it has been proposed that dynamin hydrolyses GTP in order to generate the force needed to sever vesicles from the plasma membrane. A mechanism is now described in which self-assembly of dynamin is coordinated by a domain of dynamin with a GTPase-activating function. Unexpectedly, when dynamin mutants defective in self-assembly-stimulated GTPase activity are overexpressed, receptor-mediated endocytosis is accelerated. The results indicate that dynamin, like other members of the GTPase superfamily, functions as a molecular regulator in receptor-mediated endocytosis, rather than as a force-generating GTPase.     

一种新型的紫杉烷生物素共轭物的制备

生物素与肿瘤细胞表面的生物素受体结合,通过受体介导的内吞作用将共轭物摄入细胞内,二硫键在肿瘤细胞中能够开链,释放出活性物质,实现靶向性给药,从而大大降低传统化疗药物因缺乏对肿瘤细胞的选择性而引起的毒副作用。用巯基置换的方式,合成含有二硫键的连接链;用将羧酸基团转化为酰肼基团的方法,将生物素转化为生物素酰肼;选用具有抗多药耐药性的含氟紫杉烷SB-T-12854作为细胞毒分子;通过含有二硫键的连接链将含氟紫杉烷化合物SB-T-12854与生物素酰肼进行偶连,形成一种新型的紫杉烷生物素共轭物。     

Internalization of the human immunodeficiency virus does not require the cytoplasmic domain of CD4

Binding of the human immunodeficiency virus (HIV) to infectable host cells, such as B and T lymphocytes, monocytes and colorectal cells, is mediated by a high-affinity interaction between the gp120 component of the viral envelope glycoprotein and the CD4 receptor. Upon binding, it is thought that the second component of the envelope, gp41, mediates fusion between the viral envelope and host cell membranes. However, the early steps of HIV infection have not yet been thoroughly elucidated. Viral entry was first reported to be mediated by pH-dependent receptor-mediated endocytosis; subsequent studies have shown entry to be pH-independent. Although direct fusion of virus to plasma membranes of infected cells has been observed by electron microscopy, it is still formally possible that the infectious path of the virus involves receptor-mediated endocytosis. To gain a better understanding of receptor function in viral entry, we have analysed the ability of several altered or truncated forms of CD4 to serve as effective viral receptors. Our results indicate that domains beyond the HIV-binding region of CD4 are not required for viral infection. Some of the altered forms of CD4 that serve as effective HIV receptors are severely impaired in their ability to be endocytosed. These experiments therefore support the notion that viral fusion to the plasma membrane is sufficient for infection.     

A protein on Plasmodium falciparum-infected erythrocytes functions as a transferrin receptor

Several observations suggest that iron is essential for the development of malaria parasites but there is evidence that the parasites in erythrocytes do not obtain iron from haemoglobin. The total haemin level in parasitized erythrocytes does not vary during parasite development, indicating that the iron-containing moiety of haemoglobin is not detectably metabolized. Although parasite proteases can degrade the protein part of haemoglobin in red cells, no parasite enzymes that degrade haemin have been identified. In mammalian cells, haemin is degraded to carbon monoxide and bilirubin by the enzyme haeme oxygenase. This enzyme has not been found in malaria parasites. In fact haemin has been found to be toxic to parasite carbohydrate metabolism. Thus, iron apparently cannot be liberated from haemin and instead is sequestered in infected red cells as haemozoin, the characteristic pigment associated with malarial infection. If iron bound to transferrin is the source of ferric ions for malaria parasites within mature erythrocytes, then the parasite must synthesize its own transferrin receptor and localize it on the surface of the infected cell, because the receptors for transferrin are lost during erythrocyte maturation. Our results here suggest that Plasmodium falciparum synthesizes its own transferrin receptors enabling it to take up iron from transferrin by receptor-mediated endocytosis.     

Fc receptor phosphorylation during receptor-mediated control of B-cell activation

It is well known that Fc receptors for IgG (FcRII) on macrophages mediate the endocytosis of antibody-antigen complexes and signal the release of inflammatory and cytotoxic agents. FcRII are also expressed at high levels on B cells where they are less involved in endocytosis than in modulating B-cell activation by membrane immunoglobulins. Although crosslinking of membrane immunoglobulins can result in B-cell differentiation and proliferation through stimulation of phospholipase C, mobilization of intracellular Ca2+, and activation of protein kinase C, crosslinking FcR with membrane immunoglobulins confers a dominant inhibitory signal that prevents or aborts activation. This form of regulation may have a role in the induction of tolerance by IgG and in controlling the B-cell repertoire by anti-idiotypes. The different functions of FcR on B cells and macrophages may reflect the fact that these cell types express closely related but distinct FcR isoforms. We have recently found that the main lymphocyte FcR isoform, FcRII-B1, is unable to mediate endocytosis by way of coated pits and coated vesicles owing to an in-frame insertion of 47 amino acids in its cytoplasmic tail. Here we show that this insert, absent from the FcRII-B2 macrophage isoform, also contains serine phosphorylation sites that may have a role in the ability of FcR to regulate B-cell activation through membrane immunoglobulins.     

Major histocompatibility complex class I molecules internalized via coated pits in T lymphocytes

Endocytosis of the major histocompatibility complex (MHC)-encoded class I and class II molecules has been the subject of recent investigations. Class I molecules, which are key elements in T cell-mediated cytotoxicity, are differentially endocytosed by different cell types. Fibroblasts internalize their class I molecules via uncoated cell surface vesicles and tubular invaginations when these molecules are cross-linked with multivalent ligands. T lymphocytes internalize their class I molecules spontaneously, but B lymphocytes do not internalize them at all. Here we describe a morphological investigation of the mechanism by which class I molecules are endocytosed by T lymphocytes. We show that, unlike fibroblasts, T lymphocytes spontaneously internalize 20-40% of their class I molecules in a process involving coated pits and coated vesicles. Thus, the endocytic pathway of class I molecules in T lymphocytes is similar to those of other more classical cell-surface receptors involved in receptor-mediated endocytosis. In contrast, the same class I molecules remained on the cell surface in B lymphocytes. These data show that class I molecules are differentially regulated in T and B lymphocytes and fibroblasts.     

The major Fc receptor in blood has a phosphatidylinositol anchor and is deficient in paroxysmal nocturnal haemoglobinuria

Fc receptors on phagocytic cells in the blood mediate binding and clearance of immune complexes, phagocytosis of antibody-opsonized microorganisms, and potently trigger effector functions, including superoxide anion production and antibody-dependent cellular cytotoxicity. The Fc receptor type III (Fc gamma R III, CD 16), present in 135,000 sites per cell 1 on neutrophils and accounting for most of FcR in blood, unexpectedly has a phosphatidylinositol glycan (PIG) membrane anchor. Deficiency of Fc gamma R III is observed in paroxysmal nocturnal haemoglobinuria (PNH), an acquired abnormality of haematopoietic cells affecting PIG tail biosynthesis or attachment, and is probably responsible for circulating immune complexes and susceptibility to bacterial infections associated with this disease. Although a growing number of eukaryotic cell-surface proteins with PIG-tails are being described, none has thus far been implicated in receptor-mediated endocytosis or in triggering of cell-mediated killing. Our findings on the Fc gamma R III raise the question of how a PIG-tailed protein important in immune complex clearance in vivo and in antibody-dependent killing mediates ligand internalization and cytotoxicity. Together with our results, previous functional studies on Fc gamma R III and Fc gamma R II suggest that these two receptors may cooperate and that the type of membrane anchor is an important mechanism whereby the functional capacity of surface receptors can be regulated.     

Chloroquine and ammonium chloride prevent terminal glycosylation of immunoglobulins in plasma cells without affecting secretion

The generation of an acidic pH in intracellular organelles is required for several membrane and protein recycling processes. For instance, the internalization of ligands by receptor-mediated endocytosis is followed by the development of an acidic pH inside endosomes; this allows dissociation of the ligand, which is then transported to the lysosomes, from the receptor, which is recycled to the cell surface. There is evidence that part of this recycling process involves the distal region of the Golgi complex, where terminal glycosylation occurs: when the plasma membrane transferrin receptor is desialylated by neuraminidase treatment, it acquires new sialic acid molecules after endocytosis and before cell-surface re-expression. Golgi membranes have been shown to contain a proton pump and the distal Golgi cisternae appear to have an acidic content. Here, we have studied the effects of chloroquine and ammonium chloride, which raise the pH of acidic intracellular compartments, on the processing and secretion of immunoglobulins by plasma cells. Sialic acid transfer to terminal galactose residues, a reaction known to occur in the distal Golgi shortly before secretion, is completely and rapidly inhibited in the presence of these drugs, without significant modification of the secretion rate. This effect is accompanied by a dilatation of the Golgi cisternae and is not rapidly reversible.     

Transglutaminase is essential in receptor-mediated endocytosis of alpha 2-macroglobulin and polypeptide hormones

The receptor-mediated endocytosis of alpha 2-macroglobulin can be inhibited by a diverse group of chemical compounds all of which share the property of being inhibitors of one form of cellular transglutaminase. The present results strongly suggest that protein cross-linking may be essential for receptor-mediated endocytosis of some protein and polypeptide hormones.     

Murine leukaemogenesis: monoclonal antibodies to T-cell determinants arrest T-lymphoma cell proliferation

We have proposed a receptor-mediated leukaemogenesis hypothesis wherein T lymphomas would be clones of T cells bearing mitogen-linked surface receptors specific for the envelope determinants of the inducing MuLV. A prediction of the hypothesis is that T-lymphoma proliferation is dependent on continued presentation of MuLV envelope determinants to these cell-surface receptors, and that substances which interfere with receptor-virus interactions should inhibit T-lymphoma proliferation. Rat monoclonal antibodies were raised to the AKR mouse T lymphoma KKT-2, and these antibodies were screened independently for blockade of virus-binding and for cytostatic activity on KKT-2 cells. We report here that those monoclonal antibodies which block virus binding inhibit growth of KKT-2 cells in vitro, whereas monoclonal antibodies which bind to these cells but do not block virus binding are not cytostatic. Three of the four cytostatic antibodies detect determinants on the Thy-1 molecule, while none of the other (noncytostatic) antibodies detect Thy-1. Antibody inhibition of KKT-2 cell growth is precluded by saturation of KKT-2 virus receptors with the inducing leukaemia virus.     

Apolipoprotein-mediated pathways of lipid antigen presentation

Peptide antigens are presented to T cells by major histocompatibility complex (MHC) molecules, with endogenous peptides presented by MHC class I and exogenous peptides presented by MHC class II. In contrast to the MHC system, CD1 molecules bind lipid antigens that are presented at the antigen-presenting cell (APC) surface to lipid antigen-reactive T cells. Because CD1 molecules survey endocytic compartments, it is self-evident that they encounter antigens from extracellular sources. However, the mechanisms of exogenous lipid antigen delivery to CD1-antigen-loading compartments are not known. Serum apolipoproteins are mediators of extracellular lipid transport for metabolic needs. Here we define the pathways mediating markedly efficient exogenous lipid antigen delivery by apolipoproteins to achieve T-cell activation. Apolipoprotein E binds lipid antigens and delivers them by receptor-mediated uptake into endosomal compartments containing CD1 in APCs. Apolipoprotein E mediates the presentation of serum-borne lipid antigens and can be secreted by APCs as a mechanism to survey the local environment to capture antigens or to transfer microbial lipids from infected cells to bystander APCs. Thus, the immune system has co-opted a component of lipid metabolism to develop immunological responses to lipid antigens.     

ClC-5 Cl- -channel disruption impairs endocytosis in a mouse model for Dent's disease

Dent's disease is an X-linked disorder associated with the urinary loss of low-molecular-weight proteins, phosphate and calcium, which often leads to kidney stones. It is caused by mutations in ClC-5, a renal chloride channel that is expressed in endosomes of the proximal tubule. Here we show that disruption of the mouse clcn5 gene causes proteinuria by strongly reducing apical proximal tubular endocytosis. Both receptor-mediated and fluid-phase endocytosis are affected, and the internalization of the apical transporters NaPi-2 and NHE3 is slowed. At steady state, however, both proteins are redistributed from the plasma membrane to intracellular vesicles. This may be caused by an increased stimulation of luminal parathyroid hormone (PTH) receptors owing to the observed decreased tubular endocytosis of PTH. The rise in luminal PTH concentration should also stimulate the hydroxylation of 25(OH) vitamin D3 to the active hormone. However, this is counteracted by a urinary loss of the precursor 25(OH) vitamin D3. The balance between these opposing effects, both of which are secondary to the defect in proximal tubular endocytosis, probably determines whether there will be hypercalciuria and kidney stones.     

Ectopic beta-chain of ATP synthase is an apolipoprotein A-I receptor in hepatic HDL endocytosis

The effect of high-density lipoprotein (HDL) in protecting against atherosclerosis is usually attributed to its role in 'reverse cholesterol transport'. In this process, HDL particles mediate the efflux and the transport of cholesterol from peripheral cells to the liver for further metabolism and bile excretion. Thus, cell-surface receptors for HDL on hepatocytes are chief partners in the regulation of cholesterol homeostasis. A high-affinity HDL receptor for apolipoprotein A-I (apoA-I) was previously identified on the surface of hepatocytes. Here we show that this receptor is identical to the beta-chain of ATP synthase, a principal protein complex of the mitochondrial inner membrane. Different experimental approaches confirm this ectopic localization of components of the ATP synthase complex and the presence of ATP hydrolase activity at the hepatocyte cell surface. Receptor stimulation by apoA-I triggers the endocytosis of holo-HDL particles (protein plus lipid) by a mechanism that depends strictly on the generation of ADP. We confirm this effect on endocytosis in perfused rat liver ex vivo by using a specific inhibitor of ATP synthase. Thus, membrane-bound ATP synthase has a previously unsuspected role in modulating the concentrations of extracellular ADP and is regulated by a principal plasma apolipoprotein.     

Direct evidence that reverse cholesterol transport is mediated by high-density lipoprotein in rabbit

Mammalian cells obtain cholesterol for membrane synthesis mostly via the receptor-mediated endocytosis of low-density lipoprotein (LDL). Macrophages and vascular endothelium additionally have receptors that recognize certain modified forms of LDL (for example, acetyl-LDL). The process by which cholesterol returns from peripheral cells to hepatocytes (reverse cholesterol transport) has not been established; although tissue culture studies have favoured high-density lipoprotein (HDL) as the principal vehicle, the in vivo evidence for this is meagre. When cholesterol-loaded macrophages are incubated in medium containing plasma, cholesterol moves from the cells to HDL and is then esterified by lecithin/cholesterol acyltransferase. The accumulation of cholesteryl esters in the particles increases their size and decreases their density; enrichment with apoprotein E (apo E) also occurs, producing a decrease in electrophoretic mobility. We now report that similar changes occur in the circulating HDL of rabbits, when their peripheral tissues are loaded with cholesterol by intravenous (i.v.) injection of acetylated or native human LDL. This result suggests that HDL is involved in reverse cholesterol transport in vivo.     

Polyamines regulate calcium fluxes in a rapid plasma membrane response

Activation of cell-surface receptors often evokes changes in Ca2+ fluxes leading to an increase in cytosolic Ca2+, a generally accepted mediator of many cell responses. The molecular mechanisms by which surface agonists elicit these changes in Ca2+ flux have remained elusive. An increase in the polyamines putrescine, spermidine and spermine, and their rate-regulating, synthetic enzyme ornithine decarboxylase (ODC), is one of the earliest events that occur during cell growth, replication and differentiation. However, the precise physiological roles of the polyamines remain enigmatic. Recently, we found that testosterone induces an early (less than 60s), Ca2+- and receptor-dependent stimulation of endocytosis, hexose transport and amino acid transport in mouse kidney cortex involving the proximal tubules. This response is associated with increased Ca2+ fluxes and a mobilization of intracellular calcium, and is thought to represent a direct, receptor-mediated action of testosterone on the surface membrane. Polyamine synthesis was previously found to be essential for the long-term effects of testosterone on mouse kidney. We now report that testosterone evokes a rapid (less than 30 s), transient increase in ODC activity and a sustained increase in polyamines in kidney cortex. This polyamine synthesis is obligatory for stimulation of membrane transport functions and Ca2+ fluxes. These findings form the basis for a new theory of information flow in stimulus-response coupling in which the polyamines serve as messengers to generate a Ca2+ signal by increasing Ca2+ influx and mobilizing intracellular calcium via a cation-exchange reaction.     

The GTP-binding protein, Go, regulates neuronal calcium channels

In neuronal cells, opioid peptides and opiates inhibit neurotransmitter release, which is a calcium-dependent process. They also inhibit adenylyl cyclase, presumably via the membrane signal-transducing component, Gi, a guanine nucleotide-binding protein (G-protein). No causal relationship between these two events has yet been demonstrated. Besides Gi, membranes of neuronal tissues contain large amounts of Go, a G-protein with unknown function. Both G-proteins are heterotrimers consisting of alpha-, beta- and gamma-subunits; the alpha-subunits can be ADP-ribosylated by an exotoxin from Bordetella pertussis (PT), which modification inhibits receptor-mediated activation of the G-protein. It was recently shown that noradrenaline, dopamine and gamma-aminobutyric acid (GABA) inhibit the voltage-dependent calcium channels in dorsal root and sympathetic ganglia; this inhibition is mimicked by intracellular application of guanine nucleotides and blocked by PT, suggesting the involvement of a G-protein. Here we report an inhibitory effect of the opioid D-Ala2, D-Leu5-enkephalin (DADLE) on the calcium current (ICa) in neuroblastoma X glioma hybrid cells (N X G cells). Pretreatment with PT almost completely abolishes the DADLE effect. The effect is restored by intracellular application of Gi and Go. As the alpha-subunit of Go (with or without beta-gamma complex) is 10 times more potent than Gi, we propose that Go is involved in the functional coupling of opiate receptors to neuronal voltage-dependent calcium channels.     

Crystal structure of a complex between anthrax toxin and its host cell receptor

Anthrax toxin consists of the proteins protective antigen (PA), lethal factor (LF) and oedema factor (EF). The first step of toxin entry into host cells is the recognition by PA of a receptor on the surface of the target cell. Subsequent cleavage of receptor-bound PA enables EF and LF to bind and form a heptameric PA63 pre-pore, which triggers endocytosis. Upon acidification of the endosome, PA63 forms a pore that inserts into the membrane and translocates EF and LF into the cytosol. Two closely related host cell receptors, TEM8 and CMG2, have been identified. Both bind to PA with high affinity and are capable of mediating toxicity. Here, we report the crystal structure of the PA-CMG2 complex at 2.5 A resolution. The structure reveals an extensive receptor-pathogen interaction surface mimicking the non-pathogenic recognition of the extracellular matrix by integrins. The binding surface is closely conserved in the two receptors and across species, but is quite different in the integrin domains, explaining the specificity of the interaction. CMG2 engages two domains of PA, and modelling of the receptor-bound PA63 heptamer suggests that the receptor acts as a pH-sensitive brace to ensure accurate and timely membrane insertion. The structure provides new leads for the discovery of anthrax anti-toxins, and should aid the design of cancer therapeutics.     

Peptide binding to empty HLA-B27 molecules of viable human cells

Intracellular binding of antigenic peptides by polymorphic class I major histocompatibility complex molecules creates the ligands recognized by receptors of CD8+ T cells. Previously described in vitro assays of peptide binding to class I molecules have been limited by either the low proportion of accessible binding sites or the lack of allelic specificity. Here we describe a system in which the human class I molecule HLA-B27 binds considerable amounts of an influenza peptide with precise allelic discrimination. Binding requires viable cells, is stimulated by gamma-interferon and is inhibited by brefeldin A. Our results are consistent with the presence of fairly stable 'empty' HLA-B27 molecules at the cell surface. By contrast, analysis of the binding of a second influenza peptide indicates that empty HLA-Aw68 molecules are relatively short-lived. We speculate that HLA-B27 might bind extracellular peptides in vivo and that this property could underlie its association with autoimmune disease.     

A complementary DNA clone for a macrophage-lymphocyte Fc receptor

Macrophages, granulocytes and many lymphocytes express or secrete receptors for the Fc domain of immunoglobulins (Ig). These Fc receptors (FcRs) are heterogeneous and can be distinguished on the basis of their cellular distribution and specificities for different immunoglobulin isotypes. Although their functions are not completely understood, FcRs are known to be involved in triggering various effector cell functions and in regulating differentiation and development of B-cells. One of the best characterized is the mouse macrophage-lymphocyte receptor for IgG1 and IgG2b (ref. 5). On macrophages, this FcR mediates the endocytosis of antibody-antigen complexes via coated pits and coated vesicles, the phagocytosis of Ig-coated particles, and the release of various inflammatory and cytotoxic agents. It is possible that the receptor possesses an intrinsic ligand-activated ion channel activity responsible for some of these functions. The IgG1/IgG2b FcR has been isolated and shown to be a transmembrane glycoprotein of relative molecular mass (Mr) 47,000-60,000 (47-60 K) containing four N-linked oligosaccharide chains and a large (greater than 10K) cytoplasmic domain. It is also immunologically indistinguishable from the murine Ly-17 alloantigen which, in turn, is tightly linked to the Mls lymphocyte activation locus. Here we describe the isolation and characterisation of a complementary DNA clone encoding the whole of the IgG1/IgG2b FcR expressed by the mouse macrophage-like cell line P388D1. The receptor is a member of the immunoglobulin superfamily and, like Ly-17, maps to the distal portion of chromosome 1. cDNA probes detect one or two mRNA species in FcR+ macrophage and B-cell lines, but not in FcR- cells or a receptor-deficient variant derived from a FcR+ B-cell line. Finally, DNA hybridization analysis indicates the receptor gene is partially deleted or rearranged in the FcR- variant.