Glycosphingolipid behaviour in complex membranes

Glycosphingolipids, due to their tendency to form laterally separated liquid-ordered phases, possess a high potential for the creation of order in biological membranes. The formation of glycosphingolipid-rich domains within the membrane has profound consequences on the membrane organization at different levels, and on the conformational and biological properties of membrane-associated proteins and multimolecular protein complexes. In this review, we will discuss 1) how glycosphingolipids influence the lateral organization of biological membranes; 2) how glycosphingolipids influence the function of membrane-associated proteins.     

Model analysis of difference between EGF pathway and FGF pathway

The difference in time course of Ras and mitogen activated protein kinase (MAPK) cascade by different growth factors is considered to be the cause of different cellular responses. We have developed the computer simulation of Ras-MAPK signal transduction pathway containing newly identified negative feedback system, Sprouty, and adaptor molecules. Unexpectedly, negative feedback system did not profoundly affect time course of MAPK activation. We propose the key role of fibroblast growth factor receptor substrate 2 (FRS2) in NGF/FGF pathway for sustained MAPK activation. More Grb2-SOS complexes were recruited to the plasma membrane by binding to membrane-bound FRS2 in FGF pathway than in EGF pathway and caused sustained activation of ERK. The EGF pathway with high concentration of EGF receptor also induced sustained MAPK activation, which is consistent with the results in the PC12 cell overexpressing the EGF receptors. The simulated time courses of FRS2 knock-out cells were consistent with those of the reported experimental results.     

A novel biotinylated lipid raft reporter for electron microscopic imaging of plasma membrane microdomains

The submicroscopic spatial organization of cell surface receptors and plasma membrane signaling molecules is readily characterized by electron microscopy (EM) via immunogold labeling of plasma membrane sheets. Although various signaling molecules have been seen to segregate within plasma membrane microdomains, the biochemical identity of these microdomains and the factors affecting their formation are largely unknown. Lipid rafts are envisioned as submicron membrane subdomains of liquid ordered structure with differing lipid and protein constituents that define their specific varieties. To facilitate EM investigation of inner leaflet lipid rafts and the localization of membrane proteins therein, a unique genetically encoded reporter with the dually acylated raft-targeting motif of the Lck kinase was developed. This reporter, designated Lck-BAP-GFP, incorporates green fluorescent protein (GFP) and biotin acceptor peptide (BAP) modules, with the latter allowing its single-step labeling with streptavidin-gold. Lck-BAP-GFP was metabolically biotinylated in mammalian cells, distributed into low-density detergent-resistant membrane fractions, and was readily detected with avidin-based reagents. In EM images of plasma membrane sheets, the streptavidin-gold-labeled reporter was clustered in 20-50 nm microdomains, presumably representative of inner leaflet lipid rafts. The utility of the reporter was demonstrated in an investigation of the potential lipid raft localization of the epidermal growth factor receptor.     

Nuclear membrane disassembly and rupture

The nuclear envelope consists of two membranes traversed by nuclear pore complexes. The outer membrane is continuous with the endoplasmic reticulum. At mitosis nuclear pore complexes are dismantled and membranes disperse. The mechanism of dispersal is controversial: one view is that membranes feed into the endoplasmic reticulum, another is that they vesiculate. Using Xenopus egg extracts, nuclei have been assembled and then induced to breakdown by addition of metaphase extract. Field emission scanning electron microscopy was used to study disassembly. Strikingly, endoplasmic reticulum-like membrane tubules form from the nuclear surface after the addition of metaphase extracts, but vesicles were also observed. Microtubule inhibitors slowed but did not prevent membrane removal, whereas Brefeldin A, which inhibits vesicle formation, stops membrane disassembly, suggesting that vesiculation is necessary. Structures that looked like coated buds were observed and buds were labelled for beta-COP. We show that nuclear pore complexes are dismantled and the pore closed prior to membrane rupturing, suggesting that rupturing is an active process rather than a result of enlargement of nuclear pores.     

Quantitative single particle tracking of NGF-receptor complexes: transport is bidirectional but biased by longer retrograde run lengths

The retrograde transport of nerve growth factor (NGF) in neurite-like processes of living differentiated PC12 cells was studied using streptavidin-quantum dots (QDs) coupled to monobiotin-NGF. These reagents were active in differentiation, binding, internalization, and transport. Ten-35% of the QD-NGF-receptor complexes were mobile. Quantitative single particle tracking revealed a bidirectional step-like motion, requiring intact microtubules, with a net retrograde velocity of 0.054+/-0.020 microm/s. Individual runs had a mean velocity of approximately 0.15 microm/s at room temperature, and the run times were exponentially distributed. The photostability and brightness of QDs permit extended real-time analysis of individual QDbNGF- receptor complexes trafficking within neurites.     

Inhibition of Na,K-ATPase-suppressive activity of translationally controlled tumor protein by sorting nexin 6

Translationally controlled tumor protein (TCTP) has both extra- and intracellular functions. Our group recently reported that TCTP interacts with Na,K-ATPase and suppresses its activity. Our studies led to the identification of sorting nexin 6 (SNX6) which binds with TCTP as a potential negative regulator of TCTP. SNX6 does not interact directly with any cytoplasmic domains of Na,K-ATPase. However, when overexpressed, it restores the Na,K-ATPase activity suppressed by TCTP. This was confirmed by measurements of purified plasma membrane Na,K-ATPase activity after incubation with recombinant TCTP and SNX6. SNX6 alone has no effect on Na,K-ATPase activity, but activates Na,K-ATPase via inhibition of TCTP. Inhibition of endogenous TCTP by the overexpression of SNX6 or knockdown of TCTP expression by siTCTP increased Na,K-ATPase activity above the basal level. The interaction between SNX6 and TCTP thus appears to regulate Na,K-ATPase activity.     

A radioimmunoassay for human epidermal growth factor receptor

The development of a radioimmunoassay (RIA) for the human epidermal growth factor receptor solubilized with nonionic detergents which employs iodinated epidermal growth factor (125I-EGF) as the specific ligand is described. A monoclonal antibody (R1) that binds specifically to human EGF receptors [Waterfield, M. D., et al. (1982) J. Cell Biochem. 20, 149-161] was used to separate solubilized receptors saturated with 125I-EGF from free ligand by absorption to protein A-Sepharose, and the bound radioactivity was determined. The RIA was linear when increasing amounts of solubilized membrane protein were added and, when compared to the standard polyethylene glycol assay, was more reproducible. In addition, the background nonspecific binding obtained in the presence of a hundred-fold excess of unlabeled EGF was less in the RIA. Substitution of normal mouse serum for the monoclonal antibody gave very low nonspecific background ligand binding and avoided the use of large amounts of unlabeled EGF in the assay. Two major classes of binding sites for EGF were observed in membrane preparations from the cervical carcinoma cell line A431 or from normal human placental tissue. These were present in approximately equal amounts, with apparent dissociation constants of 4 X 10(-10) and 4 X 10(-9) M. Upon solubilization with the nonionic detergent Triton X-100, only one class of EGF binding sites was detected in both cases, with a dissociation constant of 3 X 10(-8) M. The RIA can be used to monitor receptor purification and for quantitation of receptor number and affinity in various cell types.     

Regulation of the MVB pathway by SCAMP3

The biogenesis of multivesicular endosomes and the sorting of activated signaling receptors into multivesicular endosomes depend on soluble protein complexes (ESCRT complexes), which transiently interact with the receptor cargo and the endosomal membrane. Previously, it was shown that the transmembrane protein secretory carrier membrane protein (SCAMP) 3, which is present on endosomes, interacts with ESCRT components. Here, we report that SCAMP3 plays a role in the biogenesis of multivesicular endosomes. We find that SCAMP3 plays a role in EGF receptor sorting into multivesicular endosomes and in the formation of intralumenal vesicles within these endosomes in vitro and thus also controls EGF receptor targeting to lysosomes. We also find that SCAMP3 regulates the EGF-dependent biogenesis of multivesicular endosomes. We conclude that the transmembrane protein SCAMP3 has a positive role in sorting into and budding of intralumenal vesicles and thereby controls the process of multivesicular endosome biogenesis.     

Ligand-induced clathrin-mediated endocytosis of the keratinocyte growth factor receptor occurs independently of either phosphorylation or recruitment of eps15

Keratinocyte growth factor receptor (KGFR) is a receptor tyrosine kinase expressed on epithelial cells. Following ligand binding, KGFR is rapidly activated and internalized by clathrin-mediated endocytosis. Among the possible receptor substrates which could be involved in the regulation of KGFR endocytosis and down-modulation, we analyzed here the eps15 protein in view of the proposed general role of eps15 in regulating clathrin-mediated endocytosis as well as that of eps15 tyrosine phosphorylation in the control of regulated endocytosis. Immunoprecipitation and Western blot analysis showed that activated KGFR was not able to phosphorylate eps15, suggesting that eps15 is not a receptor substrate. Double immunofluorescence and confocal microscopy revealed that activated KGFR, differently from epidermal growth factor receptor (EGFR), did not induce recruitment of eps15 to the cell plasma membrane. Microinjection of a monoclonal antibody directed against the C-terminal DPF domain which contains the AP2 binding region of eps15 led to inhibition of both pathways of receptor-mediated endocytosis, the EGFR ligand-induced endocytosis and the transferrin constitutive endocytosis, but did not appear to block the KGFR ligand-induced internalization. Taken together our results indicate that the clathrin-mediated uptake of KGFR is not mediated by eps15.     

The tetraspanin CD9 associates with transmembrane TGF-alpha and regulates TGF-alpha-induced EGF receptor activation and cell proliferation

Transforming growth factor-alpha (TGF-alpha) is a member of the EGF growth factor family. Both transmembrane TGF-alpha and the proteolytically released soluble TGF-alpha can bind to the EGF/TGF-alpha tyrosine kinase receptor (EGFR) and activate the EGFR-induced signaling pathways. We now demonstrate that transmembrane TGF-alpha physically interacts with CD9, a protein with four membrane spanning domains that is frequently coexpressed with TGF-alpha in carcinomas. This interaction was mediated through the extracellular domain of transmembrane TGF-alpha. CD9 expression strongly decreased the growth factor- and PMA- induced proteolytic conversions of transmembrane to soluble TGF-alpha and strongly enhanced the TGF- alpha-induced EGFR activation, presumably in conjunction with increased expression of transmembrane TGF-alpha. In juxtacrine assays, the CD9-induced EGFR hyperactivation by transmembrane TGF-alpha resulted in increased proliferation. In contrast, CD9 coexpression with transmembrane TGF-alpha decreased the autocrine growth stimulatory effect of TGF-alpha in epithelial cells. This decrease was associated with increased expression of the cdk inhibitor, p21(CIP1). These data reveal that the association of CD9 with transmembrane TGF-alpha regulates ligand-induced activation of the EGFR, and results in altered cell proliferation.     

Structure-function studies of mEGF: Probing the type Iβ-turn between residues 25 and 26

The interaction between epidermal growth factor (EGF) and its receptor molecule is not completely understood and has received much attention recently. Studies combining site-directed mutagenesis and NMR spectroscopy have identified a number of EGF residues that are required for activity and are believed to interact directly with the receptor. Instead of focusing on these residues, this study combines site-directed mutagenesis and NMR spectroscopy to probe the role of the type I-bend located between residues 25 and 26 of the N-terminal subdomain of the protein. Ser25 of murine EGF is replaced by Pro in an attempt to stabilize this turn conformation to produce a variant of mEGF with increased activity relative to that for the native protein. Ser25 is also replaced by Ala, which is found at position 25 in human EGF (hEGF), as a more conservative replacement. Receptor binding studies demonstrate that both mutations produce about a 30% reduction in binding affinity, which is shown to result from local changes within the loop or minor perturbations of residues neighboring the loop rather than from long-range perturbations of the-sheet of the N-terminal subdomain. The type I-turn appears to remain intact in both mutants; however, replacement with Pro seems to introduce more flexibility into this region of the protein. These results demonstrate that perturbation of this-turn has little effect on EGF-receptor interactions.Abbreviations EGF epidermal growth factor - h human - m murine - TGF type transforming growth factor - NMR nuclear magnetic resonance - [S2A]mEGF mEGF missing the N-terminal asparagine and with the serine at position 2 replaced by alanine - [S2A,S25A]mEGF and [S2A, S25P]mEGF replacement of serine at position 25 in [S2A]mEGF by alanine and proline, respectively - ¹²⁵I-mEGF ¹²⁵I-labeled mEGF - DMEM Dulbecco's modified Eagle's medium - FCS fetal calf serum - HEPES N-(2-hydroxyethyl)piperazine-N-2-ethanesulfonic acid - BSA bovine serum albumin - COSY correlated spectroscopy - DQCOSY double-quantum filtered COSY - NOESY nuclear Overhauser spectroscopy - NOE nuclear Overhauser effect - TOCSY total correlation spectroscopy - ³ J(H-H^N) vicinal spin-spin coupling constant between amide proton and -proton - DSS 2,2-dimethyl-2-silapentane-5-sulfonate - chemical shift in ppm - ppm parts per million,     

Transforming growth factors (TGFs): Properties and possible mechanisms of action

Transforming growth factors (TGFs) are growth-promoting polypeptides that cause phenotypic transformation and anchorage-independent growth of normal cells. They have been isolated from several human and animal carcinoma and sarcoma cells. One TGF is sarcoma growth factor (SGF) which is released hy murine sarcoma virus-transformed cells. The TGFs interact with epidermal growth factor (EGF) cell membrane receptors. TGFs are not detectable in culture fluids from cells which contain high numbers of free EGF cell membrane receptors. SGF acts as a tumor promoter in cell culture systems and its effect on the transformed phenotype is blocked by retinoids (vitamin A and synthetic analogs). The production of TGFs by transformed cells and the responses of normal cells to the addition of TGFs to the culture medium raise the possibility that cells “autostimulate” their own growth by releasing factors that rebind at the cell surface. The term “autocrine secretion” has been proposed for this type of situation where a cell secretes a hormone-like substance for which it has external cell membrane receptors. The autocrine concept may provide a partial explanation for some aspects of tumor cell progression.     

Membrane protein assembly patterns reflect selection for non-proliferative structures

Membrane proteins that regulate solute movement are often built from multiple copies of an identical polypeptide chain. These complexes represent striking examples of self-assembling systems that recruit monomers only until a prescribed level for function is reached. Here we report that three modes of assembly - distinguished by sequence and stoichiometry - describe all helical membrane protein complexes currently solved to high resolution. Using the 13 presently available non-redundant homo-oligomeric structures, we show that two of these types segregate with protein function: one produces energy-dependent transporters, while the other builds channels for passive diffusion. Given such limited routes to functional complexes, membrane proteins that self-assemble exist on the edge of aggregation, susceptible to mutations that may underlie human diseases.     

The Fluid—Mosaic Model of Membrane Structure: Still relevant to understanding the structure,function and dynamics of biological membranes after more than 40 years

In 1972 the Fluid—Mosaic Membrane Model of membrane structure was proposed based on thermodynamic principals of organization of membrane lipids and proteins and available evidence of asymmetry and lateral mobility within the membrane matrix [S. J. Singer and G. L. Nicolson, Science 175 (1972) 720–731]. After over 40 years, this basic model of the cell membrane remains relevant for describing the basic nano-structures of a variety of intracellular and cellular membranes of plant and animal cells and lower forms of life. In the intervening years, however, new information has documented the importance and roles of specialized membrane domains, such as lipid rafts and protein/glycoprotein complexes, in describing the macrostructure, dynamics and functions of cellular membranes as well as the roles of membrane-associated cytoskeletal fences and extracellular matrix structures in limiting the lateral diffusion and range of motion of membrane components. These newer data build on the foundation of the original model and add new layers of complexity and hierarchy, but the concepts described in the original model are still applicable today. In updated versions of the model more emphasis has been placed on the mosaic nature of the macrostructure of cellular membranes where many protein and lipid components are limited in their rotational and lateral motilities in the membrane plane, especially in their natural states where lipid–lipid, protein–protein and lipid–protein interactions as well as cell–matrix, cell–cell and intracellular membrane-associated protein and cytoskeletal interactions are important in restraining the lateral motility and range of motion of particular membrane components. The formation of specialized membrane domains and the presence of tightly packed integral membrane protein complexes due to membrane-associated fences, fenceposts and other structures are considered very important in describing membrane dynamics and architecture. These structures along with membrane-associated cytoskeletal and extracellular structures maintain the long-range, non-random mosaic macro-organization of membranes, while smaller membrane nano- and submicro-sized domains, such as lipid rafts and protein complexes, are important in maintaining specialized membrane structures that are in cooperative dynamic flux in a crowded membrane plane. This Article is Part of a Special Issue Entitled: Membrane Structure and Function: Relevance in the Cell's Physiology, Pathology and Therapy.     

Distinct roles for ADAM10 and ADAM17 in ectodomain shedding of six EGFR ligands

All ligands of the epidermal growth factor receptor (EGFR), which has important roles in development and disease, are released from the membrane by proteases. In several instances, ectodomain release is critical for activation of EGFR ligands, highlighting the importance of identifying EGFR ligand sheddases. Here, we uncovered the sheddases for six EGFR ligands using mouse embryonic cells lacking candidate-releasing enzymes (a disintegrin and metalloprotease [ADAM] 9, 10, 12, 15, 17, and 19). ADAM10 emerged as the main sheddase of EGF and betacellulin, and ADAM17 as the major convertase of epiregulin, transforming growth factor alpha, amphiregulin, and heparin-binding EGF-like growth factor in these cells. Analysis of adam9/12/15/17-/- knockout mice corroborated the essential role of adam17-/- in activating the EGFR in vivo. This comprehensive evaluation of EGFR ligand shedding in a defined experimental system demonstrates that ADAMs have critical roles in releasing all EGFR ligands tested here. Identification of EGFR ligand sheddases is a crucial step toward understanding the mechanism underlying ectodomain release, and has implications for designing novel inhibitors of EGFR-dependent tumors.     

小鼠胚胎发育过程中表皮生长因子受体的免疫荧光定位研究

用间接免疫荧光法研究了第12—19天小鼠胚胎的表皮生长因子受体(EGFR)在各种组织中的分布。结果表明,特异性荧光出现在EGFR阳性细胞的细胞膜上。第14.5天鼠胚鼻粘膜上皮首先显示很强的EGFR特异性荧光,此后荧光稍为减弱,直至第19天后消失。消化系统中,舌味觉上皮在第16天、胃粘膜上皮在第17—18天间、十二指肠上皮在第17.5—18.5天、直肠粘膜上皮在第15.5—16天和肛管粘膜上皮在第15天均显示强特异性荧光。肝细胞从第14.5天起有弱阳性反应,随胎龄增大强度缓慢地增强。此外,在胚胎发育不同时期,还看到若干组织呈现阳性反应,包括膀胱粘膜变移上皮、眼睑原基、腺垂体上皮、舌下腺腺泡细胞、附属腺上皮细胞、胰岛细胞、颌下腺腺上皮和导管上皮细胞、降主动脉内皮以及卵巢髓质部富含血管的疏松结缔组织中的成纤维细胞等。     

Haptoglobin Genotype-dependent Differences in Macrophage Lysosomal Oxidative Injury

The major function of the Haptoglobin (Hp) protein is to control trafficking of extracorpuscular hemoglobin (Hb) thru the macrophage CD163 receptor with degradation of the Hb in the lysosome. There is a common copy number polymorphism in the Hp gene (Hp 2 allele) that has been associated with a severalfold increased incidence of atherothrombosis in multiple longitudinal studies. Increased plaque oxidation and apoptotic markers have been observed in Hp 2-2 atherosclerotic plaques, but the mechanism responsible for this finding has not been determined. We proposed that the increased oxidative injury in Hp 2-2 plaques is due to an impaired processing of Hp 2-2-Hb complexes within macrophage lysosomes, thereby resulting in redox active iron accumulation, lysosomal membrane oxidative injury, and macrophage apoptosis. We sought to test this hypothesis in vitro using purified Hp-Hb complex and cells genetically manipulated to express CD163. CD163-mediated endocytosis and lysosomal degradation of Hp-Hb were decreased for Hp 2-2-Hb complexes. Confocal microscopy using lysotropic pH indicator dyes demonstrated that uptake of Hp 2-2-Hb complexes disrupted the lysosomal pH gradient. Cellular fractionation studies of lysosomes isolated from macrophages incubated with Hp 2-2-Hb complexes demonstrated increased lysosomal membrane oxidation and a loss of lysosomal membrane integrity leading to lysosomal enzyme leakage into the cytoplasm. Additionally, markers of apoptosis, DNA fragmentation, and active caspase 3 were increased in macrophages that had endocytosed Hp 2-2-Hb complexes. These data provide novel mechanistic insights into how the Hp genotype regulates lysosomal oxidative stress within macrophages after receptor-mediated endocytosis of Hb.     

Both clathrin-positive and -negative coats are involved in endosomal sorting of the EGF receptor

Sorting of endocytosed EGF receptor (EGFR) to internal vesicles of multivesicular bodies (MVBs) depends on sustained activation and ubiquitination of the EGFR. Ubiquitination of EGFR is mediated by the ubiquitin ligase Cbl, being recruited to the EGFR both directly and indirectly through association with Grb2. Endosomal sorting of ubiquitinated proteins further depends on interaction with ubiquitin binding adaptors like Hrs. Hrs localizes to flat, clathrin-coated domains on the limiting membrane of endosomes. In the present study, we have investigated the localization of EGFR, Cbl and Grb2 with respect to coated and non-coated domains of the endosomal membrane and to vesicles within MVBs. Both EGFR, Grb2, and Cbl were concentrated in coated domains of the limiting membrane before translocation to inner vesicles of MVBs. While almost all Hrs was in clathrin-positive coats, EGFR and Grb2 in coated domains only partially colocalized with Hrs and clathrin. The extent of colocalization of EGFR and Grb2 with Hrs and clathrin varied with time of incubation with EGF. These results demonstrate that both clathrin-positive and clathrin-negative electron dense coats exist on endosomes and are involved in endosomal sorting of the EGFR.     

表皮生长因子受体在正常大鼠眼组织中的表达

采用免疫组织化学方法观察了表皮生长因子受体在正常大鼠眼组织的表达。结果发现：角膜上皮的深层细胞和角膜缘上皮细胞、部分结膜上皮细胞和结膜下结缔组织内成纤维细胞均强烈表达表皮生长因子受体。结果显示：表皮生长因子受体阳性细胞主要分布于眼表层组织。这些细胞不仅是眼组织损伤后修复、而且是多种手术能否成功和某些疾病形成中起重要作用的细胞     

Recruitment of Uev1B to Hrs-containing endosomes and its effect on endosomal trafficking

Endocytosis of signaling receptors, such as epidermal growth factor receptor (EGFR), tightly controls the signal transduction process triggered by ligand activation of these receptors. To identify new regulators of the endocytic trafficking of EGFR an RNA interference screen was performed for genes involved in ubiquitin conjugation and down-regulation of EGFR. The screen revealed that small interfering RNAs (siRNAs) that target the conserved ubiquitin-binding domain Uev1 increased down-regulation of EGFR. Since these siRNAs simultaneously targeted multiple genes containing a Uev1 domain, we analyzed the role of these gene products by overexpressing individual Uev1-related proteins. This analysis revealed that overexpression of Uev1A (UBE2V1) has no effect on the degradation of EGFR:EGF complexes. In contrast, overexpression of Uev1B (TMEM189-UBE2V1 isoform 2) slowed the degradation of EGF:receptor complexes. The Uev1B protein was found to strongly colocalize and associate with ubiquitin and Hrs in endosomes. Moreover, overexpression of Uev1B abrogated the ability of Hrs to colocalize with EGFR. The B-domain of Uev1B, and not the UEV-domain, was mainly responsible for the observed phenotypes suggesting the presence of a novel endosomal targeting sequence within the B-domain. Together, the data show that elevated levels of Uev1B protein in cells lead to decreased efficiency of endosomal sorting by associating with ubiquitinated proteins and Hrs.