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Biomedical subjects

A Sorkin

Publications and source records attributed to A Sorkin.

At least 19 recordsLinked to original sources
PubMed2001

Cost of immunization with a locally produced, oral cholera vaccine in Viet Nam.

Policy decisions regarding whether to incorporate new vaccines into routine public health practice in developing countries will depend in part on the costs of vaccine purchase and of vaccine delivery. In March, 1997, a large-scale effectiveness trial of a locally produced, orally administered bivalent vaccine against Vibrio cholerae 01 and 0139 began in Viet Nam. Empirical data obtained from the trial was used to determine the costs of the immunization campaign from the government perspective. The study population, including the children less than one year of age and pregnant women who were ineligible for immunization, was 353926. A total of 289041 persons received two doses of vaccine, and 13340 persons received one dose of vaccine. Two-dose vaccine coverage was 83.4%. The total cost of vaccine delivery during the immunization campaign was $66527. The cost of each dose of vaccine was $0.31. Therefore, the total cost of the immunization campaign was $0.44 per dose administered, and $0.91 per fully immunized person. Attempts to reduce the cost per dose of vaccine (e.g. the use of a monovalent vaccine against serogroup 01) are likely to have a large impact on the cost of future similar immunization campaigns.

Administration, Oral↗
PubMed2001

Persistent interactions between the two transmembrane clusters dictate the targeting and functional assembly of adenylyl cyclase.

Adenylyl cyclases possess complex structures like those of the ATP binding cassette (ABC) transporter family, which includes the cystic fibrosis transmembrane regulator, the P-glycoprotein, and ATP-sensitive K(+) channels [1-4]. These structures comprise a cytosolic N terminus followed by two tandem six-transmembrane cassettes, each associated with a highly homologous (ATP binding) cytosolic loop [5-8]. The catalytic domains, which are located in the two large cytoplasmic loops, are highly conserved and well studied. The crystal structure of these domains has even been described recently [9, 10]. However, nothing is known of the function or organization of the 12 transmembrane segments. In the present study we adopted a range of strategies including live-cell fluorescence resonance energy transfer (FRET) microscopy, coimmunoprecipitation, and functional assays of various truncated and substituted, fluorescently-tagged molecules to analyze the trafficking and activity of this molecule. When expressed as individual peptides, the two transmembrane domains - largely independently of any cytosolic region - formed a tight complex that was delivered to the plasma membrane. This cooperation between the two intact transmembrane domains was essential and sufficient to target the enzyme to the plasma membrane of the cell. The extracellular loop between the ninth and tenth transmembrane segments, which contains an N-glycosylation site, was also necessary. Furthermore, the interaction between the two transmembrane clusters played a critical role in bringing together the cytosolic catalytic domains to express functional adenylyl cyclase activity in the intact cell.

Adenylyl Cyclases↗
PubMed2001

Trafficking of yellow-fluorescent-protein-tagged mu1 subunit of clathrin adaptor AP-1 complex in living cells.

Clathrin adaptor protein AP-1 complex is thought to function in forming clathrin-coated vesicles at the trans-Golgi network (TGN) and mediating transport of cargo between the TGN and endosomes. To study trafficking of AP-1 in living cells, yellow fluorescent protein (YFP) was inserted in the middle of mu1 A subunit of AP-1. When expressed in a tetracycline-dependent manner in HeLa cells, YFP-mu1 was efficiently incorporated into the AP-1 complex, replacing endogenous mu1 in most of cellular AP-1. Time-lapse imaging revealed that YFP-mu1/AP-1 departs from TGN as isolated vesicles and spherical structures, or varicosities, associated with fine tubular processes. Typically, several vesicles or varicosities were seen moving sequentially along the same 'tracks' from TGN to cell periphery. These data suggest that AP-1 may function after formation of Golgi transport intermediates in facilitating their intracellular movement. Mutagenesis of YFP-mu1 determined that the structural requirements for its binding to tyrosine-containing sequence motifs are similar to those previously defined in mu2 subunit of AP-2. Moreover, the carboxyl-terminal half of mu2 could replace the corresponding fragment of mu1 without loss of the ability of the resulting mu1-YFP-mu2 chimeric protein to incorporate into AP-1 and bind tyrosine-containing motifs. Mutations that abolish binding capacity for tyrosine motifs did not mistarget AP-1 in the cell, suggesting that AP-1 interactions with this type of sorting signals are not essential for membrane docking of AP-1 at the TGN. Altogether, this study demonstrates that YFP-tagged mu1 protein can serve as a useful tool for visualizing the dynamics of AP-1 in living cells and for the structure-function analysis of mu1-cargo interactions.

Bacterial Proteins↗
PubMed2001

Internalization of the epidermal growth factor receptor: role in signalling.

The interaction of the activated epidermal growth factor (EGF) receptor (EGFR) with the Src homology 2 (SH2) domain of Grb2 (growth-factor-receptor-bound protein 2) initiates signalling through Ras and mitogen-activated protein kinase. Grb2 can bind EGFR directly or through another SH2-containing protein, Shc. Activation of EGFRs by ligand also triggers rapid endocytosis of EGF-receptor complexes. To analyse the spatial and temporal regulation of EGFR interactions with SH2 domains in living cells, we have combined imaging microscopy with a modified method of measuring fluorescence resonance energy transfer (FRET) on a pixel-by-pixel basis using EGFR fused to cyan fluorescent protein (CFP) in pair with Grb2 or Shc fused to yellow fluorescent protein (YFP). Stimulation by EGF resulted in the recruitment of Grb2-YFP and YFP-Shc to cellular compartments that contained EGFR-CFP, and a large increase in the FRET signal. In particular, FRET measurements indicated that activated EGFR-CFP interacted with YFP-Shc and Grb2-YFP in membrane ruffles and endosomes. These results demonstrate that signalling via EGFRs can occur in the endosomal compartment. Moreover, in contrast with previous biochemical studies, FRET experiments show that a large pool of Grb2 and Shc is associated with EGFRs for a prolonged period after EGF stimulation.

3T3 Cells↗
PubMed2000

Interaction of EGF receptor and grb2 in living cells visualized by fluorescence resonance energy transfer (FRET) microscopy.

The interaction of activated epidermal growth factor receptor (EGFR) with the Src homology 2 (SH2) domain of the growth-factor-receptor binding protein Grb2 initiates signaling through Ras and mitogen-activated protein kinase (MAP kinase) [1,2]. Activation of EGFRs by ligand also triggers rapid endocytosis of EGF-receptor complexes. To analyze the spatiotemporal regulation of EGFR-Grb2 interactions in living cells, we have combined imaging microscopy with a modified method of measuring fluorescence resonance energy transfer (FRET) on a pixel-by-pixel basis using EGFR fused to cyan fluorescent protein (CFP) and Grb2 fused to yellow fluorescent protein (YFP). Efficient energy transfer between CFP and YFP should only occur if CFP and YFP are less than 50A apart, which requires direct interaction of the EGFR and Grb2 fused to these fluorescent moieties [3]. Stimulation by EGF resulted in the recruitment of Grb2-YFP to cellular compartments that contained EGFR-CFP and a large increase in FRET signal amplitude. In particular, FRET measurements indicated that activated EGFR-CFP interacted with Grb2-YFP in membrane ruffles and endosomes. These results demonstrate that signaling via EGFRs can occur in the endosomal compartment. The work also highlights the potential of FRET microscopy in the study of subcellular compartmentalization of protein-protein interactions in living cells.

Adaptor Proteins, Signal Transducing↗
PubMed2000

RLIP76, an effector of the GTPase Ral, interacts with the AP2 complex: involvement of the Ral pathway in receptor endocytosis.

RLIP76 is a modular protein that was identified as a putative effector of Ral, a GTPase activated during Ras signaling. To explore further the contribution of the Ral-RLIP76 pathway to Ras signaling, we have looked for partners of RLIP76. Mu2, the medium chain of the AP2 complex is shown to interact with RLIP76. We show also that in vivo endogenous AP2 and RLIP76 form a complex and that this in vivo interaction is independent of cells being stimulated by a growth factor. Furthermore, RLIP76 differentiates AP2 from AP1 in vivo as RLIP76 differentiates mu2 from mu1 in vitro and in two hybrid assays. We show that activated Ral interferes with both tranferrin receptor endocytosis and epidermal growth factor (EGF) receptor endocytosis in HeLa cells. We propose a model where the Ral-RLIP76 pathway connects signal transduction and endocytosis through interaction on one hand between the Ras-Ral pathway and RLIP, on the other hand between RLIP and proteins belonging to the endocytotic machinery.

ATP-Binding Cassette Transporters↗
PubMed1999

Inhibition of the receptor-binding function of clathrin adaptor protein AP-2 by dominant-negative mutant mu2 subunit and its effects on endocytosis.

Although interactions between the mu2 subunit of the clathrin adaptor protein complex AP-2 and tyrosine-based internalization motifs have been implicated in the selective recruitment of cargo molecules into coated pits, the functional significance of this interaction for endocytosis of many types of membrane proteins remains unclear. To analyze the function of mu2-receptor interactions, we constructed an epitope-tagged mu2 that incorporates into AP-2 and is targeted to coated pits. Mutational analysis revealed that Asp176 and Trp421 of mu2 are involved in the interaction with internalization motifs of TGN38 and epidermal growth factor (EGF) receptor. Inducible overexpression of mutant mu2, in which these two residues were changed to alanines, resulted in metabolic replacement of endogenous mu2 in AP-2 complexes and complete abrogation of AP-2 interaction with the tyrosine-based internalization motifs. As a consequence, endocytosis of the transferrin receptor was severely impaired. In contrast, internalization of the EGF receptor was not affected. These results demonstrate the potential usefulness of the dominant-interfering approach for functional analysis of the adaptor protein family, and indicate that clathrin-mediated endocytosis may proceed in both a mu2-dependent and -independent manner.

Adaptor Protein Complex 1↗
PubMed1999

Clathrin assembly lymphoid myeloid leukemia (CALM) protein: localization in endocytic-coated pits, interactions with clathrin, and the impact of overexpression on clathrin-mediated traffic.

The clathrin assembly lymphoid myeloid leukemia (CALM) gene encodes a putative homologue of the clathrin assembly synaptic protein AP180. Hence the biochemical properties, the subcellular localization, and the role in endocytosis of a CALM protein were studied. In vitro binding and coimmunoprecipitation demonstrated that the clathrin heavy chain is the major binding partner of CALM. The bulk of cellular CALM was associated with the membrane fractions of the cell and localized to clathrin-coated areas of the plasma membrane. In the membrane fraction, CALM was present at near stoichiometric amounts relative to clathrin. To perform structure-function analysis of CALM, we engineered chimeric fusion proteins of CALM and its fragments with the green fluorescent protein (GFP). GFP-CALM was targeted to the plasma membrane-coated pits and also found colocalized with clathrin in the Golgi area. High levels of expression of GFP-CALM or its fragments with clathrin-binding activity inhibited the endocytosis of transferrin and epidermal growth factor receptors and altered the steady-state distribution of the mannose-6-phosphate receptor in the cell. In addition, GFP-CALM overexpression caused the loss of clathrin accumulation in the trans-Golgi network area, whereas the localization of the clathrin adaptor protein complex 1 in the trans-Golgi network remained unaffected. The ability of the GFP-tagged fragments of CALM to affect clathrin-mediated processes correlated with the targeting of the fragments to clathrin-coated areas and their clathrin-binding capacities. Clathrin-CALM interaction seems to be regulated by multiple contact interfaces. The C-terminal part of CALM binds clathrin heavy chain, although the full-length protein exhibited maximal ability for interaction. Altogether, the data suggest that CALM is an important component of coated pit internalization machinery, possibly involved in the regulation of clathrin recruitment to the membrane and/or the formation of the coated pit.

Adaptor Proteins, Vesicular Transport↗
PubMed1999

Clathrin, adaptors and eps15 in endosomes containing activated epidermal growth factor receptors.

Activation of the epidermal growth factor receptor (EGFR) by EGF results in binding of clathrin adaptor protein complex AP-2 to the receptor cytoplasmic tail. The transient interaction with AP-2 is thought to be responsible for the selective recruitment of the EGFR into coated pits during endocytosis. In this study we found that EGF-induced EGFR/AP-2 association, measured by co-immunoprecipitation, persists after receptor internalization. Double-label immunofluorescence of EGF-treated A-431 and COS-1 cells revealed the presence of AP-2, clathrin and eps15, another component of the plasma membrane coated pits, in the large perinuclear endosomes loaded with EGFRs. By optical sectioning and image deconvolution, the immunoreactivities were seen to be distributed within vesicular and tubular elements of these endosomes. In addition, these compartments contained the transferrin receptors and a EEA.1 protein, markers of early endosomes. Furthermore, Golgi clathrin adaptor complex AP-1 was found in EGFR-containing endosomes and EGFR immunoprecipitates in A-431 cells. The direct interaction of the EGFR with micro1 as well as micro2 subunits of AP-1 and AP-2, correspondingly, was shown using the yeast two-hybrid assay. Brefeldin A, a drug that releases AP-1 from the trans-Golgi membranes, had no effect on AP-1 association with endosomes and its co-precipitation with EGFR. Taken together, the data suggest that endosomal EGFR-AP complexes make up a significant portion of the total amount of these complexes detectable by co-immunoprecipitation. It can be proposed that APs are capable of binding to the endosomal membrane via a mechanism that requires AP interaction with the intracellular tails of multimeric receptors like activated EGFR, which in turn allows recruitment of clathrin and eps15. The hypothesis that the competition between adaptor complexes for binding to the receptor tails in endosomes may regulate of the sorting of receptors is discussed.

Adaptor Protein Complex alpha Subunits↗
PubMed1998

Endocytosis of functional epidermal growth factor receptor-green fluorescent protein chimera.

A chimera of the epidermal growth factor receptor (EGFR) and green fluorescent protein (GFP) has been engineered by fusing GFP to the carboxyl terminus of EGFR. Data are provided to demonstrate that the GFP moiety does not affect the expected functioning of EGFR. EGFR-GFP becomes phosphorylated at tyrosine residues in response to EGF and is capable of phosphorylating endogenous substrates and initiating signaling cascades. EGF-dependent association of the chimeric receptor with the clathrin adaptor protein AP-2, involved in endocytosis, and with Shc adaptor protein, which binds in close proximity to the fusion point, is not affected by the GFP moiety. Receptor down-regulation and internalization occur at rates similar to those in cells expressing wild-type EGFR. Western blot analysis reveals that lysosomal degradation of EGFR-GFP proceeds from the extracellular domain and that GFP is not preferentially cleaved. Time-dependent co-localization of EGFR-GFP and Texas Red-conjugated EGF in living cells using digital deconvolution microscopy demonstrates the trafficking of ligand-receptor complexes through the early and multivesicular endosomes followed by segregation of the ligand and receptor at the late stages of endocytosis. Time-lapse optical analysis of the early stages of endocytosis reveals localization of EGFR-GFP in the tubular-vesicular endosomal compartments. Rapid dynamics of membrane movement and fusion within these compartments were observed. This approach and the fidelity of the biochemical properties of the EGFR-GFP demonstrate that real-time visualization of trafficking and protein interactions of tyrosine kinase receptors in the presence or absence of the ligand are feasible.

Biological Transport↗
PubMed1998

Structure and Asn-Pro-Phe binding pocket of the Eps15 homology domain.

Eps15 homology (EH) domains are eukaryotic signaling modules that recognize proteins containing Asn-Pro-Phe (NPF) sequences. The structure of the central EH domain of Eps15 has been solved by heteronuclear magnetic resonance spectroscopy. The fold consists of a pair of EF hand motifs, the second of which binds tightly to calcium. The NPF peptide is bound in a hydrophobic pocket between two alpha helices, and binding is mediated by a critical aromatic interaction as revealed by structure-based mutagenesis. The fold is predicted to be highly conserved among 30 identified EH domains and provides a structural basis for defining EH-mediated events in protein trafficking and growth factor signaling.

Amino Acid Sequence↗
PubMed1998

Treatment and vaccination strategies to control cholera in sub-Saharan refugee settings: a cost-effectiveness analysis.

CONTEXT: There is significant controversy about how best to control cholera epidemics in refugee settings. Specifically, there is marked disagreement about whether to use oral cholera vaccines in these settings, despite the improved safety and effectiveness profiles of these vaccines. OBJECTIVE: To determine the cost-effectiveness of alternative intervention strategies, including vaccination, to control cholera outbreaks in sub-Saharan refugee camps. DESIGN: A cost-effectiveness analysis based on probabilities of cholera outcomes derived from epidemiologic data compiled for refugee settings in Malawi from 1987 through 1993; data for costs were obtained from a large relief agency that provides medical care in such settings. SETTING AND PARTICIPANTS: A hypothetical refugee camp with 50000 persons in sub-Saharan Africa evaluated for a 2-year period. INTERVENTIONS: We compared the costs and outcomes of alternative strategies in which appropriate rehydration therapy for cholera is introduced preemptively (at the establishment of a camp) or reactively (once an epidemic is recognized) and in which mass immunization with oral B subunit killed whole-cell (BS-WC) cholera vaccine is added to a rehydration program either preemptively or reactively. MAIN OUTCOME MEASURES: Cost per cholera case prevented and cost per cholera death averted. RESULTS: In a situation with no available rehydration therapy suitable for the management of severe cholera, a strategy of preemptive therapy ($320 per death averted) costs less and is more effective than a strategy of reactive therapy ($586 per death averted). Adding vaccination to preemptive therapy is expensive: $1745 per additional death averted for preemptive vaccination and $3833 per additional death averted for reactive vaccination. However, if the cost of vaccine falls below $0.22 per dose, strategies combining vaccination and preemptive therapy become more cost-effective than therapy alone. CONCLUSIONS: Provision for managing cholera outbreaks at the inception of a refugee camp (preemptive therapy) is the most cost-effective strategy for controlling cholera outbreaks in sub-Saharan refugee settings. Should the price of BS-WC cholera vaccine fall below $0.22 per dose, however, supplementation of preemptive therapy with mass vaccination will become a cost-effective option.

Africa South of the Sahara↗
PubMed1998

Eps15R is a tyrosine kinase substrate with characteristics of a docking protein possibly involved in coated pits-mediated internalization.

eps15R was identified because of its relatedness to eps15, a gene encoding a tyrosine kinase substrate bearing a novel protein-protein interaction domain, called EH. In this paper, we report a biochemical characterization of the eps15R gene product(s). In NIH-3T3 cells, three proteins of 125, 108, and 76 kDa were specifically recognized by anti-eps15R sera. The 125-kDa species is a bona fide product of the eps15R gene, whereas p108 and p76 are most likely products of alternative splicing events. Eps15R protein(s) are tyrosine-phosphorylated following epidermal growth factor receptor activation in NIH-3T3 cells overexpressing the receptor, even at low levels of receptor occupancy, thus behaving as physiological substrates. A role for eps15R in clathrin-mediated endocytosis is suggested by its localization in plasma membrane-coated pits and in vivo association to the coated pits' adapter protein AP-2. Finally, we demonstrate that a sizable fraction of eps15R exists in the cell as a complex with eps15 and that its EH domains exhibit binding specificities that are partially distinct from those of eps15. We propose that eps15 and eps15R are multifunctional binding proteins that serve pleiotropic functions within the cell.

3T3 Cells↗
PubMed1997

Eps15 is constitutively oligomerized due to homophilic interaction of its coiled-coil region.

Eps15 is a member of an emerging family of proteins containing a novel protein/protein interaction domain, the EH domain, of as yet unknown function. Recent findings of Eps15 association with clathrin adaptor complex AP-2 and its localization in clathrin-coated pits have implicated Eps15 in the regulation of vesicle trafficking. Here we show that Eps15 exists in several multimeric states in vivo. When purified recombinant Eps15 or lysates of NIH 3T3 cells were treated with cross-linking reagents, covalent dimers of Eps15 and larger covalent multimers were detected in high yield. Large Eps15 oligomers co-immunoprecipitated with AP-2 at an efficiency higher than that of Eps15 dimers. Furthermore, cross-linking of the membrane-bound fraction of Eps15 in mildly permeabilized cells was as efficient as that of the cytosolic fraction. Size-exclusion column chromatography of recombinantly produced Eps15 and of total cell lysates was performed to examine the equilibrium ratio of the monomers versus the aggregated forms of Eps15. These experiments showed that essentially all the Eps15 was aggregated, whereas monomers of Eps15 could be obtained only under strong denaturing conditions. To map the region of Eps15 responsible for dimerization, fusion proteins corresponding to the three structural domains of Eps15 were prepared. Cross-linking analysis revealed that the central portion of Eps15, which possesses a coiled-coil region (residues 321-520), serves as the interacting interface. The possibility that hetero-oligomeric complexes of Eps15 dimers and AP-2 function during the recruitment of proteins into coated pits is discussed.

3T3 Cells↗
PubMed1996

Lysosomal targeting of epidermal growth factor receptors via a kinase-dependent pathway is mediated by the receptor carboxyl-terminal residues 1022-1123.

Binding of epidermal growth factor (EGF) to its receptor induces rapid internalization and degradation of both ligand and receptor via the lysosomal pathway. To study the mechanism of intracellular sorting of EGF-EGF receptor complexes to lysosomes, NIH 3T3 cells transfected with wild-type and mutant EGF receptors were employed. The kinetics of 125I-EGF trafficking was analyzed using low concentrations of the ligand to avoid saturation of the specific sorting system. The relative size of the pool of internalized 125I-EGF-receptor complexes that were capable of recycling decreased as receptors traversed the endosomal system. The rate of 125I-EGF sequestration from the recycling pathway correlated with the rate of 125I-EGF transition from early to late endosomes as measured by Percoll gradient fractionation. Deletion of the last 63 amino acids of the EGF receptor cytoplasmic tail did not inhibit the process of sequestration and targeting to the late endosomes and lysosomes. Truncation of the 123 residues, however, resulted in impaired lysosomal targeting and increased recycling of EGF. Receptor mutant in which 165 residues were deleted displayed maximal ability to recycle and a minimal extent of sorting to the late endosomes. The data suggest that two regions of the EGF receptor molecule, residues 1022-1063 and to a lesser extent residues 1063-1123, contribute in the regulation of routing of EGF receptors to the degradation pathway. The kinase-negative receptor mutant recycled EGF more intensively compared with the wild-type receptor, and the transport of this mutant to late endosomes was inhibited. These results support the view that the receptor kinase activity is important for ligand-induced sorting of EGF receptors to the pathway of lysosomal degradation.

3T3 Cells↗
PubMed1996

Eps15 is a component of clathrin-coated pits and vesicles and is located at the rim of coated pits.

Eps15, a phosphorylation substrate of the epidermal growth factor (EGF) receptor kinase, has been shown to bind to the alpha-subunit of the clathrin-associated protein complex AP-2. Here we report that in cells, virtually all Eps15 interacts with the cytosol and membrane-bound forms of AP-2. This association is not affected by the treatment of cells with EGF. Immunofluorescence microscopy reveals nearly absolute co-localization of Eps15 with AP-2 and clathrin, and analysis by immunoelectron microscopy shows that the localization of membrane-associated Eps15 is restricted to the profiles corresponding to endocytic coated pits and vesicles. Unexpectedly, Eps15 was found at the edge of forming coated pits and at the rim of budding coated vesicles. This asymmetric distribution is in sharp contrast to the localization of AP-2 that shows an even distribution along the same types of clathrin-coated structures. These findings suggest several possible regulatory roles of Eps15 during the formation of coated pits.

3T3 Cells↗