Biomedical subjects

Larry A Sklar

Publications and source records attributed to Larry A Sklar.

17 recordsLinked to original sources

PubMed2004

Real-time analysis of ternary complex on particles: direct evidence for partial agonism at the agonist-receptor-G protein complex assembly step of signal transduction.

We developed a novel and generalized approach to investigate G protein-coupled receptor molecular assemblies. We solubilized a fusion protein consisting of the beta(2)-adrenergic receptor and green fluorescent protein (GFP) for bead-based flow cytometric analysis. beta(2)-Adrenergic receptor GFP bound to dihydroalprenolol-conjugated beads, providing a K(d) for the fusion protein and, in competition with beta(2)-adrenergic receptor ligands, K(d) values for agonists and antagonists. Beads displaying chelated nickel bound purified hexahistidine-tagged G protein heterotrimers and, subsequently, the binary complex of agonist with beta(2)-adrenergic receptor GFP. The dose-response curves of ternary complex formation revealed maximal assembly for ligands previously classified as full agonists and reduced assembly for ligands previously classified as partial agonists. Guanosine 5'-3-O-(thio)triphosphate-induced dissociation rates of the ternary complex were the same for full and partial agonists. Soluble G protein, competing with ternary complexes on beads provided an affinity estimate of agonist-receptor complexes to G protein. When performed simultaneously, the two assemblies discriminated between agonist, antagonist or inactive molecule in a manner appropriate for high throughput, small volume drug discovery. The assemblies can be further generalized to other G protein coupled receptor protein-protein interactions.

Adrenergic beta-2 Receptor Agonists↗

PubMed2004

Relationship between molecular and cellular dissociation rates for VLA-4/VCAM-1 interaction in the absence of shear stress.

The rate of leukocyte recruitment to and detachment from the vasculature contributes to cellular tethering, rolling, firm adherence, and migration across an endothelium layer. The molecular rates depend on the type and number of bound integrin or selectin adhesion molecules, shear force acting on the bound adhesion molecules, and affinity state of integrins. Although little is known of the effect that the number of adhesion molecules has on leukocyte recruitment, it has been shown that firm adhesion for cells in suspension may be mediated by small numbers of bound adhesion molecules. We studied the disaggregation of aggregates composed of B78H1 cells transfected with human vascular cell adhesion molecule-1 (VCAM-1) and human monoblastoid U937 cells expressing Very Late Antigen-4 (VLA-4). Aggregate disaggregation rates were obtained and compared to dissociation rates for soluble rhVCAM-1 ligand and monoblastoid U937 cells. Under conditions without shear stress, it was found that average cellular disaggregation rates were a factor of 1.3 +/- 0.4 times slower than molecular dissociation rates for the 1 mM Mn(2+) and 1 mM Mn(2+) + 1 mM Ca(2+) conditions. A simple mathematical model was used to predict how much smaller the dissociation constant would be if the number of bonds holding an aggregate varied from one bond to N bonds under conditions without shear stress. The average number of adhesion bonds holding the cell aggregates together was found to be 1.5 +/- 0.7. This suggests that a few bonds were needed to form cellular aggregates and that increased aggregation was related to integrin affinity changes and not due to clustering or increased bond numbers.

Animals↗

PubMed2003

Release of ubiquitin-charged Cdc34-S - Ub from the RING domain is essential for ubiquitination of the SCF(Cdc4)-bound substrate Sic1.

The S. cerevisiae SCF(Cdc4) is a prototype of RING-type SCF E3s, which recruit substrates for polyubiquitination by the Cdc34 ubiquitin-conjugating enzyme. Current models propose that Cdc34 ubiquitinates the substrate while remaining bound to the RING domain. In contrast, we found that the formation of a ubiquitin thiol ester regulates the Cdc34/SCF(Cdc4) binding equilibrium by increasing the dissociation rate constant, with only a minor effect on the association rate. By using a F72VCdc34 mutant with increased affinity for the RING domain, we demonstrate that release of ubiquitin-charged Cdc34-S - Ub from the RING is essential for ubiquitination of the SCF(Cdc4)-bound substrate Sic1. Release of ubiquitin-charged E2 from E3 prior to ubiquitin transfer is a previously unrecognized step in ubiquitination, which can explain both the modification of multiple lysines on the recruited substrate and the extension of polyubiquitin chains. We discuss implications of this finding for function of other ubiquitin ligases.

Anaphase-Promoting Complex-Cyclosome↗

PubMed2003

Alpha4beta1 integrin affinity changes govern cell adhesion.

Integrin alpha4beta1 is a receptor for vascular cell adhesion molecule-1 and fibronectin. It is important in lymphopoiesis, inflammatory recruitment of leukocytes, and other situations that require cell adhesion to the vascular endothelium. The avidity of the cells expressing alpha4beta1 integrin can be rapidly changed by chemokines and chemoattractants. Different mechanisms, including changes in the number of interacting molecules due to the alteration of the receptor topology or changes in the affinity of the individual bonds, have been proposed to explain the nature of these fast changes in avidity. Recently, we described a fluorescent LDV-containing small molecule, which we used to monitor the affinity changes on live cells in real time (Chigaev, A., Blenc, A. M., Braaten, J. V., Kumaraswamy, N., Kepley, C. L., Andrews, R. P., Oliver, J. M., Edwards, B. S., Prossnitz, E. R., Larson, R. S. et al. (2001) J. Biol. Chem. 276, 48670-48678). Here we show that the affinity of the small molecule probe as well as the native ligand vascular cell adhesion molecule-1 varies in parallel when the integrin is modulated with divalent cations and that the affinity modulation leads to the changes in cell avidity. Using formyl peptide receptor-transfected U937 cells, we further show that the time course of avidity changes in response to the receptor activation coincides with the time course of the affinity changes. Taken together, these data are consistent with the idea that affinity regulation is a major factor that governs the avidity of cell adhesion mediated by the alpha4 integrin.

Animals↗

PubMed2003

Functional capabilities of an N-formyl peptide receptor-G(alpha)(i)(2) fusion protein: assemblies with G proteins and arrestins.

G protein-coupled receptors (GPCRs) must constantly compete for interactions with G proteins, kinases, and arrestins. To evaluate the interactions of these proteins with GPCRs in greater detail, we generated a fusion protein between the N-formyl peptide receptor and the G(alpha)(i2) protein. The functional capabilities of this chimeric protein were determined both in vivo, in stably transfected U937 cells, and in vitro, using a novel reconstitution system of solubilized components. The chimeric protein exhibited a cellular ligand binding affinity indistinguishable from that of the wild-type receptor and existed as a complex, when solubilized, containing betagamma subunits, as demonstrated by sucrose density sedimentation. The chimeric protein mobilized intracellular calcium and desensitized normally in response to agonist. Furthermore, the chimeric receptor was internalized and recycled at rates similar to those of the wild-type FPR. Confocal fluorescence microscopy revealed that internalized chimeric receptors, as identified with fluorescent ligand, colocalized with arrestin, as well as G protein, unlike wild-type receptors. Soluble reconstitution experiments demonstrated that the chimeric receptor, even in the phosphorylated state, existed as a high ligand affinity G protein complex, in the absence of exogenous G protein. This interaction was only partially prevented through the addition of arrestins. Furthermore, our results demonstrate that the GTP-bound state of the G protein alpha subunit displays no detectable affinity for the receptor. Together, these results indicate that complex interactions exist between GPCRs, in their unphosphorylated and phosphorylated states, G proteins, and arrestins, which result in the highly regulated control of GPCR function.

Animals↗

PubMed2003

High-throughput flow cytometry: validation in microvolume bioassays.

BACKGROUND: We recently reported an automated sample handling system, designated HyperCyt, by which samples are aspirated from microplate wells and delivered to the flow cytometer for analysis at rates approaching 100 samples per minute. In this approach, an autosampler and peristaltic pump introduce samples into a tubing line that directly connects to the flow cytometer. Air bubbles are inserted between samples to prevent sample dispersion. In the present work, we compare results of HyperCyt with those of conventional manual flow cytometric analysis in representative flow cytometric bioassays and describe a cell suspension method in which HyperCyt exploits the use of microvolume wells. METHODS: Human eosinophils and neutrophils were treated with trypsin to generate a wide (>25-fold) range of membrane P-selectin glycoprotein ligand-1 (PSGL-1) expression and then stained with fluorescent anti-PSGL-1 antibodies. Human peripheral blood mononuclear cells were stained with fluorescein isothiocyanate- and phycoerythrin-conjugated monoclonal antibodies for multiparameter immunophenotype analysis. U937 cells labeled with PKH62GL were used to assess cell settling in microplate wells. RESULTS: Differences in PSGL-1 expression levels were detected by HyperCyt autosampling of leukocytes from 96-well plates at an analysis rate of approximately 1.5 s/well. HyperCyt measurements linearly correlated with parallel manual measurements (r(2) = 0.98). Lymphocyte subpopulations were accurately distinguished and reproducibly quantified in multiparameter immunophenotyping assays performed over a range of HyperCyt analysis rates (1.4-5.5 s/sample). When assay volumes were reduced to 10 microl/well in 60-well Terasaki plates, cells could be maintained in uniform suspension for up to 30 min by periodically inverting plates on a rotating carousel before HyperCyt analysis. HyperCyt analysis of five fluorescence-level Cyto-Plex beads sampled from Terasaki plate microwells at 2.5 s/well produced highly reproducible results over a wide range of input bead concentrations (from 7 x 10(5) to 20 x 10(6) beads/ml) that linearly correlated with manual analysis results. CONCLUSIONS: The HyperCyt autosampling system enabled a 10-fold or greater increase in sample throughput compared with conventional manual flow cytometric sample analysis, with comparable analysis results. Assays were performed efficiently in 10-microl volumes to enable significant reagent cost savings, use of quantity-limited reagents at otherwise prohibitive concentrations, and maintenance of uniform suspensions of cells for prolonged periods.

Biological Assay↗

PubMed2003

FRET detection of cellular alpha4-integrin conformational activation.

Integrins are cell adhesion receptors, expressed on every cell type, that have been postulated to undergo conformational changes upon activation. Here, different affinity states were generated by exposing alpha4-integrins to divalent ions or by inside-out activation using a chemokine receptor. We probed the dynamic structural transformation of the integrin on live cells using fluorescence resonance energy transfer (FRET) between a peptide donor, which specifically binds to the alpha4-integrin, and octadecyl rhodamine B acceptors incorporated into the plasma membrane. We analyzed the data using a model that describes FRET between a random distribution of donors and acceptors in an infinite plane. The distance of closest approach was found to vary with the affinity of the integrin. The change in distance of closest approach was approximately 50 A between resting and Mn2+ activated receptors and approximately 25 A after chemokine activation. We used confocal microscopy to probe the lateral organization of donors and acceptors subsequent to integrin activation. Taken together, FRET and confocal results suggest that changes in FRET efficiencies are primarily due to the vertical extension of the integrin. The coordination between the extension of alpha4-integrin and its affinity provides a mechanism for Dembo's catch-bond concept.

Biophysical Phenomena↗

PubMed2003

Ligand-receptor-G-protein molecular assemblies on beads for mechanistic studies and screening by flow cytometry.

G protein-coupled receptors form a ternary complex of ligand, receptor, and G protein heterotrimer (LRG) during signal transduction from the outside to the inside of a cell. Our goal was to develop a homogeneous, small-volume, bead-based approach compatible with high-throughput flow cytometry that would allow evaluation of G protein coupled receptor molecular assemblies. Dextran beads were derivatized to carry chelated nickel to bind hexahistidine-tagged green fluorescent protein (GFP) and hexahistidine-tagged G proteins. Ternary complexes were assembled on these beads using fluorescent ligand with wild-type receptor or a receptor-Gialpha2 fusion protein, and with a nonfluorescent ligand and receptor-GFP fusion protein. Streptavidin-coated polystyrene beads used biotinylated anti-FLAG antibodies to bind FLAG-tagged G proteins for ternary complex assembly. Validation was achieved by showing time and concentration dependence of ternary complex formation. Affinity measurements of ligand for receptor on particles, of the ligand-receptor complex for G protein on the particles, and receptor-Gialpha2 fusion protein for Gbetagamma, were consistent with comparable assemblies in detergent suspension. Performance was assessed in applications representing the potential of these assemblies for ternary complex mechanisms. We showed the relationship for a family of ligands between LR and LRG affinity and characterized the affinity of both the wild-type and GFP fusion receptors with G protein. We also showed the potential of kinetic measurements to allow observation of individual steps of GTP-induced ternary complex disassembly and discriminated a fast step caused by RG disassembly compared with the slower step of Galphabetagamma disassembly.

Flow Cytometry↗

PubMed2002

N-formyl peptide receptor phosphorylation domains differentially regulate arrestin and agonist affinity.

Arrestins regulate the signaling and endocytosis of many G protein-coupled receptors (GPCRs). It has been suggested that the functions of arrestins are dependent upon both the number and pattern of phosphorylation sites present in an activated GPCR. However, little is currently known about the relationships between the sites of receptor phosphorylation, the resulting affinities of arrestin binding, and the ensuing mechanisms of receptor regulation for any given GPCR. To investigate these interactions, we used an active truncated mutant of arrestin (amino acids 1-382) and phosphorylation-deficient mutants of the N-formyl peptide receptor (FPR). In contrast to results with wild type arrestins, the truncated arrestin-2 protein bound to the unphosphorylated wild type FPR, although with lower affinity and a low affinity for the agonist as revealed by competition studies with heterotrimeric G proteins. Using FPR mutants, we further demonstrated that the phosphorylation status of serines and threonines between residues 328-332 is a key determinant that regulates the affinity of the FPR for arrestins. Furthermore, we found that the phosphorylation status of serine and threonine residues between amino acids 334 and 339 regulates the affinity of the receptor for agonist when arrestin is bound. These results suggest that the agonist affinity state of the receptor is principally regulated by phosphorylation at specific sites and is not simply a consequence of arrestin binding as has previously been proposed. Furthermore, this is the first demonstration that agonist affinity of a GPCR and the affinity of arrestin binding to the phosphorylated receptor are regulated by distinct receptor phosphodomains.

Amino Acid Sequence↗

PubMed2002

Biomolecular recognition on well-characterized beads packed in microfluidic channels.

We describe a new approach for the analysis of biomolecular recognition in microfluidic channels. The method involves real-time detection of soluble molecules binding to receptor-bearing microspheres, sequestered in affinity column format inside a microfluidic channel. Identification and quantitation of analytes occurs via direct fluorescence measurements or fluorescence resonance energy transfer (FRET). We establish a model system that detects the FLAG epitope. The assay can potentially detect subfemtomole quantities of antibody with a high signal-to-noise ratio and a large dynamic range spanning nearly 4 orders of magnitude in analyte concentration in microliter-to-submicroliter volumes of analyte fluid. Kinetic and equilibrium constants for the reaction of this receptor-ligand pair are obtained through modeling of kinetic responses of the affinity microcolumn and are consistent with those obtained by flow cytometry. Because of the correlation between kinetic and equilibrium data obtained for the microcolumns, quantitative analysis can be done prior to the steady-state end point of the recognition reaction. This method has the promise of combining the utility of affinity chromatography with the advantage of direct, quantitative, and real-time analysis and the cost-effectiveness of microanalytical devices. The approach has the potential to be generalized to a host of bioaffinity assay methods including analysis of protein complexes and molecular assembly and microsystem-based multianalyte determinations.

Antibodies, Monoclonal↗

PubMed2002

Nozzle design parameters and their effects on rapid sample delivery in flow cytometry.

BACKGROUND: Rapid kinetic and high throughput flow cytometry are emerging as valuable tools in biotechnology research applications ranging from mechanistic analysis of molecular assemblies to high throughput screening. Many of these new applications have been made possible by improved sample delivery capabilities, focusing increased attention on fluidic issues associated with rapid sample delivery. METHODS: Using basic fluidic premises, we derived a model that predicted the effect of nozzle parameters during rapid sample delivery. We tested the model using the rapid mix flow cytometer and modifications were made to the equipment to optimize performance. RESULTS: The model predicted that shorter nozzles with wide exit orifices decrease the delay before initial particle analysis and the fluidic stabilization time. Experimental results confirmed this prediction and model-based modifications allowed analysis of particles within 55 ms or 600 ms after mixing, with or without electronic gating, respectively. CONCLUSIONS: The model along with modifications to commercial equipment will allow rapid mix flow cytometry to analyze reactions in time frames threefold shorter than previously possible. The model allows for nozzle design predictions that should allow for analysis in the millisecond time frame. Furthermore, these findings are general for all rapid delivery applications, including high throughput flow cytometry.

Automation↗

PubMed2002

Arrestin variants display differential binding characteristics for the phosphorylated N-formyl peptide receptor carboxyl terminus.

The phosphorylation-dependent binding of arrestins to cytoplasmic domains of G protein-coupled receptors (GPCRs) is thought to be a crucial step in receptor desensitization. In some GPCR systems, arrestins have also been demonstrated to be involved in receptor internalization, resensitization, and the activation of signaling cascades. The objective of the current study was to examine binding interactions of members of the arrestin family with the formyl peptide receptor (FPR), a member of the GPCR family of receptors. Peptides representing the unphosphorylated and phosphorylated carboxyl terminus of the FPR were synthesized and bound to polystyrene beads via a biotin/streptavidin interaction. Using fluorescein-conjugated arrestins, binding interactions between arrestins and the bead-bound FPR carboxyl terminus were analyzed by flow cytometry. Arrestin-2 and arrestin-3 bound to the FPR carboxyl-terminal peptide in a phosphorylation-dependent manner, with K(d) values in the micromolar range. Binding of visual arrestin, which binds rhodopsin with high selectivity, was not observed. Arrestin-2-(1--382) and arrestin-3-(1--393), truncated mutant forms of arrestin that display phosphorylation-independent binding to intact receptors, were also observed to bind the bead-bound FPR terminus in a phosphorylation-dependent manner, but with much greater affinity than the full-length arrestins, yielding K(d) values in the 5--50 nm range. Two additional arrestin mutants, which are full-length but display phosphorylation-independent binding to intact GPCRs, were evaluated for their binding affinity to the FPR carboxyl terminus. Whereas the single point mutant, arrestin-2 R169E, displayed an affinity similar to that of the full-length arrestins, the triple point mutant, arrestin-2 I386A/V387A/F388A, displayed an affinity more similar to that of the truncated forms of arrestin. The results suggest that the carboxyl terminus of arrestin is a critical determinant in regulating the binding affinity of arrestin for the phosphorylated domains of GPCRs.

Amino Acid Sequence↗

PubMed2002

A quantitative approach for studying IgE-FcepsilonRI aggregation.

Aggregation of cell surface receptors is a ubiquitous means of initiating signal transduction in many cellular systems. In this manuscript, we describe a combined theoretical and experimental approach based on multiparameter flow cytometry for measuring the time course of ligand induced aggregation of IgE-FcepsilonRI on RBL cells. By fluorescently labeling both the ligand and surface IgE (sIgE), we have developed an assay that permits us to simultaneously measure both occupancy of sIgE combining sites and association of antigen with the cell surface. This allows for a direct calculation of the degree of receptor aggregation present on the cell. By employing new mixing technologies developed for flow cytometry, we are able to look at aggregation in the sub second time domain. To extend our work, we have synthesized a new set of chemically well defined ligands (of valences 1-3) to use as probes in our studies. We show that the magnitude of the cellular response is dramatically increased as the valence of our ligand is raised from two to three.

Animals↗

PubMed2002

Suspension array technology: evolution of the flat-array paradigm.

Suspension arrays of microspheres analyzed using flow cytometry offer a new approach to multiplexed assays for large-scale screening applications. By optically encoding micron-sized polymer particles, suspension microarrays can be created to enable highly multiplexed analysis of complex samples. Each element in the array is comprised of a subpopulation of particles with distinct optical properties and each array element bears a different surface receptor. Nucleic acids, proteins, lipids or carbohydrates can serve as receptors to support the analysis of a wide range of biomolecular assemblies, and applications in genomic and proteomic research are being developed. Coupled with recent innovations for rapid serial analysis of samples, molecular analysis with microsphere arrays holds significant potential as a general analysis platform for both research and clinical applications.

Directed Molecular Evolution↗

PubMed2002

Fluorescence biosensing strategy based on energy transfer between fluorescently labeled receptors and a metallic surface.

A new fluorescence-based biosensor is presented. The biosensing scheme is based on the fact that a fluorophore in close proximity to a metal film (<100 A) experiences strong quenching of fluorescence and a dramatic reduction in the lifetime of the excited state. By immobilizing the analyte of interest (or a structural analog of the analyte) to a metal surface and exposing it to a labeled receptor (e.g. antibody), the fluorescence of the labeled receptor becomes quenched upon binding because of the close proximity to the metal. Upon exposure to free analyte, the labeled receptor dissociates from the surface and diffuses into the bulk of the solution. This increases its separation from the metal and an increase of fluorescence intensity and/or lifetime of the excited state is observed that indicates the presence of the soluble analyte. By enclosing this system within a small volume with a semipermeable membrane, a reversible device is obtained. We demonstrate this scheme using a biotinylated self-assembled monolayer (SAM) on gold as our surface immobilized analyte analog, fluorescently labeled anti-biotin as a receptor, and a solution of biotin in PBS as a model analyte. This scheme could easily be extended to transduce a wide variety of protein-ligand interactions and other biorecognition phenomena (e.g. DNA hybridization) that result in changes in the architecture of surface immobilized biomolecules such that a change in the separation distance between fluorophores and the metal film is obtained.

Biosensing Techniques↗

PubMed2002

Regulation of human basophil adhesion to endothelium under flow conditions: Different very late antigen 4 regulation on umbilical cord blood-derived and peripheral blood basophils.

BACKGROUND: Although soluble mediators released by basophils in tissue sites contribute to the chronic injury that occurs in hypersensitivity diseases, only limited information is available about how circulating basophils are recruited to tissues. In particular, the interaction of basophils with endothelium under conditions that mimic physiologic flow has not been explored. OBJECTIVE: We sought to identify adhesion molecules regulating the attachment of human basophils to IL-4-activated human umbilical vein endothelial cells (HUVECs) under flow conditions. METHODS: A parallel-plate flow chamber and blocking mAbs were used to define the adhesion molecules involved in the interactions of peripheral blood basophils (PBBs) and cord blood-derived basophils (CBDBs) with IL-4-activated HUVECs and with Chinese hamster ovary (CHO) cell transfectants expressing specific adhesion molecules. A fluorescent ligand specific for very late antigen 4 (VLA-4) was used to directly examine the VLA-4 affinity state of basophils. RESULTS: Flowing PBBs and CBDBs attached to activated HUVECs and to CHO cells expressing P- or E-selectin. However, only CBDBs attached to vascular cell adhesion molecule 1 (VCAM-1)-transfected CHO cells under flow conditions. The attachment of CBDBs to CHO cells was blocked by mAbs directed against E-selectin, P-selectin, and VCAM-1, whereas attachment of PBBs was blocked by E-selectin and P-selectin mAbs. Activating VLA-4 with Mn(2+) on PBBs resulted in adhesion to the VCAM-1-transfected CHO cells, indicating that VLA-4 activity on PBBs can be regulated, at least in part, through affinity changes. The Mn(2+)-induced upregulation of basophil VLA-4 affinity was demonstrated directly by using a fluorescent ligand for VLA-4 and flow cytometry. CONCLUSIONS: The interaction of human CBDBs and PBBs with endothelium under flow conditions is mediated in part by both P- and E-selectin. VLA-4 additionally contributes to the adhesion of flowing CBDBs. However, the affinity of VLA-4 is too low to support the adhesion under flow conditions of unstimulated PBBs.

Animals↗

PubMed2001

Flow cytometric analysis of ligand-receptor interactions and molecular assemblies.

Flow cytometers make homogeneous real-time measurements of ligand-receptor interactions and, simultaneously, the physiological responses of cells. Their multiparameter capabilities are also useful in resolving multicomponent assemblies or in developing multiplexed assays. Recent advances suggest that these approaches can be extended in several important ways. Sample delivery in the millisecond time domain is applicable to the analysis of complex binding kinetics and reaction mechanisms. The homogeneous discrimination of free components and particle-based assemblies can be extended into the micromolar concentration range. Measurements can be made of molecular assemblies among proteins, DNA, RNA, lipids, and carbohydrates on beads. The topography and assembly of components within cells can be evaluated with resonance energy transfer. Temperature dependence can be evaluated with Peltier temperature control. Many assembly endpoints can be assessed through new tools for high-throughput flow cytometry using plate-based assay formats and small volume samples.

Animals↗