Low-affinity binding in to P2YR mediates force-dependent integrin activation during hantavirus infection.

Pathogenic hantaviruses bind to the plexin-semaphorin-integrin (PSI) domain of inactive, β integrins. Previous studies have implicated a cognate interaction between the bent conformation β/β integrins and an arginine-glycine-aspartic acid (RGD) sequence in the first extracellular loop of P2YR. With single-molecule atomic force microscopy, we show a specific interaction between an atomic force microscopy tip decorated with recombinant αβ integrins and (RGD)P2YR expressed on cell membranes.

A High-Throughput Flow Cytometry Assay for Identification of Inhibitors of 3',5'-Cyclic Adenosine Monophosphate Efflux.

Assays to identify small molecule inhibitors of cell transporters have long been used to develop potential therapies for reversing drug resistance in cancer cells. In flow cytometry, these approaches rely on the use of fluorescent substrates of transporters. Compounds which prevent the loss of cell fluorescence have typically been pursued as inhibitors of specific transporters, but further drug development has been largely unsuccessful.

Quantitative bead-based flow cytometry for assaying Rab7 GTPase interaction with the Rab-interacting lysosomal protein (RILP) effector protein.

Rab7 facilitates vesicular transport and delivery from early endosomes to late endosomes as well as from late endosomes to lysosomes. The role of Rab7 in vesicular transport is dependent on its interactions with effector proteins, among them Rab-interacting lysosomal protein (RILP), which aids in the recruitment of active Rab7 (GTP-bound) onto dynein-dynactin motor complexes to facilitate late endosomal transport on the cytoskeleton. Here we detail a novel bead-based flow cytometry assay to measure Rab7 interaction with the Rab-interacting lysosomal protein (RILP) effector protein and demonstrate its utility for quantitative assessment and studying drug-target interactions.

Discovery of regulators of receptor internalization with high-throughput flow cytometry.

We developed a platform combining fluorogen-activating protein (FAP) technology with high-throughput flow cytometry to detect real-time protein trafficking to and from the plasma membrane in living cells. The hybrid platform facilitates drug discovery for trafficking receptors such as G protein-coupled receptors and was validated with the β₂-adrenergic receptor (β₂AR) system. When a chemical library containing ∼1200 off-patent drugs was screened against cells expressing FAP-tagged β₂ARs, all 33 known β₂AR-active ligands in the library were successfully identified, together with a number of compounds that might regulate receptor internalization in a nontraditional manner.

Drug Repurposing from an Academic Perspective.

Academia and small business research units are poised to play an increasing role in drug discovery, with drug repurposing as one of the major areas of activity. Here we summarize project status for a number of drugs or classes of drugs: raltegravir, cyclobenzaprine, benzbromarone, mometasone furoate, astemizole, R-naproxen, ketorolac, tolfenamic acid, phenothiazines, methylergonovine maleate and beta-adrenergic receptor drugs, respectively. Based on this multi-year, multi-project experience we discuss strengths and weaknesses of academic-based drug repurposing research.

Simultaneous in vitro molecular screening of protein-peptide interactions by flow cytometry, using six Bcl-2 family proteins as examples.

The B-cell lymphoma-2 (Bcl-2) family contains six antiapoptotic members, each with a hydrophobic pocket in which Bcl-2 homology region 3 (BH3) helices bind. This binding quenches apoptotic signals from activated BH3 family members. Many tumor cells either have increased expression of one of these six proteins or become overexpressed under treatment.

High-throughput screen for the chemical inhibitors of antiapoptotic bcl-2 family proteins by multiplex flow cytometry.

The human Bcl-2 family includes six antiapoptotic members (Bcl-2, Bcl-B, Bcl-W, Bcl-X(L), Bfl-1, and Mcl-1) and many proapoptotic members, wherein a balance between the two determines cell life or death in many physiological and disease contexts. Elevated expression of various antiapoptotic Bcl-2 members is commonly observed in cancers, and chemical inhibitors of these proteins have been shown to promote apoptosis of malignant cells in culture, in animal models, and in human clinical trials. All six antiapoptotic members bind a helix from the proapoptotic family member Bim, thus quenching Bim's apoptotic signal.

Flow cytometry for real-time measurement of guanine nucleotide binding and exchange by Ras-like GTPases.

Ras-like small GTPases cycle between GTP-bound active and GDP-bound inactive conformational states to regulate diverse cellular processes. Despite their importance, detailed kinetic or comparative studies of family members are rarely undertaken due to the lack of real-time assays measuring nucleotide binding or exchange. Here we report a bead-based flow cytometric assay that quantitatively measures the nucleotide binding properties of glutathione-S-transferase (GST) chimeras for prototypical Ras family members Rab7 and Rho.

Estrogen receptors alpha (ESR1) and beta (ESR2) are expressed in circulating human lymphocytes.

Bone marrow thymocytes in part mediate the bone-preserving effects of estrogen by decreasing their production of osteoclast growth factors such as interleukin-1 and -6 and tumor necrosis factor alpha in the presence of physiological amounts of estradiol. Although several in vitro studies implicate the T-lymphocyte as a candidate mediator of estrogen signaling in the skeleton, whether these cells or any lymphocytes ordinarily express one or both nuclear estrogen receptors was previously unresolved. The purpose of our investigation was therefore to ascertain, by using real-time PCR, immmunoblotting, and cytometric techniques, if any of the nuclear estrogen receptors could be detected in normal peripheral blood mononuclear cells (PBMNC) collected from healthy volunteers.

Rapid-mix flow cytometry measurements of subsecond regulation of G protein-coupled receptor ternary complex dynamics by guanine nucleotides.

We have used rapid-mix flow cytometry to analyze the early subsecond dynamics of the disassembly of ternary complexes of G protein-coupled receptors (GPCRs) immobilized on beads to examine individual steps associated with guanine nucleotide activation. Our earlier studies suggested that the slow dissociation of Galpha and Gbetagamma subunits was unlikely to be an essential component of cell activation. However, these studies did not have adequate time resolution to define precisely the disassembly kinetics.

Some mechanistic insights into GPCR activation from detergent-solubilized ternary complexes on beads.

The binding of full and partial agonist ligands (L) to G protein-coupled receptors (GPCRs) initiates the formation of ternary complexes with G proteins [ligand-receptor-G protein (LRG) complexes]. Cyclic ternary complex models are required to account for the thermodynamically plausible complexes. It has recently become possible to assemble solubilized formyl peptide receptor (FPR) and beta(2)-adrenergic receptor (beta(2)AR) ternary complexes for flow cytometric bead-based assays.

Duplexed, bead-based competitive assay for inhibitors of protein kinases.

Background: Many cellular signal transduction cascades have protein kinases as critical components. Small molecule protein kinase inhibitors can be effective as laboratory probes and drugs. Methods that allow two or more kinases to be evaluated simultaneously for inhibition by a small molecule would allow unequivocal tests of specificity and selectivity of action of the small molecule.

Glutathione-S-transferase-green fluorescent protein fusion protein reveals slow dissociation from high site density beads and measures free GSH.

Background: Glutathione, a ubiquitous tripeptide, is an important cellular constituent, and measurement of reduced and oxidized glutathione is a measure of the redox state of cells. Glutathione-S-transferase (GST) fusion proteins bind naturally to beads derivatized with glutathione, and elution of such bead-bound fusion proteins with buffer containing millimolar glutathione is a commonly used method of protein purification. Many protein-protein interactions have been established by using GST fusion proteins and measuring binding of fusion protein binding partners by GST pulldown assays, usually monitored by Western blot methodology.

Dynamics of fluorescence dequenching of ostrich-quenched fluorescein biotin: a multifunctional quantitative assay for biotin.

We describe a simple and rapid quantitative assay for biotin and biotin conjugates. The assay is based on the kinetic analysis of the enhancement of fluorescence of streptavidin/fluorescein biotin complexes in the presence of biotin. The kinetic response of fluorescence enhancement is proportional to the concentration of biotin.

Techniques: GPCR assembly, pharmacology and screening by flow cytometry.

Flow cytometers are well known for their ability to analyze and sort cells at high rates based on physiological responses and expression of protein markers. The potential for flow cytometry in G-protein-coupled receptor (GPCR) research, however, is less well appreciated. Potential applications include: (i) the homogenous discrimination of free and bound ligands or proteins in both cellular and microsphere-based assays; and (ii) multiplexed ('suspension array') analysis of cell responses and protein-protein interactions.

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.

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.