Activation of Rho Family GTPases by Small Molecules.

Ras and Ras-related small GTPases are key regulators of diverse cellular functions that impact cell growth, survival, motility, morphogenesis, and differentiation. They are important targets for studies of disease mechanisms as well as drug discovery. Here, we report the characterization of small molecule agonists of one or more of six Rho, Rab, and Ras family GTPases that were first identified through flow cytometry-based, multiplexed high-throughput screening of 200000 compounds.

A selective ATP-binding cassette subfamily G member 2 efflux inhibitor revealed via high-throughput flow cytometry.

Chemotherapeutics tumor resistance is a principal reason for treatment failure, and clinical and experimental data indicate that multidrug transporters such as ATP-binding cassette (ABC) B1 and ABCG2 play a leading role by preventing cytotoxic intracellular drug concentrations. Functional efflux inhibition of existing chemotherapeutics by these pumps continues to present a promising approach for treatment. A contributing factor to the failure of existing inhibitors in clinical applications is limited understanding of specific substrate/inhibitor/pump 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.

Identification of a small molecule yeast TORC1 inhibitor with a multiplex screen based on flow cytometry.

TOR (target of rapamycin) is a serine/threonine kinase, evolutionarily conserved from yeast to human, which functions as a fundamental controller of cell growth. The moderate clinical benefit of rapamycin in mTOR-based therapy of many cancers favors the development of new TOR inhibitors. Here we report a high-throughput flow cytometry multiplexed screen using five GFP-tagged yeast clones that represent the readouts of four branches of the TORC1 signaling pathway in budding yeast.

A miniature Couette to generate shear for flow cytometry: studying real-time modulation of intracellular calcium in monocytic cells.

Extracellular hydrodynamic forces may be transmitted to the interior of cells through the alteration of integrin conformation and affinity. Integrin activation regulates leukocyte recruitment, cell activation, and transmigration. The cellular and molecular mechanisms for integrin activation are not precisely known, although intracellular calcium signaling is involved.

Real-time analysis of very late antigen-4 affinity modulation by shear.

Shear promotes endothelial recruitment of leukocytes, cell activation, and transmigration. Mechanical stress on cells caused by shear can induce a rapid integrin conformational change and activation, followed by an increase in binding to the extracellular matrix. The molecular mechanism of increased avidity is unknown.

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.

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.

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.

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.

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.