A method for determining the presence of a molecule coupled to the cytoplasmic side of a cellular membrane is disclosed. The method is implemented in a microfluidic setting and is particularly suitable for determining the presence and/or the activity of a G protein or an arrestin protein in a cell.

The invention relates to antibodies or antibody fragments capable of binding to G-protein alpha, nucleic acid sequences coding for the antibody, vectors comprising the nucleic acid sequence, cells comprising the vector or the nucleic acid sequence and a kit-of-parts comprising (i) the antibody or antibody fragment or the composition and (ii) a source of GTP labeled with a member of a pair of RET partners.

The invention relates to a method for determining the ability of a molecule to modulate the activity of a G protein-coupled receptor (GPCR), said method comprising the following steps: a) introducing, into a first container:—a first membrane preparation bearing one or more GPCRs and one or more G protein(s),—a source of GDP or a source of GTP,—a first ligand of the alpha subunit of a G protein (Galpha protein) labelled with a first member of a pair of RET partners, and a second ligand of the Galpha protein labelled with a second member of the pair of RET partners, said ligands being able to specifically bind, either individually or in combination, to the empty Galpha protein or to the full Galpha protein; b) measuring the RET signal emitted in the first container; c) introducing (i) into a second container, the same reactants as in step a) and the molecule to be tested or (ii) into the first container, the molecule to be tested; d) measuring the RET signal emitted in the second container or in the first container obtained in step c); e) comparing the signals obtained in steps b) and d).

TABLE-US-00001 AA GPCR activation BB from “full” G to “empty” G form CC Agonist DD Format 1 EE Format 2 FF G.sub.empty GG Before activation HH After activation

Resonance energy transfer (RET)- or protein-fragment complement assay (PCA)-based biosensors useful for assessing the activity of G-proteins are described. These biosensors are based on the competition between the Got subunit and a Gβγ interacting protein (βγ IP) for the binding to the Gβγ dimer. These biosensors comprises (1) a βγ IP and (2) a Gβ or Gγ protein; a GPCR; or a plasma membrane targeting domain, fused to suitable RET or PCA tags. Methods using such biosensors for different applications, including the identification of agents that modulates G-protein activity or for the characterization of GPCR signaling/regulation, such as G-protein preferences and activation profiles of GPCRs, are also described.

Diagnostic, prognostic, and treatment methods, compositions, and kits for enhancing insulin secretion and beta cell numbers and functions, and controlling glycemia associated with diabetes, obesity, atherosclerosis, stroke, myocardial infarction, and other cardiovascular diseases, by modulating FKN/CX3CR1 expression levels or activities, and its downstream signaling pathways in a subject in need or at risk.

Disclosed herein is a G protein-coupled receptor (GPCR) assay platform comprised of two complementary systems that equate dynamic intermolecular interactions between a receptor and transducer with more complex stimulus-response cascades in living cells. In the disclosed in vitro ADSoRB method, the forced dissociation of transducers like G protein heterotrimers from receptors alters receptor conformations and ligand interactions to simulate pathway activation in a cell. In the disclosed TRUPATH method, measuring the extent of engineered G protein heterotrimer complex dissociation provides single transducer resolution in a cell.

Described herein are systems for measuring enzymatic activities. Also described herein are methods for measuring or screening enzymatic activities utilizing the systems described herein.

The invention relates to a method for determining the ability of a molecule to modulate the activation of a G protein-coupled receptor (GPCR), said method comprising the following steps: a) introducing, in a first container: a membrane preparation bearing one or more GPCRs and one or more alpha G-proteins, a source of nonhydrolyzable or slowly hydrolyzable GTP labeled with a first member of a pair of RET partners, a ligand of the alpha subunit of a G protein (alpha G-protein) labeled with a second member of the pair of RET partners, said ligand being capable of binding to the full alpha G-protein bound to the nonhydrolyzable or slowly hydrolyzable GTP labeled with the first member of a pair of RET partners, optionally a GPCR agonist; b) measuring the RET signal emitted in the first container; c) introducing (i) in a second container, the same reagents as in step a) and the molecule to be assayed or (ii) in the first container, the molecule to be assayed; d) measuring the RET signal emitted in the second container or in the first container obtained in step c); e) comparing the signals obtained in steps b) and d), a modulation of the signal obtained in step d) relative to that obtained in step b) indicating that the molecule to be tested is capable of modulating the activation of the GPCR.

Disclosed herein are methods for providing a molecular efficacy of a ligand, especially when utilizing single-molecule fluorescence resonance energy transfer (“smFRET”) imaging, as well as compounds useful in such methods.

An object of the present invention is to provide a method and a kit that can accurately measure autoantibody activity against a TSH receptor in a short time without pretreating a blood sample (blood specimen). The autoantibody activity against a TSH receptor in a blood sample can be measured by using a kit for measurement of autoantibody activity against a TSH receptor in a mammal cell, comprising a mammal cell expressing both a cAMP biosensor and the TSH receptor, and a substrate capable of visualizing and/or quantifying the cAMP biosensor as constituents, and performing a method comprising the steps of: (a) incubating a mammal cell expressing both a cAMP biosensor and the TSH receptor in the presence of a blood sample collected from a test subject; (b) measuring an activation level of the cAMP biosensor after the step (a); and (c) comparing the activation level measured in the step (b) with an activation level in a control to calculate the autoantibody activity. This method eliminates the need of pretreatment of a blood sample (specimen), can complete measurement itself in a short time (e.g., within 3 hours) by convenient means, and further eliminates the need of a special apparatus except for a commercially available apparatus measuring light emission. Therefore, the method is sufficiently available in testing laboratories in hospitals.