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.

The current invention is methods and assays for detecting and/or measuring the modification, i.e., activation or inhibition, of a receptor of interest, without the need to modify the receptor with a label or a tag. The invention uses proteins that move, translocate, are recruited and/or bind to the receptor of interest when the receptor is modified, and proteins in proximity to and/or in the same compartment as the receptor of interest. The proteins are attached or linked to polypeptides that result from the unique cleavage of a modified Oplophorus luciferase. When the proteins are in proximity allowing the unique polypeptides to recombine, a luminescent signal is generated. The invention also includes vector encoding these proteins, cells expressing these proteins, compositions, system and kits.

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 G subunit and a G interacting protein ( IP) for the binding to the G dimer. These biosensors comprises (1) a IP and (2) a G or G 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.

An purpose of the present invention is to provide a construct for expressing a G protein-coupled receptor, and the use of said construct. The construct is used for expressing a G protein-coupled receptor protein or a subunit thereof, wherein the construct is characterized by including a nucleic acid that encodes G protein subunit protein; a nucleic acid that comprises an IRES sequence; and a nucleic acid that encodes the G protein-coupled receptor protein or a subunit thereof in the stated order from the 5 side in a single vector.

The present invention relates to the discovery that the T1R receptors assemble to form functional taste receptors. Particularly, it has been discovered that co-expression of T1R1 and T1R3 results in a taste receptor that responds to umami taste stimuli, including monosodium glutamate. Also, it has been discovered that co-expression of the T1R2 and T1R3 receptors results in a taste receptor that responds to sweet taste stimuli including naturally occurring and artificial sweeteners. Also the present invention relates to the use of hetero-oligomeric taste receptors comprising T1R1/T1R3 and T1R2/T1R3 in assays to identify compounds that respectively respond to umami taste stimuli and sweet taste stimuli. Further, the invention relates to the constitutive of cell lines that stably or transiently co-express a combination of T1R1 and T1R3; or T1R2 and T1R3; under constitutive or inducible conditions. The use of these cells lines in cell-based assays to identify umami and sweet taste modulatory compounds is also provided, particularly high throughput screening assays that detect receptor activity by use of fluorometric imaging. Finally, the invention relates to the discovery that some compounds, e.g., lactisole, inhibit both the activities of human T1R2/T1R3 and T1R1/T1R3 receptors, and accordingly the sweet and umami taste, suggesting that these receptors may be the only sweet and umami receptors.

The current invention is methods and assays for detecting and/or measuring the modification, i.e., activation or inhibition, of a receptor of interest, without the need to modify the receptor with a label or a tag. The invention uses proteins that move, translocate, are recruited and/or bind to the receptor of interest when the receptor is modified, and proteins in proximity to and/or in the same compartment as the receptor of interest. The proteins are attached or linked to polypeptides that result from the unique cleavage of a modified Oplophorus luciferase. When the proteins are in proximity allowing the unique polypeptides to recombine, a luminescent signal is generated. The invention also includes vector encoding these proteins, cells expressing these proteins, compositions, system and kits.

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

A highly sensitive method of detecting GASP-1 or a fragment thereof in a sample is provided. The method comprises (a) exposing a surface to a sample comprising GASP-1 or a fragment thereof; (b) immobilizing an anti-GASP-1 antibody to the surface in the presence of the GASP-1 or a fragment thereof; (c) measuring the amount of the anti-GASP-1 antibody immobilized to the surface; and (d) determining the presence of the GASP-1 or a fragment thereof in the sample based on the amount of the anti-GASP-1 antibody immobilized to the surface. A coating agent may be immobilized to the surface in step (a), and the anti-GASP-1 antibody may be immobilized to the surface via the coating agent. The coating agent may be selected from the group consisting of a conjugate of bovine serum albumin (BSA) and a GASP-1 peptide (BSA-GASP-1 conjugate) a capture antibody against a microvesicle or exosomal surface biomarker, and a combination thereof. Also provided are kits for detecting GASP-1 or its fragment.

The present invention relates to the discovery that the T1R receptors assemble to form functional taste receptors. Particularly, it has been discovered that co-expression of T1R1 and T1R3 results in a taste receptor that responds to umami taste stimuli, including monosodium glutamate. Also, it has been discovered that co-expression of the T1R2 and T1R3 receptors results in a taste receptor that responds to sweet taste stimuli including naturally occurring and artificial sweeteners.

Also the present invention relates to the use of hetero-oligomeric taste receptors comprising T1R1/T1R3 and T1R2/T1R3 in assays to identify compounds that respectively respond to umami taste stimuli and sweet taste stimuli.

Further, the invention relates to the constitutive of cell lines that stably or transiently co-express a combination of T1R1 and T1R3; or T1R2 and T1R3;

under constitutive or inducible conditions. The use of these cells lines in cell-based assays to identify umami and sweet taste modulatory compounds is also provided, particularly high throughput screening assays that detect receptor activity by use of fluorometric imaging.

Finally, the invention relates to the discovery that some compounds, e.g., lactisole, inhibit both the activities of human T1R2/T1R3 and T1R1/T1R3 receptors, and accordingly the sweet and umami taste, suggesting that these receptors may be the only sweet and umami receptors.

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