Abstract: An intramolecular bioluminescence resonance energy transfer (BRET), biosensor for monitoring receptor activity and signalling cascades is disclosed. The “double-brilliance” biosensor sandwiches ?-arrestin (?-arr) between Renilla luciferase (Luc) and the yellow fluorescent protein (YFP). ?-arr associates with G-protein coupled receptors GPCR following receptor activation, bringing Luc and YPF into close proximity that favours energy transfer. In addition to providing new insights into the agonist-induced conformational rearrangements of ?-arr in living cells, the double-brilliance ?-arr offers a universal biosensor for GPCR activation, allowing the study of native receptors in large-scale screening analysis. The activity of other signalling molecules known to interact with ? arrestin could also be monitored by double brilliance ? arr.
Abstract: To measure a receptor-mediated increase in intracellular calcium concentration, use is made of a calcium-responding luminescent protein localized in the cytoplasm or mitochondria and a reporter gene having a promoter responding to the second messenger in cells and Renilla-origin luciferase attached to the downstream thereof. By using a coelenterazine derivative, which can exist in the cells in a stable state over a long time, as the substrate of these proteins, a transient increase in the intracellular calcium concentration observed within several seconds is measured by using the calcium-responding luminescent protein. Subsequently, the cells are allowed to survive and cultured for several hours as such without adding a fresh substrate. During this period, an increase/decrease in the gene expression is continuously monitored by the reporter assay method and it is measured and evaluated, if necessary, whether or not the inhibition of the activity is caused by cytotoxicity.
Abstract: The claimed invention comprises a single molecule-format bioluminescent probe for detecting a target-specific ligand in a living cell, which comprises, a ligand-binding molecule of which conformation is changed upon binding to the ligand, wherein the ligand-binding molecule comprises a ligand-binding domain (LBD) of a nuclear receptor and an LBD-interacting domain that is a co-activator peptide of said nuclear receptor, and an N-terminal polypeptide and a C-terminal polypeptide of a click beetle luciferase (N-CBLuc and C-CBLuc), which flank each end of the ligand-binding molecule, respectively, wherein the N-CBLuc and the C-CBLuc self-complement to generate a luminescent signal only upon binding of the ligand to the ligand-binding molecule.
Type:
Grant
Filed:
January 15, 2008
Date of Patent:
February 28, 2012
Assignee:
National Institute of Advanced Industrial Science and Technology
Inventors:
Yoshio Umezawa, Moritoshi Sato, Hiroaki Tao, SungBae Kim
Abstract: The invention provides methods and compositions for screening for pharmacological agents which regulate satiety, fat metabolism and/or the type II diabetes mellitus in mammals, and in particular, agents active at regulating the level of ob gene expression. An exemplary assay involves (a) contacting a mammalian adipocyte comprising a mutant of a native ob allele encoding a reporter of ob gene expression, wherein the expression of the reporter is under the control of the gene expression regulatory sequences of the native ob allele, with a candidate agent under conditions whereby but for the presence of the agent, the reporter is expressed at a first expression level; and, (b) measuring the expression of the reporter to obtain a second expression level, wherein a difference between the first and second expression levels indicates that the candidate agent modulates ob gene expression.
Type:
Grant
Filed:
September 4, 1996
Date of Patent:
July 14, 1998
Assignee:
Tularik, Inc
Inventors:
Fabienne Charles de la Brousse, Jin-long Chen