1. Field of the Invention
The present invention relates, in general, to a method of identifying ligands and antagonists of ligands. In particular, the present invention relates to a method of identifying ligands and antagonists of ligands which bind to cloned G.sub.s - or G.sub.i -coupled receptors. The present invention also relates to a cell comprising a recombinant cyclic AMP sensitive reporter construct.
2. Background Information
In the last several years, numerous cDNAs or genes for hormones and neurotransmitters that couple to G-proteins have been cloned. With only two exceptions--the receptors for insulin-like growth factor II and glutamic acid--these G-coupled receptors are rather similar, and they are thought to comprise a large "superfamily" of proteins. By analogy to bacterial rhodopsin, the members of this superfamily are thought to contain seven membrane-spanning domains and have a number of highly conserved amino acids. As a consequence, oligonucleotide probes directed at shared domains of cloned receptors have been used at low stringency to screen cDNA libraries for novel receptor candidates. More recently, primers directed at conserved domains of these receptors have been used in the polymerase chain reaction to clone additional candidates. The cDNAs obtained as described above, which encode "orphan receptors" for which the endogenous ligand is not known, must be expressed and the ligands which the receptors bind must be identified. This is not as straightforward as it seems at first glance. Screening with radiolabelled ligands is expensive. It requires many (unstable) ligands and large amounts of transfected cells. Functional screens, on the other hand, are hampered by the fact that one cannot guess from the structure of a receptor which second messenger system it activates. Some G-protein coupled receptors activate phospholipase C or phospholipase A.sub.2 and increase inositol phosphates and arachidonic acid, respectively. Others activate (G.sub.s -coupled) or inhibit the activation (G.sub.i -coupled) of adenylate cyclase, increasing or decreasing the level of cyclic AMP in cells. The present invention provides simple and rapid methods for discovering the agonists which act on orphan G.sub.s - or G.sub.i -coupled receptors.
The assay that has been developed employs a novel mouse L cell line, LVIP2.OZc. These L cells contain a stably integrated fusion gene pLVIP2.OZ plasmid (Riabowol, K.T. et al., (1988) Nature 336:83-86), consisting of the Escherichia coli lac Z gene under the transcriptional control of 2 kb fragment derived from the vasoactive intestinal polypeptide (VIP) gene. This DNA segment includes the VIP promoter and a cyclic AMP responsive enhancer element (CRE). Forskolin increases cellular cyclic AMP levels and thus can be used to induce the .beta.-galactosidase enzyme in the LVIP2.OZc. reporter cells. After the cells are lysed, the enzyme activity can be detected by addition of a chromogenic substrate, o-nitrophenyl .beta.-D-galactopyranoside (ONPG). The product of the reaction, o-nitrophenol, is yellow in color and can be seen with the naked eye or measured spectrophotometrically at a wavelength of 405 nm. The assay can be performed in 96 well tissue culture plates and a commercially available plate reader can be used to measure the levels of o-nitrophenol and record the results.
Transfection of cells with a putative G.sub.s -coupled receptor allows one to induce the enzyme by adding the appropriate ligand. For example, cells transfected with a .beta..sub.2 -adrenergic receptor cDNA increase their .beta.-galactosidase levels in response to isoproterenol. G.sub.i -coupled receptors, on the other hand, inhibit the activation of adenylate cyclase. In the presence of the appropriate ligands the dopamine D.sub.2L, muscarinic cholinergic m.sub.2, or cannabinoid transfected receptors significantly reduce the forskolin-stimulated increase in .beta.-galactosidase normally seen in LVIP2.OZc cells. Thus, LVIP2.OZc cells provide a rapid, convenient and semi-automated system in which a large number of putative ligands may be screened for binding to an "orphan receptor" transiently expressed in these cells.
USPTO Backgrounds
USPTO Backgrounds