Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or...
Patent
1997-01-14
1999-12-14
Wax, Robert A.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
4352522, 43525421, 435232, C12Q 100, C12N 114, C12N 988
Patent
active
060015535
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
CROSS REFERENCE TO RELATED APPLICATIONS
A commonly owned application, U.S. Ser. No. 08/322,137, filed Oct. 13, 1994, incorporated by reference herein, relates to use of engineered yeast cells in screening for substances which modulate the activity of a mammalian surrogate of a yeast pheromone system protein, e.g., the yeast pheromone receptor, a G protein-coupled receptor.
1. Field of the Invention
The invention relates inter alia, to expression of a mammalian adenylyl cyclase in yeast, the transformed yeast cells, and their use, e.g., in identifying potential inhibitors or activators of the mammalian adenylyl cyclase, or of other proteins which are natively or artificially coupled to the mammalian adenylyl cyclase in the engineered yeast cell.
2. Description of the Background Art
In some instances, for a drug to cure a disease or alleviate its symptoms, the drug must be delivered to the appropriate cells, and trigger the proper "switches." The cellular switches are known as "receptors." Hormones, growth factors, neurotransmitters and many other biomolecules normally act through interaction with specific cellular receptors. Drugs may activate or block particular receptors to achieve a desired pharmaceutical effect. Cell surface receptors mediate the transduction of an "external" signal (the binding of a ligand to the receptor) into an "internal" signal (the modulation of a pathway in the cytoplasm or nucleus involved in the growth, metabolism or apotosis of the cell).
In many cases, transduction is accomplished by the following signaling cascade: cell surface. change which activates a transducing protein in the cell membrane. "second messenger molecules." the cell that have the potential to "switch on" or "off" specific genes or alter some metabolic process.
This series of events is coupled in a specific fashion for each possible cellular response. The response to a specific ligand may depend upon which receptor a cell expresses. For instance, the response to adrenalin in cells expressing .alpha.-adrenergic receptors may be the opposite of the response in cells expressing .beta.-adrenergic receptors.
The above "cascade" is idealized, and variations on this theme occur. For example, a receptor may act as its own transducing protein, or a transducing protein may act directly on an intracellular target without mediation by a "second messenger".
Signals initiated by a variety of mammalian hormones and neurotransmitters are received by seven transmembrane domain receptors in the plasma membrane of cells and are transduced to intracellular effectors via heterotrimeric G proteins. Many different G proteins are known to interact with receptors. G protein signaling systems include three components: the receptor itself, a GTP-binding protein (G protein), and an intracellular target usually a protein.
The cell membrane acts as a switchboard. Messages arriving through different receptors can produce a single effect if the receptors acts on the same type of G protein. On the other hand, signals activating a single receptor can produce more than one effect if the receptor acts on different kinds of G proteins, or if the G proteins can act on different effectors.
The heterotrimeric G protein is composed of a guanine nucleotide-binding a subunit together with a tight complex of .beta. and .gamma. subunits. In their resting state, the G proteins, which consist of alpha (.alpha.), beta (.beta.) and gamma (.gamma.) subunits, are complexed with the nucleotide guanosine diphosphate (GDP) and are in contact with receptors. When a hormone or other first messenger binds to receptor, the receptor changes conformation and this alters its interaction with the G protein. This spurs the a subunit to release GDP, and the more abundant nucleotide guanosine tri-phosphate (GTP), replaces it, activating the G protein. The G protein then dissociates to separate the .alpha. subunit from the still complexed beta and gamma subunits. The free G.alpha. and the G.beta..gamma. subunits both may be capable of influen
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Broach James R.
Manfredi John P.
Trueheart Joshua
Cadus Pharmaceutical Corporation
DeConti, Jr. Giulio A.
Lauro Peter C.
Wax Robert A.
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