Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-10-25
2001-06-26
Schwartzman, Robert A. (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S254200, C435S254210, C435S255100, C435S255200
Reexamination Certificate
active
06251605
ABSTRACT:
BACKGROUND OF THE INVENTION
Cell surface receptors are an important class of proteins involved in cellular functioning because they are the primary mediators of cell to cell communication. For example, G protein coupled receptors (GPCRs) are an important category of cell surface receptors. The medical importance of these receptors is evidenced by the fact that more than 60% of all commercially available prescription drugs work by interacting with known GPCRs.
In their resting state, the G proteins, which consist of alpha (&agr;), beta (&bgr;) and gamma (&ggr;) subunits, are complexed with the nucleotide guanosine diphosphate (GDP) and are in contact with the receptors to which they are coupled. When a hormone or other first messenger binds to receptor, the receptor changes conformation and this alters its interaction with the G protein. This causes the &agr; subunit to release GDP, and the more abundant nucleotide guanosine triphosphate (GTP) displaces it, activating the G protein. The G protein then dissociates to separate the &agr; subunit from the still complexed beta and gamma subunits. Either the G&agr; subunit, or the G&bgr;&ggr; complex, depending on the pathway, interacts with an effector. The effector (which is often an enzyme) in turn converts an inactive precursor molecule into an active “second messenger,” which may diffuse through the cytoplasm or may be associated with downstream signal molecules, triggering a signal cascade. After a few seconds, the G&agr; converts the GTP to GDP, thereby becoming inactive. The inactivated G&agr; may then reassociate with the G&bgr;&ggr; complex.
Hundreds, if not thousands, of receptors convey messages through heterotrimeric G proteins, of which at least 17 distinct forms have been isolated. Most G protein-coupled receptors are comprised of a single protein chain that is threaded through the plasma membrane seven times. Such receptors are often referred to as seven-transmembrane domain receptors (STRs). More than a hundred different GPCRs have been found, including many distinct receptors that bind the same ligand, and there are likely many more GPCRs awaiting discovery.
The mating factor receptors of yeast cells (STE2 and STE3) also span the membrane of the yeast cell seven times and are coupled to yeast G proteins. Heterologous GPCRs can be expressed in yeast cells and can be made to couple to yeast G proteins resulting in the transduction of signals via the endogenous yeast pheromone system signaling pathway which is normally activated by STE2 or STE3. In some cases, such heterologous receptors can be made to couple more effectively to the yeast pheromone system signaling pathway by coexpressing a heterologous G protein a subunit (e.g. U.S. Pat. No. 5,482,835 of King et al.), by expressing a chimeric G protein subunit (e.g. WO 94/23025), or by expressing a chimeric G protein coupled receptor (e.g., U.S. Pat. No. 5,576,210 of Sledziewski et al.).
The &bgr;&ggr; subunits of the activated G protein stimulate the downstream elements of the pheromone system pathway, including the Ste20p protein kinase, and a set of kinases that are similar to MEK kinase, MEK (MAP kinase kinase), and MAP kinase of mammalian cells and are encoded by the STE11, STE7, and FUS3 genes, respectively (Whiteway et al. 1995. Science. 269:1572).
In recent years drug discovery has been advanced by expression of heterologous receptors in living cells. However, due to the complexities inherent in such heterologous expression studies, the development of reliable assays to search for modulators of these receptors has presented particular challenges. For example, it is often difficult to obtain sufficient expression of heterologous G protein coupled receptors. The development of new means of optimizing heterologous receptor expression or enhancing the sensitivity of yeast based functional assays would be of tremendous benefit in the development of improved drug screening assays.
SUMMARY OF THE INVENTION
The present invention provides an important advance in drug screening methodologies for identifying modulators of G protein coupled receptors by providing, inter alia, a means of enhancing signaling through G protein coupled receptors in the membrane of yeast host cells.
In one embodiment, the invention pertains to an isolated yeast cell having a mutation that renders an endogenous yeast Cav1 protein nonfunctional such that the cell exhibits enhanced signaling via the pheromone response pathway. In preferred embodiments, the yeast cell of the present invention are Saccharomyces cells.
In another embodiment, the invention pertains to a recombinant yeast cell having an endogenous yeast pheromone system pathway and further expressing a heterologous G protein coupled receptor which functionally couples to the endogenous yeast pheromone system pathway and having a mutation which renders an endogenous yeast Cav1 protein nonfunctional. In a further embodiment, such a yeast cell further comprises a heterologous or chimeric G protein subunit. In yet another embodiment, such a yeast cell also has a reporter gene construct which produces a detectable signal upon stimulation of the yeast pheromone system pathway. In preferred embodiments, the yeast cell of the present invention are Saccharomyces cells.
In one embodiment, a heterologous G protein coupled receptor (e.g., a mammalian GPCR) is expressed in a yeast cell using a native leader sequence of the heterologous G protein coupled receptor. Alternatively, a heterologous G protein coupled receptor that naturally lacks a leader sequence can be expressed in the yeast cell. Still further, an unrelated leader sequence (i.e., a leader sequence that is heterologous to the GPCR, such as a yeast leader sequence with a mammalian GPCR such as &agr;-factor sequence) can be used to express the heterologous G protein coupled receptor. This unrelated leader sequence can be, for example, added to a heterologous GPCR that does not itself contain a leader sequence, or can replace the native leader sequence of a heterologous GPCR that itself contains a leader sequence, or can be added in tandem to a heterologous GPCR that itself contains a leader sequence (such that the resultant construct contains both the unrelated leader and the native leader). In one embodiment, the heterologous G protein coupled receptor is expressed in the yeast cell using a leader sequence other than an &agr;-factor leader sequence. In another embodiment, the heterologous G protein coupled receptor is expressed in the yeast cell using an &agr;-factor leader sequence.
In a preferred embodiment, the heterologous G protein coupled receptor is a mammalian G protein coupled receptor. In a particularly preferred embodiment, the heterologous G protein coupled receptor is a human G protein coupled receptor.
In a preferred embodiment, the heterologous G protein coupled receptor expressed by a yeast cell is a human nociceptin receptor. In another preferred embodiment, the heterologous G protein coupled receptor is a human melanocortin receptor. For example, the melanocortin receptor can be hMCR4 or hMCR5. In yet another preferred embodiment the heterologous G protein coupled receptor is a human somatostatin receptor. For example, the somatostatin receptor can be hSSTR2 or hSSTR3. In another preferred embodiment, the heterologous G protein coupled receptor is the human neuropeptide Y1 receptor. In another preferred embodiment, the heterologous G protein coupled receptor is the human neuropeptide Y2 receptor.
In another embodiment, the invention pertains to a method of identifying compounds which modulate a G protein coupled receptor, comprising the steps of:
a) providing a yeast cell that:
(i) expresses a heterologous G protein coupled receptor which functionally couples to the yeast pheromone response pathway; and
(ii) has a mutation which renders an endogenous yeast Cav1 protein nonfunctional;
b) contacting the yeast cell with a compound; and
c) identifying compounds which induce a change in a detectable signal in the yeast cell, wherein said detectable signal indicates that th
Ostanin Kirill
Silverman Lauren
Cadus Pharmaceutical Corporation
DeConti, Jr. Esq. Giulio A.
Lahive & Cockfield LLP
Lauro, Esq. Peter C.
Schwartzman Robert A.
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