Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2001-01-30
2003-02-18
Kemmerer, Elizabeth (Department: 1646)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S006120, C435S007100, C435S007920
Reexamination Certificate
active
06521414
ABSTRACT:
FIELD OF THE INVENTION
The invention relates in general to the N-methyl-D-aspartate (NMDA) receptor and its signaling activity. The invention provides methods for identifying agonists and antagonists of NMDA receptor signaling, as well as compositions and methods useful for treating physiologic and pathologic conditions mediated by the NMDA receptor. The invention finds application in the biomedical sciences.
BACKGROUND OF THE INVENTION
In the majority of mammalian excitatory synapses, glutamate (Glu) mediates rapid chemical neurotransmission by binding to three distinct types of glutamate receptors on the surfaces of brain neurons. Although cellular responses mediated by glutamate receptors are normally triggered by exactly the same excitatory amino acid (EAA) neurotransmitters in the brain (e.g., glutamate or aspartate), the different subtypes of glutamate receptors have different patterns of distribution in the brain, and mediate different cellular signal transduction events. One major class of glutamate receptors is referred to as N-methyl-D-aspartate receptors (NMDA-Rs), since they bind preferentially to N-methyl-D-aspartate (NMDA). NMDA is a chemical analog of aspartic acid; it normally does not occur in nature, and NMDA is not present in the brain. When molecules of NMDA contact neurons having NMDA-Rs, they strongly activate the NMDA-R (i.e., they act as a powerful receptor agonist), causing the same type of neuronal excitation that glutamate does. It has been known that excessive activation of NMDA-R plays a major role in a number of important central nervous system (CNS) disorders, while hypoactivity of NMDA-R has been implicated in several psychiatric diseases.
NMDA-Rs contain an NR1 subunit and at least one of four different NR2 subunits (designated as NR2A, NR2B, NR2C, and NR2D). NMDA-Rs are “ionotropic” receptors since they control ion channels. These ion channels allow ions to flow into a neuron, thereby activating (depolarizing) the neuron, when the receptor is activated by glutamate, aspartate, or an agonist drug.
Protein tyrosine phosphorylation plays an important role in regulating diverse cellular processes. The regulation of protein tyrosine phosphorylation is mediated by the reciprocal actions of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). NMDA-Rs are regulated by protein tyrosine kinases and phosphatases. Phosphorylation of NMDA-R by protein tyrosine kinases results in enhanced NMDA-R responsiveness in neurons (Wang et al., Nature 369:233-235, 1994). NR2B and NR2A have been shown to be the main sites of phosphorylation by protein tyrosine kinases. Protein tyrosine phosphatases, on the other hand, exert opposing effects on the responsiveness of NMDA-R in the neurons (Wang et al, Proc. Natl. Acad. Sci. U.S.A. U.S.A. 93:1721-1725, 1996). It is believed that members of the Src family of protein tyrosine kinases mediate the NMDA-R tyrosine phosphorylation. On the other hand, the identity of the enzyme responsible for the counter dephosphorylation of NMDA-R has been elusive.
SUMMARY OF THE INVENTION
In one aspect, the present invention provides methods for identifying a modulator of N-methyl-D-aspartate receptor (NMDA-R) signaling by detecting the ability of an agent to modulate the phosphatase activity of a protein tyrosine phosphatase (PTP), e.g., on a NMDA-R substrate, or to modulate the binding of the PTP to NMDA-R. In one embodiment, the modulator is identified by detecting its ability to modulate the phosphatase activity of the PTP. In another embodiment, the modulator is identified by detecting its ability to modulate the binding of the PTP and the NMDA-R.
In a related aspect, the invention provides methods for identifying an agent as a modulator of NMDA-R signaling. The methods contain the steps of (a) contacting the agent with a composition containing PTPL1 and NMDA-R; (b) measuring the tyrosine phosphorylation level of the NMDA-R in the composition; and (c) comparing the NMDA-R tyrosine phosphorylation level thus obtained to the tyrosine phosphorylation level of the NMDA-R in the composition obtained in the absence of the agent. In an aspect, the invention provides a method for identifying an agent as a modulator of NMDA-R signaling, by contacting the (i) agent, (ii) PTPL1 (or a functional derivative thereof), and (iii) NMDA-R, NMDA-R subunit (or a functional derivative thereof), wherein either or both of (ii) and (iii) is substantially pure or recombinantly expressed; measuring the tyrosine phosphorylation level of NMDA-R (or functional derivative thereof) and comparing the tyrosine phosphorylation level in the presence of the agent with the tyrosine phosphorylation level in the absence of the agent, where a difference in tyrosine phosphorylation levels identifies the agent as a modulator of NMDA-R signaling. In one embodiment, the agent is identified by detecting its ability to enhance the PTPL1 dephosphorylation of the NMDA-R. In another embodiment, the agent is identified by detecting its ability to inhibit PTPL1 dephosphorylation of NMDA-R. In some related embodiments, the agent is screened for its ability to modulate binding of the PTPL1 to the NMDA-R. In one embodiment, the agent promotes or enhances the binding. In another embodiment, the agent disrupts or inhibits the binding. In certain other embodiments, the NMDA-R and the PTPL1 are present in a PTPL1/NMDA-R-containing protein complex.
In another related aspect, methods for identifying a nucleic acid molecule that modulates NMDA-R signaling are provided. Such methods contain the steps of introducing a nucleic acid molecule encoding a gene product into cells co-expressing the NMDA-R and PTPL1; culturing the cells harboring the nucleic acid molecule under conditions in which the gene product is expressed; measuring the tyrosine phosphorylation level of the NMDA-R in the cells containing the gene product; and comparing the NMDA-R tyrosine phosphorylation level thus obtained to NMDA-R tyrosine phosphorylation level in cells that do not harbor the nucleic acid molecule. Thus, the invention provides a method for identifying a nucleic acid molecule that modulates NMDA-R signaling, by (a) obtaining a cell culture coexpressing the NMDA-R (or functional derivative thereof) and PTPL1 (or functional derivative thereof), (b) introducing a nucleic acid molecule encoding a gene product into a portion of the cells; thereby producing cells comprising the nucleic acid molecule; (c) culturing the cells in (b) under conditions in which the gene product is expressed; (d) measuring the tyrosine phosphorylation level of the NMDA-R in the cells in (c) and comparing the level with that of control cells into which the nucleic acid molecule has not been introduced, wherein a difference in tyrosine phosphorylation levels identifies the nucleic acid molecule as a modulator of NMDA-R signaling.
In another aspect, the invention provides methods for treating a disease mediated by abnormal NMDA-R-signaling by administering a modulator of a PTPL1 activity that modulate the tyrosine phosphorylation level of the NMDA-R. In some embodiments, the modulator modulates the ability of PTPL1 to dephosphorylate NMDA-R. In some related embodiments, the modulator modulates the ability of PTPL1 to bind to NMDA-R. In certain embodiments, the modulator is a PTPL1 agonist and the disease to be treated is mediated by excessive NMDA-R signaling. In some other embodiments, the modulator is a PTPL1 antagonist and the disease to be treated is mediated by NMDA-R hypofunction.
In another aspect, the invention provides a method for isolating a polypeptide containing the PDZ2 domain of PTPL1 from a biological preparation containing the polypeptide.
In another aspect, the invention provides the use of an agent that modulates PTPL1 phosphatase activity in the treatment of a disease or condition mediated by NMDA-receptor activity or signaling.
In another aspect, the invention provides the use of a modulator (e.g. agonist or antagonist) of PTPL1 phosphatase activity in the manufacture of a medicament for treatment of a di
Kask Kalev
Melcher Thorsten
AGY Therapeutics, Inc.
Bozicevic Field & Francis LLP
Kemmerer Elizabeth
Li Ruixiang
Sherwood Pamela J.
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