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Compositions and methods for identifying modulators of transducisomes, a new class of therapeutic targets

a technology of transducisomes and modulators, applied in the field of compositions and methods for identifying modulators of signal transduction in cells, can solve the problems of reducing the specificity of signal, affecting the normal function of receptors when heterologously expressed, and affecting the normal function of receptors, so as to avoid signal cross talk, enhance signal response time, specificity and selectivity

Inactive Publication Date: 2007-06-28
LIFE TECH CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0006] Cells respond to a wide variety of external signals mediated by cell surface receptors that transduce extracellular stimuli into an intracellular response. Although receptors that recognize different ligands are known to interact with the same intracellular signaling molecules, the specificity of signaling, often essential to a cell's physiological role, is maintained. How is this specificity maintained, or rather, how is signal cross talk avoided? One solution involves organizing different signaling cascades into physically and functionally distinct signaling units. Such assemblies can permit or enhance signal response time, specificity and selectivity while minimizing cross talk. Until the advent of present invention, know little was known about the architectural organization of the corresponding signaling machinery or how it can be used to discover useful modulators of signal transduction.

Problems solved by technology

Currently available assays of signal transduction are often hampered by low or non-specific signals.
Receptor activation pathways can cross talk, leading to a loss in signal specificity.
In addition, some receptors when heterologously expressed may not function in the normal fashion due to the absence of protein(s) integral to signal transduction function.
Additionally, assay tools for monitoring protein-protein interactions of signal transduction are few and cumbersome, such as antibody labeling and not well suited for high throughput screening.

Method used

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  • Compositions and methods for identifying modulators of transducisomes, a new class of therapeutic targets
  • Compositions and methods for identifying modulators of transducisomes, a new class of therapeutic targets
  • Compositions and methods for identifying modulators of transducisomes, a new class of therapeutic targets

Examples

Experimental program
Comparison scheme
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example 1

and Methods Used in the Examples

Mutant Screens and Western blots

[0140] Males of cn bw genotype were aged for 5 days, treated with EMS, and crossed en masse to flies carrying the dominant temperature sensitive DTS91 allele. Single F1 males were collected and crossed in single vials to CyO / DTS91 virgin females. The vials were then shifted to 29° C. for 72 hrs to eliminate any eggs or larvae carrying the DTS allele. The parents were then removed and the vials were incubated at 29° C. for an additional 48 hrs before returning to 25° C. The progeny from this cross were transferred to fresh food, and their homozygous white-eyed offspring (cn bw) were subjected to a protein immunoblot screen for the loss of the INAD antigen33.

Antibodies

[0141] To generate antibodies specific to INAD, we generated a T7-fusion protein consisting of the last 300 residues of the protein. All antibodies were checked for specificity and affinity using wild type, mutant, and transgenic controls. For immunosta...

example 2

Transducisome Structure

[0145] To investigate a putative transducisome and its structure, the inventors analyzed the protein structure of INAD (SEQ. ID NO.: 1). INAD was selected as a candidate transducisome because mutants in the gene, InaD, produce a dominant negative phenotype for photoreceptor activation. The primary structure of INAD was analyzed using BLAST. INAD is a modular protein composed of five closely linked PDZ domains (see FIG. 2). Each of these domains contains the structural hallmarks of a prototypical PDZ motif, including the conserved amino acid region target binding. Each PDZ domains, however, is different and displays sufficient differences in amino acid sequence to permit binding of different signal transduction proteins and to allow distinct protein-protein interactions. This finding was quite surprising, as previous studies had shown that INAD contains two PDZ domains instead of five.

[0146]FIG. 2A shows a diagram of INAD protein with the locations of and size...

example 3

somes Organize Signal Transduction in a Membranes

[0149] To investigate the ability of transducisomes to organize signal transduction proteins in membranes, the inventors measured binding of signal transduction proteins to INAD. The inventors assayed for binding of different phototransduction cascade proteins to INAD. Immunoprecipitations and GST-INAD protein fusions were used to identify INAD binding entities or targets.

[0150]FIG. 3A shows that INAD antibodies co-immunoprecipitate TRP, eye-PKC and PLCβ from retinal extracts38. Rhodopsin and Gqa failed to immunoprecipate despite the fact that both proteins are extremely abundant in photoreceptor cells. Membranes prepared from the heads of wt flies (IP) or trp, norpA and inaC mutants were immunoprecipitated (100 ug) with anti-INAD antibody as described in Example 1. The immunoprecipitated proteins were separated by SDS-PAGE, transferred to nitrocellulose and separately probed with antibodies specific for TRP, PLC, eye-PKC, G alpha su...

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Abstract

The invention provides cells and methods for identifying modulators of signal transduction, based on transducisome proteins that coordinate and assemble many types of signal transduction proteins. A transducisome is a PDZ domain containing protein that binds at least one signal transduction protein or a PDZ domain containing protein with at least one signal transduction protein bound. Examples of transducisome proteins include INAD, GRIP and other recently identified multi-PDZ domain proteins. Examples of signal transduction proteins include GPCRs, tyrosine kinase receptors, tyrosine phosphatase receptors, ion channels, phospholipases, adenylate cyclases, kinases and G-proteins. Also provided are methods for identifying modulators of signal transduction, proteins (and polynucleotides encoding the same) corresponding to transducisomes, modified transducisomes or defective transducisomes to use in assays of signal transduction, and a screening assay system for detecting protein-protein interactions.

Description

FIELD OF THE INVENTION [0001] The invention relates to compositions and methods for identifying modulators of signal transduction in cells, particularly signal transduction related to cell surface receptors and ion channels. BACKGROUND [0002] Many physiological signals (e.g., sensory, hormonal and neurotransmitter signals) are transduced from extracellular to intracellular environments by cell surface receptors. For example, G-protein coupled receptors (GPCRs) (for a review, see Neer, 1995, Cell 80:249-257), tyrosine kinase receptors and tyrosine phosphatase receptors are involved in signal transduction. [0003] As an example, GPCRs mediate signal transduction across a cell membrane upon the binding of a ligand to an extracellular portion of a GPCR. The intracellular portion of a GPCR interacts with a G-protein to modulate signal transduction from outside to inside a cell. A GPCR is therefore said to be “coupled” to a G-protein. G-proteins are composed of three polypeptide subunits: ...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A01K67/033C07H21/04C12N5/06C07K14/705C12N9/12C12N9/16
CPCG01N33/5041G01N2333/726G01N2333/912G01N2333/916
Inventor ZUKER, CHARLESMENDLEIN, JOHNSUN, YUMEITSUNODA, SUSANSIERRALTA, JIMENA
Owner LIFE TECH CORP
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