Identification of activated receptors and ion channels

Abstract

The present invention related to methods and reagents for generating and using activating mutations of receptors and ion channels.

1 . A method for identifying constitutively activating mutations in a receptor or an ion channel, comprising: (A) providing a library of coding sequences for potentially activating mutations of a candidate receptor or ion channel, which library is generated by replacing coding sequences for small or medium side-chain amino acids with coding sequences for large side-chain amino acids, wherein said small or medium side-chain amino acids are located in or proximate transmembrane segment(s) of the receptor or ion channel; (B) expressing said library in host cells; (C) measuring the activity of the encoded receptor or ion channels in said host cells; (D) identifying those coding sequence(s) which encoded activated receptors or ion channels. 
     
     
         2 . A method for identifying a target second messenger or downstream signaling component of a receptor or ion channel, comprising: (A) identifying an activating mutation of a receptor or ion channel using the method of  claim 1 ; (B) expressing said activating mutation of a receptor or ion channel in host cells; and, (C) identifying one or more second messenger molecules or downstream signaling components whose level is higher or lower or which is modified as a consequence to expression of said activating mutation of said receptor or ion channel. 
     
     
         3 . The method of  claim 2 , wherein the receptor is selected from the group consisting of: a growth factor receptor, a cytokine receptor, a chemokine receptor, and an multisubunit immune recognition receptor (MIRR). 
     
     
         4 . The method of  claim 2 , wherein the receptor is a multipass transmembrane receptor. 
     
     
         5 . The method of  claim 4 , wherein the receptor is a 7TM receptor selected from the group consisting of: a G-protein coupled receptor, a chemoattractant peptide receptor, a neuropeptide receptor, a light receptor, a neurotransmitter receptor, and a polypeptide hormone receptor. 
     
     
         6 . The method of  claim 2 , wherein the receptor is a receptor tyrosine kinase (RTK). 
     
     
         7 . A method for identifying an antagonist of an activating mutation of a receptor or ion channel, comprising: (A) translationally providing a constitutively active mutant of a receptor or ion channel in an environment, which active mutant includes one or more point mutation(s) in or proximate a transmembrane segment(s) of the receptor that replace a small or medium amino acid residue with a large amino acid residue; (B) contacting the receptor or ion channel with a test agent; (C) comparing the activity of the receptor or ion channel in the presence of the test agent with the activity of the receptor or ion channel in the absence of the test agent; and, (D) identifying the test agent as an antagonist of the activated receptor or ion channel if the activity of the receptor or ion channel in the presence of the test agent is lower than the activity of the receptor or ion channel in the absence of the test agent. 
     
     
         8 . The method of  claim 7 , wherein the translationally providing step is performed in a cell. 
     
     
         9 . The method of  claim 8 , wherein the cell is a prokaryotic cell. 
     
     
         10 . The method of  claim 8 , wherein the cell is a eukaryotic cell. 
     
     
         11 . The method of  claim 10 , wherein the cell is selected from the group consisting of: a mammalian cell, an avian cell, an insect cell, a yeast cell, and a plant cell. 
     
     
         12 . The method of  claim 10 , wherein the cell is a pigment cell capable of dispersing or aggregating its pigment in response to an activated receptor or ion channel. 
     
     
         13 . The method of  claim 7 , wherein the test agent is a member of a library. 
     
     
         14 . The method of  claim 13 , wherein the library is selected from the group consisting of: a randomly synthesized polypeptide library, a semi-randomly synthesized polypeptide library, a cDNA encoded polypeptide library, a genomic DNA encoded polypeptide library, a synthetic chemical library, and a natural chemical compound library. 
     
     
         15 . A method for generating a non-human transgenic animal which expresses at least one activating mutant(s) of a receptor or an ion channel, comprising the steps of: (A) identifying an activating mutant of a receptor or an ion channel using the method of  claim 1 ; and, (B) generating a non-human transgenic animal expressing said activating mutant. 
     
     
         16 . The method of  claim 15 , wherein the transgenic animal is selected from the group consisting of: a mammal, an insect, and a yeast. 
     
     
         17 . A method for identifying a modulator of a receptor or ion channel, comprising: (A) identifying an activating mutation of a receptor or ion channel using the method of  claim 1 ; (B) identifying one or more second messenger molecules or downstream signaling components whose level is higher or lower or which is modified as a consequence to expression of said activating mutation of said receptor or ion channel; (C) contacting an environment expressing a wild-type receptor or ion channel with a test agent; (D) comparing the level of target second messenger molecules or down stream signaling components identified in step (B) in the presence or absence of the test agent; and, (E) determining whether the test agent increases or decreases the level of the target second messenger. 
     
     
         18 . A method of conducting a pharmaceutical business, comprising: (A) by the method of  claim 7 , identifying one or more agents which effects signaling by a cell-surface receptor or ion channel; (B) conducting therapeutic profiling of said identified agent(s), or further analogs thereof, for efficacy and toxicity in animals; and, (C) formulating a pharmaceutical preparation including one or more agents identified in step (B) as having an acceptable therapeutic profile. 
     
     
         19 . A method of conducting a pharmaceutical business, comprising: (A) by the method of  claim 17 , identifying one or more agents which effects signaling by a cell-surface receptor or ion channel; (B) conducting therapeutic profiling of said identified agent(s), or further analogs thereof, for efficacy and toxicity in animals; and, (C) formulating a pharmaceutical preparation including one or more agents identified in step (B) as having an acceptable therapeutic profile. 
     
     
         20 . The business method of  claim 18 , further comprising an additional step of establishing a distribution system for distributing the pharmaceutical preparation for sale. 
     
     
         21 . The business method of  claim 18 , further including establishing a sales group for marketing the pharmaceutical preparation. 
     
     
         22 . The business method of  claim 20 , further including establishing a sales group for marketing the pharmaceutical preparation. 
     
     
         23 . A method of conducting a pharmaceutical business, comprising: (A) by the method of  claim 7 , identifying one or more agents which effects signaling by a cell-surface receptor or ion channel; (B) (optionally) conducting therapeutic profiling of the agent for efficacy and toxicity in animals; and, (C) licensing, to a third party, the rights for further drug development of the target agent. 
     
     
         24 . A method of conducting a pharmaceutical business, comprising: (A) by the method of  claim 17 , identifying one or more agents which effects signaling by a cell-surface receptor or ion channel; (B) (optionally) conducting therapeutic profiling of the agent for efficacy and toxicity in animals; and, (C) licensing, to a third party, the rights for further drug development of the target agent.