Hemipteran muscarinic receptor
Novel nucleic acid sequences encoding hemipteran muscarinic receptors, and recombinant expressions and host cells comprising the same are disclosed. Isolated hemipteran muscarinic receptors, host cells expressing hemipteran muscarinic receptors, methods of producing the hemipteran muscarinic receptors and antibodies specific for hemipteran muscarinic receptors are also disclosed. Methods of identifying modulators of hemipterana muscarinic receptors are disclosed.
1 . A substantially pure protein having the amino acid sequence selected from the group consisting of: SEQ ID NO: 2, a mutant thereof, a fragment thereof, SEQ ID NO: 4, a mutant thereof, and a fragment thereof.
2 . The protein of claim 1 wherein said protein has the amino acid sequence selected from the group consisting of: SEQ ID NO: 2, a mutant thereof, and a fragment thereof.
3 . An isolated nucleic acid molecule that comprises a nucleic acid sequence that encodes the protein of claim 1 .
4 . A recombinant expression vector comprising the nucleic acid molecule of claim 3 .
5 . A host cell comprising the recombinant expression vector of claim 4 .
6 . An isolated nucleic acid molecule having a nucleic acid sequence selected from the group consisting of: SEQ ID NO: 1, a fragment thereof having at least 10 nucleotides, SEQ ID NO: 3, and a fragment thereof having at least 10 nucleotides.
7 . An isolated nucleic acid molecule consisting of SEQ ID NO: 1 or a fragment thereof having at least 10 nucleotides.
8 . A recombinant expression vector comprising the nucleic acid molecule of claim 6 .
9 . A host cell comprising the recombinant expression vector of claim 8 .
10 . An isolated antibody which binds to an epitope on SEQ ID NO: 2 or an epitope on SEQ ID NO: 4.
11 . A method of identifying a modulator of a hemipteran muscarinic receptor protein activity comprising the steps of:
performing a test assay by contacting a test cell, which comprises a recombinant expression vector that contains a nucleic acid sequence that encodes said hemipteran muscarinic receptor, wherein said test cell expresses hemipteran muscarinic receptor, with a solution containing calcium in the presence of a test compound, and detecting the amount of intracellular calcium in said test cell;
performing a negative control assay by contacting a negative control cell which comprises a recombinant expression vector that contains a nucleic acid sequence that encodes said hemipteran muscarinic receptor, wherein said negative control cell expresses hemipteran muscarinic receptor, with a solution containing calcium in the absence of said test compound, and detecting the amount of intracellular calcium in said negative control cell;
comparing the amount of intracellular calcium in said test cell to the amount of intracellular calcium in said negative control cell
wherein a change in the amount of and a hemipteran muscarinic receptor agonist calcium in said test cell compared to the amount of intracellular calcium in said negative control cell indicates the test compound is a modulator of a hemipteran muscarinic receptor protein activity.
12 . The method of claim 11 wherein the intracellular calcium is detected by using an assay in which fluorescence generated by dye inside of said cell interacting with intracellular calcium is measured.
13 . The method of claim 11 further comprising performing a positive control assay by contacting a positive control cell which comprises a recombinant expression vector that contains a nucleic acid sequence that encodes said hemipteran muscarinic receptor, wherein said positive control cell expresses hemipteran muscarinic receptor, with a solution containing calcium in the absence of said test compound and in the presence of a hemipteran muscarinic receptor agonist, and detecting the amount of intracellular calcium in said positive control cell.
14 . The method of claim 11 further comprising the step of
performing a second-type negative control assay by contacting a hemipteran muscarinic receptor negative control cell that does not express hemipteran muscarinic receptor with a solution containing calcium in the absence of said test compound, and detecting the amount of calcium taken up by said hemipteran muscarinic receptor negative control cell; and/or
performing a third-type negative control assay by contacting a hemipteran muscarinic receptor negative control cell that does not express hemipteran muscarinic receptor with a solution containing calcium in the absence of said test compound and in the presence of a hemipteran muscarinic receptor agonist, and detecting the amount of calcium taken up by said hemipteran muscarinic receptor negative control cell.
15 . The method of claim 11 wherein said hemipteran muscarinic receptor protein has an amino acid sequence selected from the group consisting of: SEQ ID NO: 2, a mutant thereof, a fragment thereof, SEQ ID NO: 4, a mutant thereof, and a fragment thereof.
16 . The method of claim 15 wherein said hemipteran muscarinic receptor protein has an amino acid sequence selected from the group consisting of: SEQ ID NO: 2, a mutant thereof, and a fragment thereof.
17 . A method of identifying an inhibitor of a hemipteran muscarinic receptor protein activity comprising the steps of:
performing a test assay by contacting a test cell, which comprises a recombinant expression vector that contains a nucleic acid sequence that encodes said hemipteran muscarinic receptor, wherein said test cell expresses hemipteran muscarinic receptor, with a solution containing calcium and a hemipteran muscarinic receptor agonist in the presence of a test compound, and detecting the amount of intracellular calcium in said test cell;
performing a control assay by contacting a negative control cell, which comprises a recombinant expression vector that contains a nucleic acid sequence that encodes said hemipteran muscarinic receptor, wherein said test cell expresses hemipteran muscarinic receptor, with a solution containing calcium and a hemipteran muscarinic receptor agonist in the absence of said test compound, and detecting the amount of intracellular calcium in said negative control cell;
comparing the amount of intracellular calcium in said test cell to the amount of intracellular calcium in said control cell
wherein a decrease in the amount of intracellular calcium in said test cell compared to the amount of intracellular calcium in said control cell indicates the test compound is an inhibitor of a hemipteran muscarinic receptor protein activity.
18 . The method of claim 17 wherein the intracellular calcium is detected by using an assay in which fluorescence generated by dye inside of said cell interacting with intracellular calcium is measured.
19 . The method of claim 17 wherein said hemipteran muscarinic receptor protein has an amino acid sequence selected from the group consisting of: SEQ ID NO: 2, a mutant thereof, a fragment thereof, SEQ ID NO: 4, a mutant thereof, and a fragment thereof.
20 . A method of preparing an isolated protein having the amino acid sequence selected from the group consisting of: SEQ ID NO: 2, a mutant thereof, a fragment thereof, SEQ ID NO: 4, a mutant thereof, and a fragment thereof comprising the step of isolating said protein from a host cell of claim 8.