Non-endogenous, constitutively activated human G protein-coupled receptors
First Claim
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1. A method for creating a non-endogenous, constitutively active version of an endogenous human G protein coupled receptor (GPCR), said endogenous GPCR comprising a transmembrane 6 region and an intracellular loop 3 region, the method comprising:
- (a) selecting an endogenous human GPCR comprising a proline residue in the transmembrane-6 region, wherein the GPCR is not the β
2-adrenergic receptor or the α
1B-adrenergic receptor;
(b) identifying the endogenous 16th amino acid residue from the proline residue of step (a), in a carboxy-terminus to amino-terminus direction;
(c) altering the identified amino acid residue of step (b) to a non-endogenous amino acid residue to create a non-endogenous version of the endogenous human GPCR; and
(d) determining if the non-endogenous version of the endogenous human GPCR of step (c) is constitutively active by measuring a difference in an intracellular signal measured for the non-endogenous version as compared with a signal induced by the endogenous human GPCR.
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Abstract
Disclosed herein are constitutively activated, non-endogenous versions of endogenous human G protein-coupled receptors comprising (a) the following amino acid sequence region (C-terminus to N-terminus orientation) and/or (b) the following nucleic acid sequence region (3′ to 5′ orientation) transversing the transmembrane-6 (TM6) and intracellular loop-3 (IC3) regions of the GPCR:
- (a) P1 AA15X
and/or - (b) pcodon (AA-codon)15 Xcodon,
respectively. In a most preferred embodiment, P1 and Pcodon are endogenous proline and an endogenous nucleic acid encoding region encoding proline, respectively, located within TM6 of the non-endogenous GPCR; AA15 and (AA-codon)15 are 15 endogenous amino acid residues and 15 codons encoding endogenous amino acid residues, respectively; and X and Xcodon are non-endogenous lysine and a non-endogenous nucleic acid encoding region encoding lysine, respectively, located within IC3 of the non-endogenous GPCR. Because it is most preferred that the non-endogenous human GPCRs which incorporate these mutations are incorporated into mammalian cells and utilized for the screening of candidate compounds, the non-endogenous human GPCR incorporating the mutation need not be purified and isolated per se (i.e., these are incorporated within the cellular membrane of a mammalian cell), although such purified and isolated non-endogenous human GPCRs are well within the purview of this disclosure.
- (a) P1 AA15X
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Citations
10 Claims
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1. A method for creating a non-endogenous, constitutively active version of an endogenous human G protein coupled receptor (GPCR), said endogenous GPCR comprising a transmembrane 6 region and an intracellular loop 3 region, the method comprising:
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(a) selecting an endogenous human GPCR comprising a proline residue in the transmembrane-6 region, wherein the GPCR is not the β
2-adrenergic receptor or the α
1B-adrenergic receptor;(b) identifying the endogenous 16th amino acid residue from the proline residue of step (a), in a carboxy-terminus to amino-terminus direction; (c) altering the identified amino acid residue of step (b) to a non-endogenous amino acid residue to create a non-endogenous version of the endogenous human GPCR; and (d) determining if the non-endogenous version of the endogenous human GPCR of step (c) is constitutively active by measuring a difference in an intracellular signal measured for the non-endogenous version as compared with a signal induced by the endogenous human GPCR. - View Dependent Claims (2, 3, 4, 5)
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6. A method for creating a non-endogenous, constitutively active version of an endogenous human G protein coupled receptor (GPCR), said endogenous GPCR comprising a transmembrane 6 region and an intracellular loop 3 region, the method comprising:
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(a) providing a polynucleotide;
said polynucleotide encoding an endogenous human GPCR, said endogenous GPCR comprising a transmembrane 6 region and an intracellular loop 3 region, said transmembrane 6 region comprisin a roline residue, wherein the GPCR is not the β
2-adrenergic receptor or the α
1B-adernergic receptor;(b) identifying the codon of said polynucleotide corresponding to the endogenous 16th amino acid residue from said proline residue of said GPCR of step (a), in a carboxy-terminus to amino-terminus direction; (c) altering said identified codon of step (b) to encode a non-endogenous amino acid residue, to provide a non-endogenous polynucleotide; (d) expressing said non-endogenous polynucleotide in a host cell, thereby providing a non-endogenous version of the endogenous human GPCR; and (e) determining if the non-endogenous version of the endogenous human GPCR of step (d) is constitutively active by measuring a difference in an intracellular signal measured for the non-endogenous version as compared with a signal induced by the endogenous human GPCR. - View Dependent Claims (7, 8, 9, 10)
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Specification