| 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:n (a) P 1 AA 15 Xn nand/orn (b) p codon (AA-codon) 15 X codon ,n nrespectively. In a most preferred embodiment, P 1 and P codon are endogenous proline and an endogenous nucleic acid encoding region encoding proline, respectively, located within TM6 of the non-endogenous GPCR; AA 15 and (AA-codon) 15 are 15 endogenous amino acid residues and 15 codons encoding endogenous amino acid residues, respectively; and X and X codon 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. |