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Site‐directed Mutagenesis in tandem with selective ligands to better understand the tertiary structure of GPCRs
Author(s) -
Pei Ying,
Kastal Arpad,
Mercier Richard,
Makriyannis Alexandros
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.5.a613-d
Subject(s) - pharmacophore , chemistry , cysteine , g protein coupled receptor , covalent bond , mutagenesis , site directed mutagenesis , amino acid , biochemistry , stereochemistry , receptor , mutant , enzyme , gene , organic chemistry
The cannabinoid CB2 receptor is an increasingly important therapeutic target for neuropathic pain, inflammation, cancer treatment, as well a variety of other physiological disorders. In lieu of the significance of this G‐protein coupled receptor, it is essential for researchers to investigate the manor in which cannabinergic ligands interact with the receptor¡ ¯ s binding domain(s). A number of high affinity covalent ligands (pharmacophores conjugated to isothiocyanate (NCS)) have been designed and synthesized in order to biochemically map the receptor¡ ¯ s binding site(s) including: AM‐841, AM‐1336, AM‐4772 as well as other novel high affinity covalent probes. These probes bind irreversibly to specific amino residues which incorporate sulfhydryl or unprotonated amino side chains by nucleophilic addition. Cysteine is the most preferred, however at pH above 10, well above the physiological pH, unprotonated lysine may couple with NCS. Using a global set of CB2 cysteine substitution (C to S) mutant transgenic cell lines, we are attempting to identify key cysteine residue in CB2 which covalently attach to specific ligands. These data will provide detail as to how the compound docks into its specific binding domain.