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Mapping substance P binding sites on the neurokinin-1 receptor using genetic incorporation of a photoreactive amino acid.

ABSTRACT: Substance P (SP) is a neuropeptide that mediates numerous physiological responses, including transmission of pain and inflammation through the neurokinin-1 (NK1) receptor, a G protein-coupled receptor. Previous mutagenesis studies and photoaffinity labeling using ligand analogues suggested that the binding site for SP includes multiple domains in the N-terminal (Nt) segment and the second extracellular loop (ECLII) of NK1. To map precisely the NK1 residues that interact with SP, we applied a novel receptor-based targeted photocross-linking approach. We used amber codon suppression to introduce the photoreactive unnatural amino acid p-benzoyl-l-phenylalanine (BzF) at 11 selected individual positions in the Nt tail (residues 11-21) and 23 positions in the ECLII (residues 170(C-10)-193(C+13)) of NK1. The 34 NK1 variants were expressed in mammalian HEK293 cells and retained the ability to interact with a fluorescently labeled SP analog. Notably, 10 of the receptor variants with BzF in the Nt tail and 4 of those with BzF in ECLII cross-linked efficiently to SP, indicating that these 14 sites are juxtaposed to SP in the ligand-bound receptor. These results show that two distinct regions of the NK1 receptor possess multiple determinants for SP binding and demonstrate the utility of genetically encoded photocross-linking to map complex multitopic binding sites on G protein-coupled receptors in a cell-based assay format.

SUBMITTER: Valentin-Hansen L

PROVIDER: S-EPMC4140293 | biostudies-literature | 2014 Jun

REPOSITORIES: biostudies-literature

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Mapping substance P binding sites on the neurokinin-1 receptor using genetic incorporation of a photoreactive amino acid.

Valentin-Hansen Louise L Park Minyoung M Huber Thomas T Grunbeck Amy A Naganathan Saranga S Schwartz Thue W TW Sakmar Thomas P TP

The Journal of biological chemistry 20140515 26

Substance P (SP) is a neuropeptide that mediates numerous physiological responses, including transmission of pain and inflammation through the neurokinin-1 (NK1) receptor, a G protein-coupled receptor. Previous mutagenesis studies and photoaffinity labeling using ligand analogues suggested that the binding site for SP includes multiple domains in the N-terminal (Nt) segment and the second extracellular loop (ECLII) of NK1. To map precisely the NK1 residues that interact with SP, we applied a nov ...[more]

PMID: 24831006

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Mapping substance P binding sites on the neurokinin-1 receptor using genetic incorporation of a photoreactive amino acid.

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Mapping substance P binding sites on the neurokinin-1 receptor using genetic incorporation of a photoreactive amino acid.

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