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J Biol Chem. 2014 Jun 27;289(26):18045-54. doi: 10.1074/jbc.M113.527085. Epub 2014 May 15.

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

Author information

1
From the Laboratory for Molecular Pharmacology, Department of Neuroscience and Pharmacology, University of Copenhagen, The Panum Institute, Blegdamsvej 3, 2200 Copenhagen, Denmark, Section for Metabolic Receptology and Enteroendocrinology, Novo Nordisk Foundation Center for Basic Metabolic Research, University of Copenhagen, Blegdamsvej 3, 2200 Copenhagen, Denmark, and.
2
Section for Metabolic Receptology and Enteroendocrinology, Novo Nordisk Foundation Center for Basic Metabolic Research, University of Copenhagen, Blegdamsvej 3, 2200 Copenhagen, Denmark, and Laboratory of Chemical Biology and Signal Transduction, The Rockefeller University, New York, New York 10065.
3
Laboratory of Chemical Biology and Signal Transduction, The Rockefeller University, New York, New York 10065.
4
Section for Metabolic Receptology and Enteroendocrinology, Novo Nordisk Foundation Center for Basic Metabolic Research, University of Copenhagen, Blegdamsvej 3, 2200 Copenhagen, Denmark, and Laboratory of Chemical Biology and Signal Transduction, The Rockefeller University, New York, New York 10065 sakmar@rockefeller.edu.

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.

KEYWORDS:

Amber Codon Suppression; G Protein-coupled Receptor (GPCR); Homology Modeling; Membrane Protein; Photoactive Unnatural Amino Acid; Protein Cross-linking; Structural Biology

PMID:
24831006
PMCID:
PMC4140293
DOI:
10.1074/jbc.M113.527085
[Indexed for MEDLINE]
Free PMC Article

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