Mapping Substance P Binding Sites on the Neurokinin-1 Receptor Using Genetic Incorporation of a Photoreactive Amino Acid

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Louise Valentin-Hansen, Minyoung Park, Thomas Huber, Amy Grunbeck, Saranga Naganathan, Thue W. Schwartz, Thomas P Sakmar

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.

Original languageEnglish
JournalThe Journal of Biological Chemistry
Volume289
Issue number26
Pages (from-to)18045-54
Number of pages10
ISSN0021-9258
DOIs
Publication statusPublished - 27 Jun 2014

    Research areas

  • Amino Acid Motifs, Amino Acid Sequence, Binding Sites, Crystallography, X-Ray, Humans, Models, Molecular, Photoaffinity Labels, Protein Binding, Protein Structure, Tertiary, Receptors, Neurokinin-1, Substance P, Ultraviolet Rays

ID: 137293697