Mapping Substance P Binding Sites on the neurokinin-1 receptor Using Genetic Incorporation of a Photoreactive Amino Acid [Protein Structure and Folding]

May 15th, 2014 by Valentin-Hansen, L., Park, M., Huber, T., Grunbeck, A., Naganathan, S., Schwartz, T. W., Sakmar, T. P.

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 (GPCR). 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 photo-crosslinking approach. We used amber codon suppression to introduce the photoreactive unnatural amino acid p-benzoyl-L-phenylalanine (BzF) at eleven 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 HEK-293 cells and retained the ability to interact with a fluorescently labeled SP analogue. Notably, ten of the receptor variants with BzF in the Nt tail and four of those with BzF in ECLII crosslinked 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 photo-crosslinking to map complex multitopic binding sites on GPCRs in a cell-based assay format.
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