Valentin-Hansen, Louise4; Park, Minyoung3; Huber, Thomas3; Grunbeck, Amy3; Naganathan, Saranga3; Schwartz, Thue W.5; Sakmar, Thomas P3
1 Section for Metabolic Receptology, Novo Nordisk Foundation Center for Basic Metabolic Research, Faculty of Health and Medical Sciences, Københavns Universitet2 Molpharm Lab, Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences, Københavns Universitet3 unknown4 Section for Metabolic Receptology, Novo Nordisk Foundation Center for Basic Metabolic Research, Faculty of Health and Medical Sciences, Københavns Universitet5 Molpharm Lab, Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences, Københavns Universitet
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.
Journal of Biological Chemistry, 2014, Vol 289, Issue 26, p. 18045-54