Mapping substance P binding sites on the neurokinin-1 receptor using genetic incorporation of a photoreactive amino acid.
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.
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.
Louise
L
Valentin-Hansen
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.
Minyoung
M
Park
Laboratory of Chemical Biology and Signal Transduction, The Rockefeller University, New York, New York 10065.
Thomas
T
Huber
Laboratory of Chemical Biology and Signal Transduction, The Rockefeller University, New York, New York 10065.
Amy
A
Grunbeck
Laboratory of Chemical Biology and Signal Transduction, The Rockefeller University, New York, New York 10065.
Saranga
S
Naganathan
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.
Thue W
TW
Schwartz
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.
Thomas P
TP
Sakmar
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001665:Binding Sites; D018360:Crystallography, X-Ray; D006801:Humans; D008958:Models, Molecular; D019920:Photoaffinity Labels; D011485:Protein Binding / Q000528:radiation effects; D017434:Protein Structure, Tertiary; D018040:Receptors, Neurokinin-1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013373:Substance P / Q000737:chemistry / Q000378:metabolism*; D014466:Ultraviolet Rays
D019920:Photoaffinity Labels; D018040:Receptors, Neurokinin-1; D013373:Substance P
Amber Codon Suppression; G Protein-coupled Receptor (GPCR); Homology Modeling; Membrane Protein; Photoactive Unnatural Amino Acid; Protein Cross-linking; Structural Biology