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

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.

FIGURE 1.

Interaction between substance P and the NK1 receptor. A, amino acid sequences for part of the N terminus and the ECLII of the NK1 receptor. Residues marked in green are found to be important for SP binding by mutational analysis. Residues colored in red are residues or fragments of NK1 that cross-linked to photoreactive analogues of SP. The asterisk represents one of the conserved cysteines bridging ECLII to TM-III. B, sequence of the endogenous peptide substance P, the conserved amidated C-terminal, FXGLM-NH₂, is shown in italics. C, binding properties of SP (black), F-SP (blue), and F-SP(BzF8) (green) to NK1^1D4, measured in competition binding against ¹²⁵I-labeled Lys-3]-SP. Functionality of the NK1^1D4 receptor, measured as SP (black), F-SP (blue), and F-SP(BzF8) (green), induced IP₃ production in HEK293T cells transiently expressing the NK1^1D4 receptor.

FIGURE 2.

Expression and labeling of the NK1^1D4 receptor. Western blot analysis of cell lysate from HEK293T cells expressing the NK1^1D4 receptor. NK1^1D4 receptor was incubated for 10 min with 100 nm F-SP(BzF8) unexposed (lane 1) or exposed (lanes 2 and 3) to UV light. UV exposure was also performed in the presence of a 100-fold excess of unlabeled SP (F-SP) (lane 4). No unspecific binding was observed in lane 5 where CCR5^1D4 was stimulated with 100 nm F-SP(BzF8) and subsequently exposed to UV light for 15 min. Lanes 6–9 are equivalent to lanes 1–4 but display NK1^1D4 receptor expressed in an N-acetylglucosaminyltransferase I-negative (GnTI⁻) HEK293 cell line that prevents formation of complex N-glycans. IP, immunoprecipitate; IB, immunoblot.

FIGURE 3.

Interaction between F-SP and N-terminal NK1^1D4-BzF mutants. A, HEK293 cells transiently expressed NK1^1D4-BZF mutants in positions 11–21 of the N terminus. NK1^1D4 WT receptor was either stimulated with 100 nm F-SP (lane 1) or F-SP(BzF8) (lane 2) for 10 min before 15 min of UV exposure. All NK1^1D4-BZF mutants were incubated with 100 nm F-SP before 15 min of UV exposure. B, functional analysis of the NK1^1D4-BZF mutants of the N terminus as measured by IP₃ accumulation without stimulation (Media) or after 45 min incubation with either SP, F-SP, or F-SP(BzF8). C, functional analysis of the S20BzF NK1^1D4 mutant in the absence of BzF but stimulated with 100 nm SP (−BzF), with BzF present but without ligand stimulation (Media), or with both BzF present in the media and stimulation of 100 mm SP. IP, immunoprecipitate; IB, immunoblot.

FIGURE 4.

Interaction between F-SP/F-SP(BzF8) and ECLII NK1^1D4-BzF mutants. HEK293 cells were transiently expressing NK1^1D4-variants in position 172(C-8) to 183(C+3) of the ECLII. A, WT receptor was either stimulated with 100 nm F-SP (lane 1) or F-SP(BzF8) (lane 2) for 10 min before 15 min of UV exposure. All NK1^1D4-BZF mutants were incubated with 100 nm F-SP before 15 min of UV exposure. IP, immunoprecipitate; IB, immunoblot. B, WT NK1^1D4 was either stimulated with F-SP (lane 1) or F-SP(BzF8) (lane 2) for 10 min before 15 min of UV exposure. The NK1^1D4-BZF mutants were incubated for 10 min with F-SP(BzF8) before 15 min of UV exposure. C, functional analysis of the NK1^1D4-BZF mutants of the ECLII as measured by IP₃ accumulation without stimulation or after 45 min of stimulation with either SP, F-SP, or F-SP(BzF8).

FIGURE 5.

Structural model of the NK1 receptor in complex with the SP ligand. SP is shown in yellow. All 100 SP structural conformations resulting from the modeling exercise are shown. NK1 residues highlighted in shades of blue indicate the sites of cross-linking between NK1 and SP using site-specific introduction of BzF. Residues shown in green represent sites that when mutated caused loss of SP binding. The N-terminal residues 15–21 of the receptor are not structurally defined due to a lack of homology with the template structure and are rendered in a transparent representation. The Ct six amino acid residues of SP are well ordered and bind in a pocket on the extracellular surface of the receptor. The rest of SP is not well constrained, so multiple conformations are possible. The high mobility of both the amino portion of SP and the Nt tail of NK1 explains the observation that multiple sites on the Nt tail of NK1 photocross-link to bound SP.