Involvement of the Second Extracellular Loop (E2) of the Neurokinin-1 Receptor in the Binding of Substance P

Abstract

Substance P (SP) interacts with the neurokinin-1 (NK-1) G-protein-coupled receptor, which has been cloned in several species. In the present study, the domains of the NK-1 receptor involved in the binding of SP and SP-(7-11) C-terminal fragment have been analyzed using two peptide analogs containing the photoreactive amino acid para-benzoylphenylalanine

Methionine acts as a “magnet” in photoaffinity crosslinking experiments

“…Many photoaffinity crosslinking reports (including studies from us and others working on different ligand-receptor systems) which identified methionine as the crosslinked residue [1,3,15,16], may now warrant interpretation in a new light. The identified sites of interaction might have been significantly shifted by the presence of methionine.…”

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

“…1) is critical for NK1-SP interaction (33). Furthermore, the two loop residues Met-174(C-6) and Met-181(Cϩ1) are close to the ligand in NK1-SP complexes (15,17,19,21). Specifically, the conserved disulfide bridge positions Met-181(Cϩ1) approximately one helical turn above His-108 (colored green in Fig.…”

“…In addition, the substitution of Phe-8 of SP, SP(BzF8), resulted in cross-linking to Met-181(Cϩ1) and possibly Met-174(C-6) (highlighted in red, Fig. 1A) (15,17,19,21). Taken together, the previous ligand binding studies indicated that the SP peptide presumably interacts with epitopes presented on the outer part of the extracellular portion of the NK1 receptor, but the overall results have not led to a satisfactory refined model of how SP binds to the NK1 receptor.…”

“…cross-linking to Met-174(C-6) and Met-181(Cϩ1) in the ECLII of NK1 had previously been suggested (15,17,19,21), we decided to further investigate the ECLII region. It has been established that Cys-180 forms an intramolecular disulfide bridge with Cys-105 that is essential for SP binding (33).…”

“…1, highlighted in green) (8 -11). A photocross-linking approach using synthetic artificial SP analogues containing an unnatural amino acid, p-benzoyl-L-phenylalanine (BzF), was also employed to investigate how SP interacts with the NK1 receptor (12)(13)(14)(15)(16)(17)(18)(19). To identify the cross-linking site of the SP analog on the NK1 receptor, this method required that receptor residues or fragments that cross-linked to the SP analog were determined using proteolytic digestion and/or mass spectrometry of the cross-linked complex.…”