Photoaffinity labeling of mutant neurokinin-1 receptors reveals additional structural features of the substance P/NK-1 receptor complex.
Photoaffinity labeling, receptor site-directed mutagenesis, and high-resolution NMR spectroscopy have been combined to further define the molecular details of the binding of substance P (SP) to the rat neurokinin-1 (NK-1) receptor. Mutant NK-1 receptors were constructed by substituting Ala for Met174 and/or Met181: residues previously identified as the sites of covalent attachment of radioiodinated, photoreactive derivatives of SP containing p-benzoyl-L-phenylalanine (Bpa) in positions 4 and 8, respectively. Photoaffinity labeling of the M181A mutant using radioiodinated Bpa8-SP resulted in a marked reduction in photoincorporation efficiency compared to the wild-type receptor. In contrast, photoaffinity labeling of the M174A mutant using radioiodinated Bpa4-SP gave the unexpected result of an increase in the efficiency of photoincorporation compared to the wild-type receptor. Enzymatic and chemical fragmentation analysis of the photolabeled receptor mutants established that the sites of covalent attachment were not the substituted alanine, but rather the other methionine on the second extracellular (E2) loop sequence, that is not the primary site of attachment in the wild-type receptor. The results thus suggest a close spatial relationship between Met174 and Met181 on the NK-1 receptor. To evaluate this structural disposition, NMR analyses were performed on a synthetic peptide with a sequence corresponding to the entire E2 loop and segments of the adjoining transmembrane helices to anchor the peptide in the lipids used to mimic a membrane. The structural features of the E2 loop include a centrally located alpha-helix, extending from Pro175 to Glu183, as well as smaller alpha-helices at the termini, corresponding to the transmembrane regions. The two methionine residues are located on the same face of the central alpha-helix, approximately 11 A apart from each other, and are therefore consistent with the conclusions of the photoaffinity labeling results.
Macdonald
D
D
Department of Pharmacology and Experimental Therapeutics, Boston University School of Medicine, Boston, Massachusetts 02118, USA.
Mierke
D F
DF
Li
H
H
Pellegrini
M
M
Sachais
B
B
Krause
J E
JE
Leeman
S E
SE
Boyd
N D
ND
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D007457:Iodine Radioisotopes / Q000378:metabolism; D008024:Ligands; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010649:Phenylalanine / Q000031:analogs & derivatives* / Q000378:metabolism; D019920:Photoaffinity Labels / Q000378:metabolism*; D017354:Point Mutation; D017433:Protein Structure, Secondary / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D018040:Receptors, Neurokinin-1 / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D013373:Substance P / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000378:metabolism; D013388:Succinimides / Q000378:metabolism; D014162:Transfection
D007457:Iodine Radioisotopes; D008024:Ligands; D046911:Macromolecular Substances; D010446:Peptide Fragments; D019920:Photoaffinity Labels; D018040:Receptors, Neurokinin-1; D013388:Succinimides; C101870:substance P, Bpa(8)-; D013373:Substance P; D010649:Phenylalanine; C022581:Bolton-Hunter reagent; C053764:benzoylphenylalanine