ABSTRACT
An azido derivative of [3H2](2S, 3S)-cis-2-(diphenylmethyl)-N-((2-methoxyphenyl) methyl)-1-azabicyclo[2.2.2]octon-3-amine (CP-96,345), a potent nonpeptide antagonist of the substance P (SP) (neurokinin-1) receptor, was synthesized and shown to have an affinity for the human SP receptor similar to that of the parent compound, CP-96,345. When Chinese hamster ovary cells expressing the human SP receptor were photolabeled with this compound and analyzed with the use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography, several radioactive bands were observed, including a major band centered at molecular mass 80 kDa, the expected value for the SP receptor expressed in Chinese hamster ovary cells. Only the labeling of the 80-kDa protein was specific: nonradiolabeled CP-96,345 but not its optical enantiomer, CP-96,344 was a potent inhibitor of photoincorporation. SP prevented photolabeling only at concentrations higher than expected from its binding affinity but similar to those shown in a competition binding assay to displace radioiodinated analogue of CP-96,345. Antiserum generated against a synthetic peptide corresponding to the carboxyl terminus of the human SP receptor immunoprecipitated only the 80-kDa photoaffinity labeled protein, confirming that it is the human SP receptor. Interestingly, a second antiserum that was generated against the third extracellular loop of the G protein-coupled receptor no longer immunoprecipitated the receptor when covalently labeled with [3H2]azido-CP-96,345. This result indicates either that attachment of the antagonist modified the antigenic region directly, suggesting involvement of this domain in the binding of CP-96,345, or that the loss of recognition by the antiserum is secondary to a change in conformation induced by the covalent attachment of the antagonist at a different site.