Probing the steroid binding domain-like I (SBDLI) of the sigma-1 receptor binding site using N-substituted photoaffinity labels

Biochemistry. 2008 Jul 8;47(27):7205-17. doi: 10.1021/bi800564j. Epub 2008 Jun 12.


Radioiodinated photoactivatable photoprobes can provide valuable insights regarding protein structure. Previous work in our laboratory showed that the cocaine derivative and photoprobe 3-[ (125)I]iodo-4-azidococaine ([ (125)I]IACoc) binds to the sigma-1 receptor with 2-3 orders of magnitude higher affinity than cocaine [Kahoun, J. R. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 1393-1397]. Using this photoprobe, we demonstrated the insertion site for [ (125)I]IACoc to be Asp188 [Chen, Y. (2007) Biochemistry 46, 3532-3542], which resides in the proposed steroid binding domain-like II (SBDLII) region (amino acids 176-194) [Pal, A. (2007) Mol. Pharmacol. 72, 921-933]. An additional photoprobe based on the sigma-1 receptor ligand fenpropimorph, 1- N-(2-3-[ (125)I]iodophenyl)propane ([ (125)I]IAF), was found to label a peptide in both the SBDLII and steroid binding domain-like I (SBDLI) (amino acids 91-109) [Pal, A. (2007) Mol. Pharmacol. 72, 921-933]. In this report, we describe two novel strategically positioned carrier-free, radioiodinated photoaffinity labels specifically designed to probe the putative "nitrogen interacting region" of sigma-1 receptor ligands. These two novel photoprobes are (-)-methyl 3-(benzoyloxy)-8-2-(4-azido-3-[ (125)I]iodobenzene)-1-ethyl-8-azabicyclo[3.2.1]octane-2-carboxylate ([ (125)I]-N-IACoc) and N-propyl- N-(4-azido-3-iodophenylethyl)-3-(4-fluorophenyl)propylamine ([ (125)I]IAC44). In addition to reporting their binding affinities to the sigma-1 and sigma-2 receptors, we show that both photoaffinity labels specifically and covalently derivatized the pure guinea pig sigma-1 receptor (26.1 kDa) [Ramachandran, S. (2007) Protein Expression Purif. 51, 283-292]. Cleavage of the photolabeled sigma-1 receptor using Endo Lys C and cyanogen bromide (CNBr) revealed that the [ (125)I]-N-IACoc label was located primarily in the N-terminus and SBDLI-containing peptides of the sigma-1 receptor, while [ (125)I]IAC44 was found in peptide fragments consistent with labeling of both SBDLI and SBDLII.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Autoradiography
  • Binding Sites
  • Cocaine / analogs & derivatives
  • Cocaine / chemical synthesis
  • Cocaine / chemistry
  • Cyanogen Bromide / metabolism
  • Guinea Pigs
  • Metalloendopeptidases / metabolism
  • Molecular Weight
  • Peptides / metabolism
  • Photoaffinity Labels / metabolism*
  • Protein Structure, Tertiary
  • Rats
  • Receptors, sigma / chemistry*
  • Receptors, sigma / metabolism


  • Peptides
  • Photoaffinity Labels
  • Receptors, sigma
  • sigma-1 receptor
  • sigma-2 receptor
  • Metalloendopeptidases
  • peptidyl-Lys metalloendopeptidase
  • Cocaine
  • Cyanogen Bromide