Probing the steroid binding domain-like I (SBDLI) of the sigma-1 receptor binding site using N-substituted photoaffinity labels.
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.
Department of Pharmacology, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin 53706, USA.
Dominique
D
Fontanilla
Abdol R
AR
Hajipour
Arindam
A
Pal
Uyen B
UB
Chu
Marty
M
Arbabian
Arnold E
AE
Ruoho
D000818:Animals; D001345:Autoradiography; D001665:Binding Sites; D003042:Cocaine / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000737:chemistry; D003488:Cyanogen Bromide / Q000378:metabolism; D006168:Guinea Pigs; D008666:Metalloendopeptidases / Q000378:metabolism; D008970:Molecular Weight; D010455:Peptides / Q000378:metabolism; D019920:Photoaffinity Labels / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D051381:Rats; D017480:Receptors, sigma / Q000737:chemistry* / Q000378:metabolism; D000097605:Sigma-1 Receptor
D003042:Cocaine; D003488:Cyanogen Bromide; D008666:Metalloendopeptidases; D010455:Peptides; D019920:Photoaffinity Labels; D017480:Receptors, sigma; D000097605:Sigma-1 Receptor; C106107:sigma-2 receptor; C039830:peptidyl-Lys metalloendopeptidase