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J. Biol. Chem., Vol. 283, Issue 28, 19646-19656, July 11, 2008

This Article Full Text Full Text (PDF) All Versions of this Article: 283/28/19646    most recent M802192200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Pal, A. Articles by Ruoho, A. E. Search for Related Content PubMed PubMed Citation Articles by Pal, A. Articles by Ruoho, A. E. Social Bookmarking                      What's this?

Juxtaposition of the Steroid Binding Domain-like I and II Regions Constitutes a Ligand Binding Site in the -1 Receptor^*

Arindam Pal, Uyen B. Chu, Subramaniam Ramachandran, David Grawoig, Lian-Wang Guo, Abdol R. Hajipour, and Arnold E. Ruoho¹

From the Department of Pharmacology, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin 53705 and the Pharmaceutical Research Laboratory, College of Chemistry, Isfahan University of Technology, Isfahan 84156, Iran

-1 receptors represent unique binding sites that are capable of interacting with a wide range of compounds to mediate different cellular events. The composition of the ligand binding site of this receptor is unclear, since no NMR or crystal structures are available. Recent studies in our laboratory using radiolabeled photoreactive ligands suggested that the steroid binding domain-like I (SBDLI) (amino acids 91-109) and the steroid binding domain-like II (SBDLII) (amino acids 176-194) regions are involved in forming the ligand binding site(s) ( Chen, Y., Hajipour, A. R., Sievert, M. K., Arbabian, M., and Ruoho, A. E. (2007) Biochemistry 46, 3532-3542[CrossRef][Medline] [Order article via Infotrieve] ; Pal, A., Hajipour, A. R., Fontanilla, D., Ramachandran, S., Chu, U. B., Mavlyutov, T., and Ruoho, A. E. (2007) Mol. Pharmacol. 72, 921-933[Abstract/Free Full Text] ). In this report, we have further addressed this issue by utilizing our previously developed sulfhydryl-reactive, cleavable, radioiodinated photocross-linking reagent: methanesulfonothioic acid, S-((4-(4-amino-3-[¹²⁵I]iodobenzoyl) phenyl)methyl) ester (Guo, L. W., Hajipour, A. R., Gavala, M. L., Arbabian, M., Martemyanov, K. A., Arshavsky, V. Y., and Ruoho, A. E. (2005) Bioconjugate Chem. 16, 685-693). This photoprobe was shown to derivatize the single cysteine residues as mixed disulfides at position 94 in the SBDLI region of the wild type guinea pig -1 receptor (Cys⁹⁴) and at position 190 in the SBDLII region of a mutant guinea pig -1 receptor (C94A,V190C), both in a -ligand (haloperidol or (+)-pentazocine)-sensitive manner. Significantly, photocross-linking followed by Endo Lys-C cleavage under reducing conditions and intramolecular radiolabel transfer from the SBDLI to the SBDLII region in the wild type receptor and, conversely, from the SBDLII to the SBDLI region in the mutant receptor were observed. These data support a model in which the SBDLI and SBDLII regions are juxtaposed to form, at least in part, a ligand binding site of the -1 receptor.

Received for publication, March 19, 2008 , and in revised form, April 23, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Grant R01 MH065503 (to A. E. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Dept. of Pharmacology, University of Wisconsin School of Medicine and Public Health, 1300 University Ave., Madison, WI 53706. Tel.: 608-262-5382; Fax: 608-262-1257; E-mail: aeruoho{at}wisc.edu.

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