Involvement of non-conserved residues important for PGE2 binding to the constrained EP3 eLP2 using NMR and site-directed mutagenesis

Chillar, Annirudha; Wu, Jiaxin; So, Shui-Ping; Ruan, Ke-He

doi:10.1016/j.febslet.2008.07.018

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Volume 582, Issue 19 , Pages 2863-2868, 20 August 2008

Involvement of non-conserved residues important for PGE₂ binding to the constrained EP3 eLP₂ using NMR and site-directed mutagenesis

Edited by Miguel De la Rosa

Center for Experimental Therapeutics and Pharmacoinformatics, and Department of Pharmacological and Pharmaceutical Sciences, S&R-2, College of Pharmacy, University of Houston, Houston, TX 77204-5037, United States

Received 3 June 2008; received in revised form 6 July 2008; accepted 14 July 2008. published online 22 July 2008.

Abstract

A peptide constrained to a conformation of second extracellular loop of human prostaglandin-E₂ (PGE₂) receptor subtype3 (hEP3) was synthesized. The contacts between the peptide residues at S211 and R214, and PGE₂ were first identified by NMR spectroscopy. The results were used as a guide for site-directed mutagenesis of the hEP3 protein. The S211L and R214L mutants expressed in HEK293 cells lost binding to [³H]PGE₂. This study found that the non-conserved S211 and R214 of the hEP3 are involved in PGE₂ recognition, and implied that the corresponding residues in other subtype receptors could be important to distinguish the different configurations of PGE₂ ligand recognition sites.

Abbreviations: NOESY, nuclear overhauser effect spectroscopy, TOCSY, total correlation spectroscopy, NOE, nuclear overhauser effect

Keywords: Extracellular loop 2, Prostaglandin E₂, EP3 receptor