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

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

Journal: FEBS letters 2008;582(19):2863-8

PMID: 18652829

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Abstract

A peptide constrained to a conformation of second extracellular loop of human prostaglandin-E(2) (PGE(2)) receptor subtype3 (hEP3) was synthesized. The contacts between the peptide residues at S211 and R214, and PGE(2) 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 [(3)H]PGE(2). This study found that the non-conserved S211 and R214 of the hEP3 are involved in PGE(2) recognition, and implied that the corresponding residues in other subtype receptors could be important to distinguish the different configurations of PGE(2) ligand recognition sites.

Address: 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, USA.

DOI: 10.1016/j.febslet.2008.07.018

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MeSH Terms: Amino Acid Sequence, Cell Line, Conserved Sequence, Dinoprostone, Humans, Molecular Sequence Data, Mutagenesis, Site-Directed, Nuclear Magnetic Resonance, Biomolecular, Peptides, Point Mutation, Protein Conformation, Radioligand Assay, Receptors, Prostaglandin E, Receptors, Prostaglandin E, EP3 Subtype, Spectrometry, Fluorescence