The rat adenine receptor: pharmacological characterization and mutagenesis studies to investigate its putative ligand bi
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The rat adenine receptor: pharmacological characterization and mutagenesis studies to investigate its putative ligand binding site Melanie Knospe & Christa E. Müller & Patrizia Rosa & Aliaa Abdelrahman & Ivar von Kügelgen & Dominik Thimm & Anke C. Schiedel
Received: 5 January 2013 / Accepted: 23 January 2013 # Springer Science+Business Media Dordrecht 2013
Abstract The rat adenine receptor (rAdeR) was the first member of a family of G protein-coupled receptors (GPCRs) activated by adenine and designated as P0-purine receptors. The present study aimed at gaining insights into structural aspects of ligand binding and function of the rAdeR. We exchanged amino acid residues predicted to be involved in ligand binding (Phe110 3.24 , Asn115 3.29 , Asn173 4.60 , Phe17945.39, Asn1945.40, Phe1955.41, Leu2015.47, His2526.54, and Tyr2687.32) for alanine and expressed them in Spodoptera frugiperda (Sf9) insect cells. Membrane preparations subjected to [3H]adenine binding studies revealed only minor effects indicating that none of the exchanged amino acids is part of the ligand binding pocket, at least in the inactive state of the receptor. Furthermore, we coexpressed the rAdeR and its mutants with mammalian Gi proteins in Sf9 insect cells to
Electronic supplementary material The online version of this article (doi:10.1007/s11302-013-9355-6) contains supplementary material, which is available to authorized users. M. Knospe : C. E. Müller (*) : A. Abdelrahman : D. Thimm : A. C. Schiedel (*) PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, An der Immenburg 4, 53121 Bonn, Germany e-mail: [email protected] e-mail: [email protected] P. Rosa CNR—Institute of Neuroscience and Department of Medical Biotechnologies and Translational Medicine (BIOMETRA), University of Milan, Milan, Italy I. von Kügelgen PharmaCenter Bonn, Department of Pharmacology, University of Bonn, Sigmund-Freud-Straße 25, 53127 Bonn, Germany
probe receptor activation. Two amino acid residues, Asn1945.40 and Leu2015.47, were found to be crucial for activation since their alanine mutants did not respond to adenine. Moreover we showed that—in contrast to most other rhodopsin-like GPCRs—the rAdeR does not contain essential disulfide bonds since preincubation with dithiothreitol neither altered adenine binding in Sf9 cell membranes, nor adenineinduced inhibition of adenylate cyclase in 1321N1 astrocytoma cells transfected with the rAdeR. To detect rAdeRs by Western blot analysis, we developed a specific antibody. Finally, we were able to show that the extended N-terminal sequence of the rAdeR constitutes a putative signal peptide of unknown function that is cleaved off in the mature receptor. Our results provide important insights into this new, poorly investigated family of purinergic receptors. Keywords Antibody . G protein-coupled receptor . Mutagenesis . Radioligand binding . Rat adenine receptor . Sf9 cells . Signal peptide
Introduction Nucleotides and nucleosides, such as ATP, ADP, and adenosine, have lon
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