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The rat adenine receptor: pharmacological characterization and mutagenesis studies to investigate its putative ligand binding site

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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 (Phe1103.24, Asn1153.29, Asn1734.60, 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 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 adenine-induced 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.

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  1. Thimm D, Knospe M, Abdelrahman A, Moutinho M, Alsdorf BBA, von Kügelgen I, Schiedel A, Müller CE (2013) Characterization of new G protein-coupled adenine receptors in mouse and hamster, submitted

  2. Seibt et al. (2013) The second extracellular loop of GPCRs determines subtype-selectivity and controls efficacy as evidenced by loop exchange study at A2 adenosine receptors, submitted

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Acknowledgments

A.C.S., C.E.M, D.T., and M.K. were supported by the state of NRW (NRW International Graduate Research Schools Chemical Biology and BIOTECH-PHARMA). We would like to thank Prof. Dr. Alfred G. Gilman (University of Texas Medical Center, Dallas, TX, USA) for providing Gαi2 baculoviruses and Prof. Dr. Peter Gierschik (Institute of Pharmacology, University of Ulm) for Gβ1γ2 baculoviruses. We are particularly grateful to Prof. Dr. Roland Seifert (Medizinische Hochschule Hannover) for valuable advice regarding the GTPγS experiments and for helpful comments on the manuscript.

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Authors and Affiliations

  1. PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, An der Immenburg 4, 53121, Bonn, Germany

    Melanie Knospe, Christa E. Müller, Aliaa Abdelrahman, Dominik Thimm & Anke C. Schiedel

  2. CNR—Institute of Neuroscience and Department of Medical Biotechnologies and Translational Medicine (BIOMETRA), University of Milan, Milan, Italy

    Patrizia Rosa

  3. PharmaCenter Bonn, Department of Pharmacology, University of Bonn, Sigmund-Freud-Straße 25, 53127, Bonn, Germany

    Ivar von Kügelgen

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  1. Melanie Knospe
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Supplemental Fig. 1

[3H]Adenine binding (10 nM) to membrane preparations of rat cortical membranes (100 μg protein) in the presence of different DMSO concentrations. Data represent means ± SEM of three independent experiments performed in triplicates or in duplicates. Results of one-way ANOVA: non-significantly different p = 0.673 (JPEG 35 kb)

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Supplemental Fig. 2

Multiple sequence alignment of 26 human and rodent Mrg receptors, including Mas and all known adenine receptors, generated using Clustal W. (*) identical amino acid residues; (:) conserved amino acid substitution; (.) semi-conserved amino acid substitution. Amino acid residues are given in the one-letter code (DOCX 29 kb)

Supplemental Fig. 3

Results from homologous competition binding of adenine to wt and mutant rat AdeRs expressed in Sf9 cells vs [3H]adenine (10 nM). Presented pIC50 values are means ± SEM from 3–7 independent experiments performed in triplicates (also see Table 1). The level of significance was determined by an unpaired t-test, ns: non-significant; p > 0.05; * p < 0.05; ***p < 0.001 (JPEG 26 kb)

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Supplemental Fig. 4

[35S]GTPγS binding studies at wt and mutant rat adenine receptors coexpressed with mammalian G proteins. Adenine-induced effects on [35S]GTPγS binding at the wt rat adenine receptor and its mutants F110A, N115A, N173A, F179A, F195A, H252A, and Y268A. The radioligand [35S]GTPγS was used at a concentration of 0.5 nM. The results shown represent means ± SEM from 3–5 independent experiments performed in triplicates. EC50 and Emax values are listed in Table 2 (JPEG 23 kb)

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(TIFF 4153 kb)

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Knospe, M., Müller, C.E., Rosa, P. et al. The rat adenine receptor: pharmacological characterization and mutagenesis studies to investigate its putative ligand binding site. Purinergic Signalling 9, 367–381 (2013). https://doi.org/10.1007/s11302-013-9355-6

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