The rat adenine receptor: pharmacological characterization and mutagenesis studies to investigate its putative ligand binding site
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DOI: 10.1007/s11302-013-9355-6
- Cite this article as:
- Knospe, M., Müller, C.E., Rosa, P. et al. Purinergic Signalling (2013) 9: 367. doi:10.1007/s11302-013-9355-6
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.
Keywords
Antibody G protein-coupled receptor Mutagenesis Radioligand binding Rat adenine receptor Sf9 cells Signal peptideSupplementary material
[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)
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)
[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)