Functional Impact of the G279S Substitution in the Adenosine A₁-Receptor (A₁R-G279S^7.44), a Mutation Associated with Parkinson's Disease

In medium-size, spiny striatal neurons of the direct pathway, dopamine D₁- and adenosine A₁-receptors are coexpressed and are mutually antagonistic. Recently, a mutation in the gene encoding the A₁-receptor (A₁R), A₁R-G279S^7.44, was identified in an Iranian family: two affected offspring suffered from early-onset l-DOPA-responsive Parkinson's disease. The link between the mutation and the phenotype is unclear. Here, we explored the functional consequence of the G279S substitution on the activity of the A₁-receptor after heterologous expression in HEK293 cells. The mutation did not affect surface expression and ligand binding but changed the susceptibility to heat denaturation: the thermodynamic stability of A₁R-G279S^7.44 was enhanced by about 2 and 8 K when compared with wild-type A₁-receptor and A₁R-Y288A^7.53 (a folding-deficient variant used as a reference), respectively. In contrast, the kinetic stability was reduced, indicating a lower energy barrier for conformational transitions in A₁R-G279S^7.44 (73 ± 23 kJ/mol) than in wild-type A₁R (135 ± 4 kJ/mol) or in A₁R-Y288A^7.53 (184 ± 24 kJ/mol). Consistent with this lower energy barrier, A₁R-G279S^7.44 was more effective in promoting guanine nucleotide exchange than wild-type A₁R. We detected similar levels of complexes formed between D₁-receptors and wild-type A₁R or A₁R-G279S^7.44 by coimmunoprecipitation and bioluminescence resonance energy transfer. However, lower concentrations of agonist were required for half-maximum inhibition of dopamine-induced cAMP accumulation in cells coexpressing D₁-receptor and A₁R-G279S^7.44 than in those coexpressing wild-type A₁R. These observations predict enhanced inhibition of dopaminergic signaling by A₁R-G279S^7.44 in vivo, consistent with a pathogenic role in Parkinson's disease. SIGNIFICANCE STATEMENT: Parkinson's disease is caused by a loss of dopaminergic input from the substantia nigra to the caudate nucleus and the putamen. Activation of the adenosine A₁-receptor antagonizes responses elicited by dopamine D₁-receptor. We show that this activity is more pronounced in a mutant version of the A₁-receptor (A₁R-G279S^7.44), which was identified in individuals suffering from early-onset Parkinson's disease.