Functional impact of the G279S substitution in the adenosine A1-receptor (A1R-G279S), a mutation associated with Parkinson's disease.

In medium-size, spiny striatal neurons of the direct pathway, dopamine D1- and adenosine A1-receptors are co-expressed and are mutually antagonistic. Recently, a mutation in the gene encoding the A1-receptor (A1R-G279S7.44) was identified in an Iranian family: two affected off-springs 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-G279S7.44was enhanced by about 2 and 8 K when compared to wildtype A1-receptorand A1R-Y288A7.53 (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-G279S7.44(73 {plus minus} 23 kJ/mol) than in wildtype A1R(135 {plus minus} 4 kJ/mol) or in A1R-Y288A7.53 (184 {plus minus} 24 kJ/mol). Consistent with this lower energy barrier, A1R-G279S7.44was more effective in promoting guanine nucleotide-exchange than wildtype A1R. We detected similar levels of complexes formed between D1-receptors and wildtype A1R or A1R-G279S7.44by co-immunoprecipitation and bioluminescence resonance energy transfer (BRET). However, lower concentrations of agonist were required for half-maximum inhibition of dopamine-induced cAMP accumulation in cells co-expressing D1-receptor and A1R-G279S7.44than in those co-expressing wildtype A1R. These observations predict enhanced inhibition of dopa-minergic signaling by A1R-G279S7.44 in vivo consistent with a pathogenic role in Parkinson's disease. Significance Statement Parkinson's diseaseis caused by a loss of dopaminergic input from the substantia nigrato the caudate nucleus and the putamen. Activation of the adenosine A1-receptor antagonizes respon-ses elicited by dopamine D1-receptors. We show that this activity is more pronounced in a mutant version of the adenosine A1-receptor (A1R-G279S7.44), which was identified in individuals suffering from early onset Parkinson's disease.