Abstract

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-G279S^7.44) 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-G279S^7.44was enhanced by about 2 and 8 K when compared to wildtype A₁-receptorand 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 {plus minus} 23 kJ/mol) than in wildtype A₁R(135 {plus minus} 4 kJ/mol) or in A₁R-Y288A^7.53 (184 {plus minus} 24 kJ/mol). Consistent with this lower energy barrier, A₁R-G279S^7.44was 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-G279S^7.44by 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-G279S^7.44than in those co-expressing wildtype 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 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 respon­ses elicited by dopamine D₁-receptors. We show that this activity is more pronounced in a mutant version of the adenosine A₁-receptor (A₁R-G279S^7.44), which was identified in individuals suffering from early onset Parkinson's disease.