G protein-coupled receptor kinase-2 confers isoform-specific calcium sensitivity to dopamine D₂ receptor...

The dopamine D₂ receptor (D₂ R) functions as an autoreceptor on dopaminergic cell bodies and terminals and as a postsynaptic receptor on a variety of neurons in the central nervous system. As a result of alternative splicing, the D₂ R is expressed as two isoforms: long (D_2L R) and short (D_2S R) differing by a stretch of 29 residues in the third intracellular loop, with D_2S R being the predominant presynaptic isoform. Recent reports described a Ca²⁺ sensitivity of the desensitization time course of potassium currents elicited via D_2S R, but not via D_2L R, when either isoform was selectively expressed in dopaminergic neurons. Here, we aimed to study the mechanism behind this subtype-specific Ca²⁺ sensitivity. Thus, we measured the desensitization of potassium channel responses evoked by D_2L R and D_2S R using two-electrode voltage clamp in Xenopus oocytes in the absence and presence of different amounts of β-arrestin2 and G protein-coupled receptor kinase-2 (GRK2), both of which are known to play important roles in D₂ R desensitization in native cells. We found that co-expression of both GRK2 and β-arrestin2 was necessary for reconstitution of the Ca²⁺ sensitivity of D_2S R desensitization, while D_2L R did not display Ca²⁺ sensitivity under these conditions. The effect of Ca²⁺ chelation by BAPTA-AM to slow the rate of D_2S R desensitization was mimicked by the GRK2 inhibitor, Cmpd101, and by the kinase-inactivating GRK2 mutation, K220R, but not by the PKC inhibitor, Gö6976, nor by the calmodulin antagonist, KN-93. Thus, Ca²⁺ -sensitive desensitization of D_2S R appears to be mediated via a GRK2 phosphorylation-dependent mechanism.