Quantification of human dopamine D2s receptor interactions with G alpha(i,1,2)- and G alpha(o)-proteins

A simple and rapid in vitro method for qualitative and quantitative estimation of the G alpha-subunits interaction with the third intracellular loop of human D2s dopamine receptor has been developed. For this purpose, D2s-CL3 was cloned in pGEX-2T vector and expressed in E. coli BL21 DE3 as a fusion protein with glutathione-S-transferase (D2s-CL3-GST). The resulting soluble construct was purified by affinity chromatography on glutathione-Sepharose. G alpha-subunits were expressed and purified as His-tagged proteins. For the assay of G alpha/D2s-CL3-GST interactions, varying concentrations of pure His-tagged G alpha-proteins were immobilized on His-Bind Resin and titrated with D2s-CL3-GST fusion protein. G alpha/D2s-CL3-GST interactions were quantified by GST activity determination assay. It was shown that the fusion protein interacts specifically with different G alpha proteins, especially with G alpha(i) proteins. Based on saturation binding analyses, Kd values were determined revealing the highest affinity of His-G alpha(i,2) binding to the fusion protein. The affinities for G alpha(i)/D2s-CL3-GST protein interactions estimated in this way were in nanomolar range of concentrations.