Interaction of intracellular loops of dopamine D₁ receptor with G protein subunits

A simple and rapid method for qualitative and quantitative estimation of Gα subunit interactions with the second and the third intracellular loop, as well as with C-terminal part of human D₁ dopamine receptor has been developed. For this purpose, D₁-ICL₂ and D₁-ICL₃ were cloned in pGEX-2T vector and expressed in E. coli BL21 as fusion proteins with glutathione-S-transferase (D₁-ICL₂-GST and D₁-ICL₃-GST). C-terminal part was cleaved into two fragments which were cloned in pGEX-2T and expressed in E. coli BL21 DE3 as fusion proteins with glutathione-S-transferase (D1-CTSF-GST and D1-CTLF-GST). The resulting soluble constructs were purified by affinity chromatography on glutathione-Sepharose. Gα subunits were expressed and purified as His-tagged proteins (Gαo and Gαi₁ in E. coli BL21 DE3 and Gαs in E. coli JM 109). For quantitative assay, varying concentrations of pure His-tagged Gα subunits were immobilized on His-Bind resin and titrated with fusion proteins and the interactions were estimated by a colorimetric assay for GST activity determination. Similar assay was employed to qualitatively demonstrate the interactions. For this purpose pure fusion proteins were immobilized on glutathione-Sepharose in known concentrations and treated with known concentrations of pure His-tagged Gα subunits. Thus created complexes were eluted from glutathione-Sepharose and analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). It was shown that D₁-CTSF interacts specifically with Gαs subunit, and D₁-CTLF with Gαo. No other interactions were observed. Based on saturation binding analyses, Kd values in nanomolar range of concentrations demonstrated the highest binding affinity of His-Gαs for D₁-CTSF-GST and of His-Gαo for D₁-CTLF-GST.