Interaction of intracellular loops of dopamine d1 receptor with g protein subunits

Summary: A simple and rapid method for qualitative and quantitative estimation of Ga 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. Ga subunits were expressed and purified as His-tagged proteins (Gao and Gai1 in E.coli BL21 DE3 and Gas in E. coli JM 109). For quantitative assay, varying concentrations of pure His-tagged Ga 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 Ga 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 Gas subunit, and D1CTLF with Gao. No other interactions were observed. Based on saturation binding analyses, Kd values in nanomolar range of concentrations demonstrated the highest binding affinity of His-Gas for D1-CTSF-GST and of His-Gao for D1-CTLF-GST.