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- W1972259271 abstract "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 D1- CTLF 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." @default.
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- W1972259271 date "2002-01-01" @default.
- W1972259271 modified "2023-10-18" @default.
- W1972259271 title "Interaction of intracellular loops of dopamine d1 receptor with g protein subunits" @default.
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- W1972259271 doi "https://doi.org/10.2298/jmh0203255d" @default.
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