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.
Simonović
M
M
Institute of Chemistry, Technology and Metallurgy, Belgrade, Yugoslavia.
Soskić
V
V
Joksimović
J
J
D004926:Escherichia coli / Q000235:genetics; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D005982:Glutathione Transferase / Q000235:genetics; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006639:Histidine; D006358:Hot Temperature; D006801:Humans; D011489:Protein Denaturation; D017448:Receptors, Dopamine D2 / Q000235:genetics / Q000502:physiology*; D011993:Recombinant Fusion Proteins
D017448:Receptors, Dopamine D2; D011993:Recombinant Fusion Proteins; D006153:Guanosine Diphosphate; D006639:Histidine; D006160:Guanosine Triphosphate; D005982:Glutathione Transferase; D019204:GTP-Binding Proteins; D019206:GTP-Binding Protein alpha Subunits, Gi-Go