Boundy V A, Luedtke R R, Gallitano A L, Smith J E, Filtz T M, Kallen R G, Molinoff P B
Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia.
J Pharmacol Exp Ther. 1993 Feb;264(2):1002-11.
A baculovirus expression system provided an enriched source of biologically and immunologically active D3 dopamine receptors. Receptors expressed in Spodoptera frugiperda insect (Sf9) cells at a density of 5 to 15 pmol/mg of protein displayed high affinity for the antagonists, eticlopride, fluphenazine and spiroperidol, and the agonist, N-propylnorapomorphine. The binding of agonists was not sensitive to GTP. Antisera raised against synthetic peptides in the third intracellular loop of the D3 dopamine receptor immunoprecipitated binding sites for (S)-3-[125I]-iodo-2-hydroxy-5,6-dimethoxy-N-[(1-ethyl-2-pyrrolidinyl)- methyl]-benzamide from solubilized extracts of infected Sf9 cells and detergent extracts of rat caudate. These antisera specifically recognized a single band on immunoblots of Sf9 cells infected with recombinant D3 baculovirus. Both the immunoprecipitation and immunoblot reactions were blocked by preincubation of the antisera with the immunization peptide. These results suggest that the D3 receptor protein is expressed in rat brain.
杆状病毒表达系统提供了一种富含生物活性和免疫活性的D3多巴胺受体来源。在草地贪夜蛾昆虫（Sf9）细胞中以5至15 pmol/mg蛋白质的密度表达的受体，对拮抗剂依替必利、氟奋乃静和螺哌啶醇以及激动剂N-丙基去甲阿扑吗啡表现出高亲和力。激动剂的结合对GTP不敏感。针对D3多巴胺受体第三细胞内环中的合成肽产生的抗血清，从感染的Sf9细胞的溶解提取物和大鼠尾状核的去污剂提取物中免疫沉淀了（S）-3-[125I]-碘-2-羟基-5,6-二甲氧基-N-[(1-乙基-2-吡咯烷基）-甲基]-苯甲酰胺的结合位点。这些抗血清在重组D3杆状病毒感染的Sf9细胞的免疫印迹上特异性识别一条单一的条带。免疫沉淀和免疫印迹反应都通过将抗血清与免疫原性肽预孵育而被阻断。这些结果表明D3受体蛋白在大鼠脑中表达。
