David C, Fishburn C S, Monsma F J, Sibley D R, Fuchs S
Department of Chemical Immunology, Weizmann Institute of Science, Rehovot, Israel.
Biochemistry. 1993 Aug 17;32(32):8179-83. doi: 10.1021/bi00083a018.
Dopamine receptors belong to a superfamily of neurotransmitter receptors that are functionally coupled to guanine nucleotide binding proteins. In this study, we have used Chinese hamster ovary (CHO) cells stably transfected with the rat D2L receptor, in conjunction with specific anti-peptide antibodies that we have developed, in order to visualize this protein and the course of its synthesis. The newly synthesized receptor exists as a 45-kDa protein which undergoes further processing to a 75-kDa glycosylated receptor in the CHO cells. In pulse-chase experiments it was noticed that a 35-kDa precursor was present which disappeared after 30 min. In order to determine whether this 35-kDa protein represents an unprocessed form of the receptor, we have employed an in vitro translation system with cDNA constructs coding for both the murine D2 and D3 dopamine receptor isoforms. In the absence of processing, the D2 and D3 receptors have an apparent molecular mass of 35 kDa. The translated proteins were shown to be the full length receptors by immunoprecipitation with various anti-peptide antibodies and by the demonstration that they can undergo glycosylation to apparent molecular masses of approximately 45 kDa in an in vitro system.
多巴胺受体属于神经递质受体超家族，其在功能上与鸟嘌呤核苷酸结合蛋白偶联。在本研究中，我们使用稳定转染了大鼠D2L受体的中国仓鼠卵巢（CHO）细胞，并结合我们开发的特异性抗肽抗体，以可视化该蛋白及其合成过程。新合成的受体以45 kDa的蛋白形式存在，在CHO细胞中进一步加工为75 kDa的糖基化受体。在脉冲追踪实验中，注意到存在一个35 kDa的前体，30分钟后消失。为了确定这个35 kDa的蛋白是否代表受体的未加工形式，我们采用了体外翻译系统，该系统带有编码小鼠D2和D3多巴胺受体亚型的cDNA构建体。在没有加工的情况下，D2和D3受体的表观分子量为35 kDa。通过用各种抗肽抗体进行免疫沉淀，以及证明它们在体外系统中可以糖基化至约45 kDa的表观分子量，表明翻译的蛋白是全长受体。
