COCAINE RECEPTOR--BIOCHEMICAL AND MOLECULAR STUDIES

这个项目的目标是阐明生物化学特性。 可卡因受体或多巴胺转运体。我们之前已经 研究表明，大鼠纹状体和核内的多巴胺转运体 伏安在分子质量上是不均匀的。当运输机 经N-葡聚糖酶处理后，两种转运蛋白的相对分子质量 显著减少，明显的差异是 缺少的或最小化的。这表明，异质性的因素是 糖基化的差异，尽管不能排除 异质性的其他来源也是如此。我们还准备了反 抗多巴胺转运蛋白的多肽抗体和其中一些 转运蛋白抗体可用于免疫沉淀和免疫印迹 吸墨水。 在克隆的多巴胺cdna中存在物种差异。 传送者。相应地，我们检测了其分子量 转运蛋白、死后变化以及糖基化差异 在不同的物种之间。分子量千差万别。 预期基于CDNA序列预测的差异。多巴胺 转运蛋白与可卡因及其类似物结合的两个不同部位 亲和力。这引发了一个问题，即是否存在更多 不只是一种类型的多巴胺转运体，或者是否由于异质性 可能是糖基化作用导致了不同的位点。 因此，多巴胺转运体的cDNA被导入COS 这些细胞表达可卡因的两个结合部位。这些 数据表明，这种异质结合发生在单个蛋白质上 它必须以不同的方式进行修改。 我们检测了多巴胺转运体在大脑中的分布。 通过原位杂交分析。我们发现在细胞群中发现的 中脑含有高水平的信使核糖核酸，而中脑以外的细胞团 中脑转运蛋白的信使核糖核酸水平很低。 我们在理解问题的本质方面取得了实质性进展 多巴胺转运蛋白，它是如何加工的，它在哪里 制作。

The goal of this project is to elucidate the biochemical properties of the cocaine receptor or the dopamine transporter. We have previously shown that the dopamine transporter in the rat striatum and nucleus accumbens are heterogeneous in molecular weight. When the transporters were treated with N-glycanase, the molecular weight of both transporters were reduced significantly and the apparent differences were either absent or minimized. This suggests that the factor in heterogeneity is differences in glycosylation although it is not possible to rule out other sources of heterogeneity as well. We have also prepared anti- peptide antibodies to the dopamine transporter and some of these transporter antibodies are useful for immunoprecipitation and western blotting. There are species differences in the cloned CDNAS for dopamine transporters. Accordingly, we examined the molecular weight of transporters, post-mortem changes as well as glycosylation differences among the different species. The molecular weights differed as one would expect based on differences predicted by the CDNA sequences. Dopamine transporters bind cocaine and its analogues with two sites of differing affinities. This has raised the issue of whether or not there are more than one type of dopamine transporter or whether heterogeneity due possibly to glycosylation could account for the different sites. Accordingly, a CDNA for the dopamine transporter was transfected into COS cells and, these cells expressed two binding sites for cocaine. These data indicate that the heterogeneous binding occurs from a single protein which must be modified in different ways. We have examined the distribution of dopamine transporter MRNA in brain by in situ hybridization analysis. We find that cell groups found in the midbrain contain high levels of MRNA while cell groups outside of the midbrain contain very low levels of MRNA for the transporter. We have made substantial progress in understanding the nature of the dopamine transporter protein, how it is processed and where it is produced.