COCAINE RECEPTOR--BIOCHEMICAL AND MOLECULAR STUDIES

可卡因受体——生物化学和分子研究

• 批准号: 3775033
• 负责人: M J KUHAR
• 金额: --
• 依托单位: NATIONAL INSTITUTE ON DRUG ABUSE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3775033
• 关键词: analog; antireceptor antibody; cocaine; complementary DNA; dopamine; drug receptors; gene expression; glycosylation; in situ hybridization; membrane transport proteins; mesencephalon; messenger RNA; molecular site; molecular weight; postmortem; protein structure function; receptor binding; species difference; tissue /cell culture; transfection

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.

该项目的目标是阐明的生化特性， 可卡因受体或多巴胺转运蛋白。 我们先前已经 表明大鼠纹状体和核团中的多巴胺转运体 分子量不均匀。 当传送者 用N-聚糖酶处理，这两种转运蛋白的分子量 显著降低，明显差异是 不存在或者最小化。这表明异质性的因素是 糖基化的差异，尽管不可能排除 也是异质性的来源。我们还准备了反- 多巴胺转运蛋白的肽抗体 转运蛋白抗体可用于免疫沉淀和Western印迹。 印迹 克隆的多巴胺CDNAS存在物种差异 运输机 因此，我们检查了 转运蛋白、死后变化以及糖基化差异 在不同的物种之间。 分子量的不同， 根据cDNA序列预测的差异进行预测。 多巴胺 转运蛋白结合可卡因及其类似物的两个不同位点， 亲和力 这就提出了一个问题， 或者是否是由于 可能与糖基化有关，可以解释不同的位点。 因此，将多巴胺转运蛋白的cDNA转染到COS中， 这些细胞表达两个可卡因结合位点。 这些 数据表明，异质结合发生在单一蛋白质上 其必须以不同的方式进行修改。 我们研究了多巴胺转运体mRNA在脑内的分布， 通过原位杂交分析。 我们发现，细胞群中发现， 中脑含有高水平的mRNA，而中脑外的细胞群 中脑含有非常低水平的转运蛋白mRNA。 我们在理解这一问题的性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。