Wolfgang W J, Quan F, Goldsmith P, Unson C, Spiegel A, Forte M
Vollum Institute For Advanced Biomedical Research, Oregon Health Sciences University, Portland 97201.
J Neurosci. 1990 Mar;10(3):1014-24. doi: 10.1523/JNEUROSCI.10-03-01014.1990.
In order to uncover the role of G proteins in the integrative functioning and development of the nervous system, we have begun a multidisciplinary study of the G proteins present in the fruit fly, Drosophila melanogaster. In this report, we describe the distribution of 3 different G protein alpha-subunits in the adult Drosophila CNS as determined by immunocytochemical localization using affinity-purified antibodies generated to synthetic oligopeptide sequences unique to each alpha-subunit. Western blot analysis of membranes prepared from Drosophila heads indicates that antibodies specific for the Drosophila Go alpha and Gs alpha homologs recognize the appropriate protein species predicted by molecular cloning (Quan et al., 1989; Thambi et al., 1989). The Gi alpha homolog could not be detected in head membranes by Western blotting, consistent with the negligible levels of expression observed for Gi alpha on Northern blots of head mRNA (Provost et al., 1988). However, a Drosophila Gi alpha fusion protein could be detected by these antibodies following expression in E. coli. Immunolocalization studies revealed that the Go alpha and Gs alpha homologs are expressed at highest levels in neuropils and at intermediate levels in the cortex of all brain and thoracic ganglion areas. Only the lamina contained low levels of these alpha-subunits in the CNS. Additionally, Gs alpha appears to be associated with the cell membranes of neuronal cell bodies, while Go alpha has a more diffuse distribution, suggesting its presence in the cytoplasm as well as cell membranes. In contrast to the wide distribution of Go alpha and Gs alpha, Gi alpha has a surprisingly restricted distribution in the CNS. It is present at high levels only in photoreceptor cell terminations, glomerulae of the antennal lobes, and the ocellar retina. Little or no Gi alpha was detected in other brain regions or in the thoracic ganglion. Gi alpha, then, appears to be uniquely associated with some primary sensory afferents and their terminations, suggesting the presence of specific receptor and/or effector systems which mediate the transmission of primary sensory information in Drosophila.
为了揭示G蛋白在神经系统的整合功能及发育中的作用，我们已开始对果蝇（黑腹果蝇）体内存在的G蛋白进行多学科研究。在本报告中，我们描述了成年果蝇中枢神经系统中3种不同G蛋白α亚基的分布情况，这些分布是通过免疫细胞化学定位来确定的，所用抗体是针对每个α亚基独特的合成寡肽序列产生的亲和纯化抗体。对果蝇头部制备的膜进行的蛋白质印迹分析表明，针对果蝇Goα和Gsα同源物的特异性抗体识别出了分子克隆预测的相应蛋白质种类（Quan等人，1989年；Thambi等人，1989年）。通过蛋白质印迹法在头部膜中未检测到Giα同源物，这与在头部mRNA的Northern印迹中观察到的Giα表达水平可忽略不计一致（Provost等人，1988年）。然而，在大肠杆菌中表达后，这些抗体能够检测到果蝇Giα融合蛋白。免疫定位研究表明，Goα和Gsα同源物在神经纤维网中表达水平最高，在所有脑区和胸神经节区域的皮质中表达水平中等。在中枢神经系统中，只有神经层含有低水平的这些α亚基。此外，Gsα似乎与神经元细胞体的细胞膜相关，而Goα分布更为弥散，表明它存在于细胞质以及细胞膜中。与Goα和Gsα的广泛分布形成对比的是，Giα在中枢神经系统中的分布出人意料地受限。它仅在光感受器细胞终末、触角叶的小球以及单眼视网膜中高水平存在。在其他脑区或胸神经节中几乎未检测到Giα。因此，Giα似乎与一些初级感觉传入纤维及其终末独特相关，这表明在果蝇中存在介导初级感觉信息传递的特定受体和/或效应器系统。
