Immunolocalization of multiple Gα subunits in mammalian spermatozoa and additional evidence for Gαs
Immunolocalization of multiple Gα subunits in mammalian spermatozoa and additional evidence for Gαs
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DOI：
10.1002/mrd.10295
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发表时间：
2003-05-01
影响因子：
2.5
通讯作者：
Fraser, LR
中科院分区：
文献类型：
--
作者：
Baxendale, RW;Fraser, LR
与体细胞一样，哺乳动物精子似乎含有几种不同的异源三聚体G蛋白α亚基，可以介导特化细胞反应。然而，确切的Ga亚基存在，它们的亚细胞位置和它们可能的作用仍然不完全确定。在这项研究中，使用市售的特异性抗体，我们已经通过免疫印迹表明，Galalpha（S）存在于人类和小鼠精子裂解物中。使用完整的精子从这两个物种的免疫定位显示，这种蛋白质是在顶体帽区和鞭毛，特别是主要的一块。用霍乱毒素处理透化的小鼠精子，导致与G α（S）大小相同的蛋白质的ADP核糖基化增强，以及cAMP增加，为G α（S）提供了进一步的证据。还获得了Galalpha（i2）、Galalpha（i3）、Galalpha（0）、Galalpha（q/11）和Galalpha（olf）的存在和不同定位的证据。特别令人感兴趣的是Ga（i2），其与Ga（S）一样存在于顶体帽区和鞭毛中，所述顶体帽区和鞭毛是刺激性和抑制性腺苷受体定位的相同区域。这些观察结果与我们的假设一致，即G蛋白介导腺苷酸环化酶的腺苷受体调节，从而改变cAMP的产生，显然是至关重要的精子的收购和维护的可移植性。
Like somatic cells, mammalian spermatozoa appear to contain several different heterotrimeric G protein alpha-subunits that could mediate specialized cell responses. However, the precise Galpha subunits present, their subcellular location and their possible roles are still incompletely defined. In this study, using commercially available specific antibodies, we have shown by immunoblotting that Galpha(S) is present in human and mouse sperm lysates. Immunolocalization using intact spermatozoa from both species revealed this protein to be in the acrosomal cap region and the flagellum, particularly the principal piece. Treatment of permeabilized mouse spermatozoa with cholera toxin led to enhanced ADP-ribosylation of a protein the same size as Galpha(S), as well as an increase in cAMP, providing further proof for Galpha(S). Evidence for the presence and distinct localizations of Galpha(i2), Galpha(i3), Galpha(0), Galpha(q/11), and Galpha(olf) was also obtained. Of particular interest was Galpha(i2) which, like Galpha(S), was present in the acrosomal cap region and flagellum, the same regions where stimulatory and inhibitory adenosine receptors are localized. These observations are consistent with our hypothesis that G proteins mediate adenosine receptor modulation of adenylyl cyclase, with consequent alterations in cAMP production, apparently crucial for the spermatozoon's acquisition and maintenance of fertilizing ability.