Isolation and molecular characterization of AKAP110, a novel, sperm-specific protein kinase A-anchoring protein.
Isolation and molecular characterization of AKAP110, a novel, sperm-specific protein kinase A-anchoring protein.
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DOI：
10.1210/mend.13.5.0278
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发表时间：
1999-05
影响因子：
--
通讯作者：
Srinivasan Vijayaraghavan;Greg A. Liberty;Jag Mohan;V. Winfrey;G. E. Olson;Daniel W. Carr
中科院分区：
文献类型：
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作者：
Srinivasan Vijayaraghavan;Greg A. Liberty;Jag Mohan;V. Winfrey;G. E. Olson;Daniel W. Carr
增加细胞内 cAMP 的药物是精子活力的有效刺激剂。锚定抑制剂肽旨在破坏 cAMP 依赖性蛋白激酶 A (PKA) 与 A 激酶锚定蛋白 (AKAP) 的相互作用，是精子活力的有效抑制剂。这些数据表明 PKA 锚定是控制运动的关键生化机制。我们现在报告 AKAP110（精子裂解物中检测到的主要 AKAP）的分离、鉴定、克隆和表征。从小鼠、牛和人睾丸文库中分离并测序 AKAP110 cDNA。使用截短的突变体，鉴定出 RII 结合结构域。 AKAP110 上的 RII 结合域与其他 AKAP 上的 RII 结合域的比对表明，AKAP 在两亲性螺旋结构中包含八个功能保守的位置，负责 RII 相互作用。对八种不同组织的 Northern 分析仅在睾丸中检测到 AKAP110，原位杂交分析仅在圆形精子细胞中检测到 AKAP110，表明 AKAP110 是仅在雄性生殖细胞中发现的蛋白质。精子细胞含有主要位于头部顶体区域的 RI 和仅位于尾部的 RII，这是 PKA 的调节亚基。免疫细胞化学分析在精子头部的顶体区域和主块的整个长度上检测到 AKAP110。这些数据表明 AKAP110 与 PKA 的 RI 和 RII 亚型共享区室，并且可能充当运动和头部相关功能（例如获能和顶体反应）的调节器。
Agents that increase intracellular cAMP are potent stimulators of sperm motility. Anchoring inhibitor peptides, designed to disrupt the interaction of the cAMP-dependent protein kinase A (PKA) with A kinase-anchoring proteins (AKAPs), are potent inhibitors of sperm motility. These data suggest that PKA anchoring is a key biochemical mechanism controlling motility. We now report the isolation, identification, cloning, and characterization of AKAP110, the predominant AKAP detected in sperm lysates. AKAP110 cDNA was isolated and sequenced from mouse, bovine, and human testis libraries. Using truncated mutants, the RII-binding domain was identified. Alignment of the RII-binding domain on AKAP110 to those from other AKAPs reveals that AKAPs contain eight functionally conserved positions within an amphipathic helix structure that are responsible for RII interaction. Northern analysis of eight different tissues detected AKAP110 only in the testis, and in situ hybridization analysis detected AKAP110 only in round spermatids, suggesting that AKAP110 is a protein found only in male germ cells. Sperm cells contain both RI, located primarily in the acrosomal region of the head, and RII, located exclusively in the tail, regulatory subunits of PKA. Immunocytochemical analysis detected AKAP110 in the acrosomal region of the sperm head and along the entire length of the principal piece. These data suggest that AKAP110 shares compartments with both RI and RII isoforms of PKA and may function as a regulator of both motility- and head-associated functions such as capacitation and the acrosome reaction.