Vasta Valeria, Sonnenburg William K, Yan Chen, Soderling Scott H, Shimizu-Albergine Masami, Beavo Joseph A
Department of Pharmacology, University of Washington, Seattle, 98195, USA.
Biol Reprod. 2005 Oct;73(4):598-609. doi: 10.1095/biolreprod.104.039180. Epub 2005 May 18.
In mature sperm, cAMP plays an important role as a second messenger regulating functions that include capacitation, the acrosome reaction, motility, and, in some cases, chemosensing. We have cloned from mouse testis a novel calmodulin-stimulated cyclic nucleotide phosphodiesterase 1A isoform, Pde1a_v7 (mmPDE1A7), which arises from an alternative transcription start in the cyclic nucleotide phosphodiesterase 1A gene. The open reading frame is predicted to encode a polypeptide with a molecular mass of 52 kDa. Two further variants of this form, which contain two additional new exons, arise from alternative splicing. Analysis of testis cDNA by real-time polymerase chain reaction (PCR) indicates that the Pde1A_v7 transcript variant is the most abundant. The PDE1A_v7 protein uniquely lacks the first amino-terminal calmodulin-binding domain, but does possess an inhibitory domain and a second calmodulin-binding site shared with other variants. In vitro translation of the corresponding Pde1a_v7 cDNA produced a 52-kDa polypeptide having cyclic nucleotide hydrolytic activity, which was stimulated threefold by calcium-bound calmodulin. Immunoprecipitation of cyclic nucleotide phosphodiesterase 1 activity from detergent extracts of mouse sperm revealed a major protein of the size expected for PDE1A_v7, and the immunocytochemical staining for cyclic nucleotide phosphodiesterase 1A in mouse sperm showed intense immunoreactivity in the tail only. These observations, along with the PCR data, strongly suggest that this new variant PDE1A_v7 is the major form of cyclic nucleotide phosphodiesterase 1A expressed in mature sperm and is therefore likely to play an important role in cyclic nucleotide regulation of mature sperm function.
在成熟精子中，环磷酸腺苷（cAMP）作为第二信使发挥着重要作用，调节包括获能、顶体反应、运动能力以及在某些情况下的化学感应等功能。我们从小鼠睾丸中克隆出一种新型的钙调蛋白刺激型环核苷酸磷酸二酯酶1A亚型，即Pde1a_v7（mmPDE1A7），它源自环核苷酸磷酸二酯酶1A基因的一个可变转录起始位点。预测其开放阅读框编码一个分子量为52 kDa的多肽。该形式的另外两个变体通过可变剪接产生，它们包含另外两个新外显子。通过实时聚合酶链反应（PCR）对睾丸cDNA进行分析表明，Pde1A_v7转录变体最为丰富。PDE1A_v7蛋白独特地缺少第一个氨基末端钙调蛋白结合结构域，但确实具有一个抑制结构域和一个与其他变体共有的第二个钙调蛋白结合位点。相应的Pde1a_v7 cDNA的体外翻译产生了一种具有环核苷酸水解活性的52 kDa多肽，该活性被钙结合的钙调蛋白刺激了三倍。从小鼠精子的去污剂提取物中免疫沉淀环核苷酸磷酸二酯酶1活性，揭示了一种大小与PDE1A_v7预期相符的主要蛋白质，并且小鼠精子中环核苷酸磷酸二酯酶1A的免疫细胞化学染色仅在尾部显示出强烈的免疫反应性。这些观察结果与PCR数据一起，强烈表明这种新变体PDE1A_v7是成熟精子中表达的环核苷酸磷酸二酯酶1A的主要形式，因此可能在成熟精子功能的环核苷酸调节中发挥重要作用。
