AKAP3 selectively binds PDE4A isoforms in bovine spermatozoa
AKAP3 selectively binds PDE4A isoforms in bovine spermatozoa
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DOI：
10.1095/biolreprod.105.043588
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发表时间：
2006-01-01
影响因子：
3.6
通讯作者：
Carr, DW
中科院分区：
文献类型：
--
作者：
Bajpai, M;Fiedler, SE;Carr, DW
cAMP通过激活cAMP依赖的蛋白激酶A（PKA）在调节精子运动和顶体反应中起重要作用。磷酸二酯酶（PDE）通过催化环核苷酸的降解来调节环核苷酸的水平。尽管已知PDE 1和PDE 4特异性的PDE抑制剂可改变人类精子的活力和获能，但对PDE在精子中的作用或亚细胞分布知之甚少。PKA的定位受A-激酶锚定蛋白（AKAP）的调节，AKAP也可能控制PDE的细胞内分布。本研究旨在探讨PDE 4在精子获能过程中的作用和定位。添加Rolipram或RS 25344，PDE 4特异性抑制剂显著增加了牛精子的前向运动力。免疫定位技术在牛精子的主要部分中检测到PDE 4A和AKAP 3（以前称为AKAP 110）。PDE 4A 5亚型主要在尾部附睾精子的Triton X-100可溶性组分中检测到。然而，在射出的精子中，它主要出现在SDS可溶性组分中，表明在精子获能过程中PDE 4A 5定位到不溶性细胞器中的转变。AKAP 3仅在尾部和射精精子的SDS可溶性部分中检测到。使用AKAP 3和Pde 4a 5或Pde 4d共转染的COS细胞的免疫沉淀实验提供了PDE 4A 5而不是PDE 4D与AKAP 3相互作用的证据。使用精子细胞裂解物的下拉试验证实了这种体外相互作用。这些数据表明，AKAP 3结合PKA和PDE 4A，并作为精子中的支架蛋白来调节局部cAMP浓度和调节精子功能。
Cyclic AMP plays an important role in regulating sperm motility and acrosome reaction through activation of cAMP-dependent protein kinase A (PKA). Phosphodiesterases (PDEs) modulate the levels of cyclic nucleotides by catalyzing their degradation. Although PDE inhibitors specific to PDE1 and PDE4 are known to alter sperm motility and capacitation in humans, little is known about the role or subcellular distribution of PDEs in spermatozoa. The localization of PKA is regulated by A-kinase anchoring proteins (AKAPs), which may also control the intracellular distribution of PDE. The present study was undertaken to investigate the role and localization of PDE4 during sperm capacitation. Addition of Rolipram or RS25344, PDE4-specific inhibitors significantly increased the progressive motility of bovine spermatozoa. Immunolocalization techniques detected both PDE4A and AKAP3 (formerly known as AKAP110) in the principal piece of bovine spermatozoa. The PDE4A5 isoform was detected primarily in the Triton X-100-soluble fraction of caudal epididymal spermatozoa. However, in ejaculated spermatozoa it was seen primarily in the SDS-soluble fraction, indicating a shift in PDE4A5 localization into insoluble organelles during sperm capacitation. AKAP3 was detected only in the SDS-soluble fraction of both caudal and ejaculated sperm. Immunoprecipitation experiments using COS cells cotransfected with AKAP3 and either Pde4a5 or Pde4d provide evidence that PDE4A5 but not PDE4D interacts with AKAP3. Pulldown assays using sperm cell lysates confirm this interaction in vitro. These data suggest that AKAP3 binds both PKA and PDE4A and functions as a scaffolding protein in spermatozoa to regulate local cAMP concentrations and modulate sperm functions.