Developmentally regulated alternative splicing of densin modulates protein-protein interaction and subcellular localization
Developmentally regulated alternative splicing of densin modulates protein-protein interaction and subcellular localization
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DOI：
10.1111/j.1471-4159.2008.05280.x
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发表时间：
2008-06-01
影响因子：
4.7
通讯作者：
Colbran, Roger J.
中科院分区：
文献类型：
--
作者：
Jiao, Yuxia;Robison, A. J.;Colbran, Roger J.
Densin是富含亮氨酸重复序列（LRR）和PDZ结构域（PDZ domain）蛋白家族的成员，其通过其C-末端结构域结合几种信号分子，包括钙/钙调蛋白依赖性蛋白激酶11（CaMKII）。在这项研究中，我们确定了几个新的mRNA剪接的致密蛋白的差异表达在发展过程中的变体。新的变体共享LRR结构域，但相对于蛋白质的成熟变体（180 kDa），其被过早截短或包含内部缺失，从而去除关键的蛋白质-蛋白质相互作用结构域。例如，CaMKII α与来自转染的HEK 293细胞的裂解物的含有完整C-末端结构域的致密蛋白剪接变体共免疫沉淀，但不与仅含有N-末端结构域的变体共免疫沉淀。免疫印迹分析，使用抗体的N-和C-末端结构域的致密蛋白的肽表位是一致的剪接变体表达在大脑中的发育调控。此外，假定的剪接变体显示不同的亚细胞分馏模式在脑提取物。绿色荧光蛋白（GFP）融合的致密蛋白剪接变体在HEK 293细胞中的表达表明LRR结构域可以将致密蛋白靶向质膜相关区室，但是剪接变体是差异定位的并且对细胞形态具有潜在的不同影响。总之，这些数据表明，致密蛋白剪接变体具有不同的功能特征，表明在神经元发育过程中的多种作用。
Densin is a member of the leucine-rich repeat (LRR) and PDZ domain (LAP) protein family that binds several signaling molecules via its C-terminal domains, including calcium/calmodulin-dependent protein kinase 11 (CaMKII). In this study, we identify several novel mRNA splice variants of densin that are differentially expressed during development. The novel variants share the LRR domain but are either prematurely truncated or contain internal deletions relative to mature variants of the protein (180 kDa), thus removing key protein-protein interaction domains. For example, CaMKII alpha coimmunoprecipitates with densin splice variants containing an intact C-terminal domain from lysates of transfected HEK293 cells, but not with variants that only contain N-terminal domains. Immunoblot analyses using antibodies to peptide epitopes in the N- and C- terminal domains of densin are consistent with developmental regulation of splice variant expression in brain. Moreover, putative splice variants display different subcellular fractionation patterns in brain extracts. Expression of green fluorescent protein (GFP)-fused densin splice variants in HEK293 cells shows that the LRR domain can target densin to a plasma membrane-associated compartment, but that the splice variants are differentially localized and have potentially distinct effects on cell morphology. In combination, these data show that densin splice variants have distinct functional characteristics suggesting multiple roles during neuronal development.