IDENTIFICATION OF DYNAMIN-2, AN ISOFORM UBIQUITOUSLY EXPRESSED IN RAT-TISSUES
IDENTIFICATION OF DYNAMIN-2, AN ISOFORM UBIQUITOUSLY EXPRESSED IN RAT-TISSUES
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DOI：
10.1073/pnas.91.2.644
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发表时间：
1994-01-18
影响因子：
11.1
通讯作者：
MCNIVEN, MA
中科院分区：
文献类型：
--
作者：
COOK, TA;URRUTIA, R;MCNIVEN, MA
发动蛋白是一种最初从哺乳动物脑中分离的100-kDa微管激活的GT3，已被提出在内吞作用的早期步骤中是至关重要的。以往对发动蛋白的一级结构、生化性质和功能作用的研究表明，发动蛋白具有神经元特异性。然而，使用一种合成肽的抗体，代表大鼠脑发动蛋白（D100）的酶促区域，我们确定了一个100 kDa的蛋白质在大鼠肝脏中的双联体，这表明发动蛋白存在不同的亚型，是不同的大脑对应。然后，我们开始了独特的发动蛋白亚型的抗体和cDNA探针的搜索。一个500 bp的PCR产生的cDNA探针对应的大鼠脑发动蛋白编码基因的酶区域被用来分离六个重叠的克隆从大鼠肝脏cDNA文库，一起跨越另一个发动蛋白基因，Dyn 2的完整编码序列。序列分析显示，动力蛋白2（Dyn 2）在DNA水平上与脑动力蛋白有75%的相同性，在蛋白质水平上有79%的相同性。通过北方印迹分析和亚型特异性PCR，Dyn 2在成年大鼠组织中以3.5 kb和4 kb的两种转录本存在，在睾丸中的表达量最高。这些结果表明，发动蛋白编码的至少两个基因在哺乳动物组织中的差异表达和Dyn 2的表达，而不是脑发动蛋白，占无处不在的分布在大鼠组织中的发动蛋白。
Dynamin is a 100-kDa microtubule-activated GTPase originally isolated from mammalian brain that has been proposed to be crucial in the early steps of endocytosis. Previous studies on the primary structure, biochemical properties, and functional role of dynamin indicated that it was neuron-specific. However, using an antibody against a synthetic peptide representing an enzymatic region of rat brain dynamin (D100), we identified a 100-kDa protein doublet in rat liver, suggesting that dynamin exists as different isoforms that are distinct from the brain counterpart. We then initiated a search for distinctive dynamin isoforms with antibodies and cDNA probes. A 500-bp PCR-generated cDNA probe corresponding to the enzymatic region of the rat brain dynamin-encoding gene was used to isolate six overlapping clones from a rat liver cDNA library that together span the complete coding sequence of another dynamin gene, ''Dyn2.'' Sequence analyses reveal that dynamin 2 (Dyn2) is 75% identical to brain dynamin at the DNA level and is 79% identical at the protein level. By Northern blot analysis and isoform-specific PCR, Dyn2 was found ubiquitously in adult rat tissues as two transcripts of 3.5 kb and 4 kb; the highest levels were found in testis. These results indicate that dynamin proteins are encoded by at least two genes expressed differentially in mammalian tissues and that the expression of Dyn2, and not of brain dynamin, accounts for the ubiquitous distribution of dynamin in rat tissues.