Analysis for the Expressional Change of Caldesmon Isoforms during Phenotyptic Modulation of Smooth Muscle Cells

高Mr caldesmon （h-CaD）主要在平滑肌中表达，而低Mr caldesmon （l-CaD）广泛分布于非肌肉组织和细胞中。它们的表达变化与平滑肌细胞的表型调节密切相关。在寻找人类cad异构体多样性的过程中，从HeLa S3细胞中克隆了新的l-CaD cdna；HeLal-CaD I由558个氨基酸组成，而HeLal-CaD II中缺失了26个氨基酸（HeLal-CaD I的残基202-227）。HeLa l-CaD的短nh_2末端序列不同于成纤维细胞（WI-38） l-CaD II和人主动脉h- CaD。我们也用RT-PCR分析了在人培养细胞中表达的CaD亚型。根据这些结果，人类l- cad可根据其短的nh_2末端序列分为hela型和wi -38型两类，并可细分为插入26个氨基酸的I型和不插入26个氨基酸的II型。为了揭示CaD同工异构体表达调控的分子事件，测定了人类CaD的基因组结构。人类CaD基因至少由14个外显子组成，并被定位到一个位点，7q33-q34。26个氨基酸的插入编码在第4号外显子上，并在h-CaD和l-CaD的mrna上特异性剪接。外显子3是唯一的外显子，编码h-CaD特有的中心重复结构域（残基208-436）以及所有cad的共同结构域（残基73-207为h-CaD或WI-38 l- cad，残基68-201为HeLa l- cad）。h-或l-CaD的表达调控被认为依赖于外显子3内两个5'剪接位点的选择。因此，l-和h-CaDs之间的表达变化可能是由这种剪接途径引起的。

The high Mr caldesmon (h-CaD) is predominantly expressed in smooth muscles, whereas the low Mr caldesmon (l-CaD) is widely distributed in nonmuscle tissues and cells. Their expressional changes are closely correlated with the phenotypic modulation of smooth muscle cells. During a search for the isoform diversity of human CaDs, novel l-CaD cDNAs were cloned from HeLa S3 cell; HeLa l-CaD I is composed of 558 amino acids, whereas 26 amino acids (residues 202-227 for HeLa l-CaD I) are deleted in HeLal-CaD II. The short NH_2-terminal sequence of HeLa l-CaDs is different from that of fibroblast (WI-38) l-CaD II and human aorta h- CaD. We have also analysed the CaD isoforms expressed in human culture cells by RT-PCR. As these results, human l-CaDs are divided into two groups (Hela-and WI-38-types) by its short NH_2-terminal sequences and they are also subdivided to type I with a 26 amino acid insertion and type II without it. To reveal the molecular events of the expressional regulation of the CaD isoforms, the genomic construction of human CaD was determined. The human CaD gene is at least composed of 14 exons, and is mapped to a single locus, 7q33-q34. The 26 amino acid insertion is encoded in exon 4, and is specifically spliced in the mRNAs for both h-CaD and l-CaD Is. Exon 3 is the unique exon which encodes the central repeating domain specific to h-CaD (residues 208-436) together with the common domain in all CaDs(residues 73-207 for h-CaD or WI-38 l-CaDs, and 68-201 for HeLa l-CaDs). The expressional regulation of h-or l-CaD is thought to depend on selection of the two 5'-splice sites within exon 3.Thus, the change in expression between l-and h-CaDs might be caused by this splicing pathway.