Function and gene analysis of an endoplasmic reticlum-resident Ca^<2+>-binding proptein with multiple EF-hand motifs

Reticulocalbin是一种内质网Ca^<2+>-结合蛋白，是fe -hand Ca^<2+>-结合蛋白超家族的成员[Ozawa， M.and Muramatsu， T.(1993) j .生物化学，268,699-705]。分析了这种独特的Ca^<2+>-结合蛋白的基因组结构。小鼠基因组DNA的Southern blot分析显示，每个单倍体基因组中有一个网状定位蛋白基因拷贝。该基因跨越超过13000个碱基对，编码6个独立的外显子。因此，reticulocalbin不同于胞质Ca^<2+>-结合蛋白calbindin D28，后者也有6个EF-hand基元结构域，但其基因被分为11个外显子。网状定位蛋白与其他EF-hand蛋白的基因组织比较表明，网状定位蛋白很早就从EF-hand蛋白超家族的其他成员中分化出来。与观察到的不同类型的细胞表达reticulocalbin一致，该基因的5'-侧翼区域含有一个富含gc（超过80%）的区域，包括两个Spl结合基元和一个CCAAT盒，但在CCAAT盒和转录起始位点之间没有明显的TATA盒，这是许多管家基因中发现的特征。我们克隆出了网状局部蛋白的人类同源物。该克隆序列揭示了一个与小鼠网状定位蛋白在氨基酸序列上具有95%同源性的新蛋白，表明该分子在哺乳动物中具有进化保守性。正如在小鼠reticulocalbin中发现的那样，人类同源物显示了一个包含EF-hand基序的结构域的6个重复。有趣的是，氨基酸序列的保守性并不局限于Ca^<2+>-结合基序，这与reticulocalbin在Ca^<2+>-结合基序之外发挥作用的可能性是一致的。正如在小鼠同源物中发现的那样，该蛋白在其羧基端具有HDEL序列，而不是KDEL序列，后者更常见地作为保留动物细胞内质网中驻留蛋白的信号。在这两个物种中，网状localbin中HDEL序列的保守性提出了该序列在该蛋白家族的功能中起一定作用的可能性。少

Reticulocalbin, an endoplasmic reticulum (ER) resident Ca^<2+>-binding protein, is a member of the EF-hand Ca^<2+>-binding protein superfamily [Ozawa, M.and Muramatsu, T.(1993) J.Biol.Chem.268,699-705]. The genomic structure of this unique Ca^<2+>-binding protein was analyzed. Southern blot analysis of mouse genomic DNA revealed that there is a single copy of the reticulocalbin gene per hapliod genome. The gene spans over 13 kilobase pairs and encodes six separate exons. Thus, reticulocalbin differs from the cytosolic Ca^<2+>-binding protein calbindin D28 which also has six EF-hand motif domains but the gene for which is divided into 11 exons. Comparison of the gene organization of reticulocalbin with that of other EF-hand proteins revealed that reticulocalbin diverged very early from other members of the EF-hand protein superfamily. Consistent with the observation that reticulocalbin is expressed by different types of cells, the 5'-flanking region of the gene contains a GC-rich (more … More than 80%) area including two Spl binding motifs and a CCAAT box, but no apparent TATA box was present between the CCAAT box and the transcription start site, a feature being found in many housekeeping genes.We have cloned the human homologue of reticulocalbin. The sequence of this clone revealed a novel protein with 95 percent identity in ammino acid sequence to the mouse reticulocalbin, indicating that this molecule has been evolutionarily conserved in mammals. As was found for the mouse reticulocalbin, the human homologue showed the six repeats of a domain containing EF-hand motifs. Interestingly, the conservation of amino acid sequence was not restricted to the Ca^<2+>-binding motifs, consistent with the possibility that reticulocalbin plays some role (s) besides Ca^<2+>-binding. As was found for the mouse homologue, the protein has the HDEL sequence at its carboxy terminus instead of the KDEL sequence, which is more common as a signal to retain resident proteins in the ER of animal cells. The conservation of the HDEL sequence in reticulocalbin in both species raises the possibility that this sequence has some roles in the function (s) of this protein family. Less