Kessler F, Falchetto R, Heim R, Meili R, Vorherr T, Strehler E E, Carafoli E
Institute of Biochemistry, Swiss Federal Institute of Technology (ETH), Zurich.
Biochemistry. 1992 Dec 1;31(47):11785-92. doi: 10.1021/bi00162a016.
The C-terminal regions of the four human plasma membrane Ca2+ pump isoforms 1a-d generated from alternatively spliced RNA have been expressed in Escherichia coli, and the recombinant proteins have been purified to a very high degree. The C-termini of isoforms 1a, 1c, and 1d contain an insert encoded by an alternatively spliced exon which is homologous to the calmodulin binding domain of isoform 1b. In isoforms 1c and 1d (29 and 38 amino acid insertions, respectively), subdomain A of the original calmodulin binding site of isoform 1b is followed by the spliced-in domain, which is then followed by subdomain B of the original calmodulin binding site. The positive charges of histidine residues at positions 27, 28, and 38 of the alternatively spliced sequence are likely to be responsible for the observed pH-dependent calmodulin binding to the novel "duplicated" binding site. The affinity of calmodulin for the C-terminal domains of isoforms 1a, 1c, and 1d, which contain the histidine-rich inserts, is much higher at pH 5.9 than at pH 7.2. A synthetic peptide (I31) containing 31 amino acids of the alternatively spliced sequence (from residue 9 to 40) also binds calmodulin with strong pH dependency. Alternative splicing in the C-terminal domain is proposed to confer pH dependence to the regulation of the activity of Ca2+ pump isoforms.
通过选择性剪接RNA产生的四种人类质膜Ca2+泵同工型1a - d的C末端区域已在大肠杆菌中表达，并且重组蛋白已被高度纯化。同工型1a、1c和1d的C末端包含一个由选择性剪接外显子编码的插入片段，该片段与同工型1b的钙调蛋白结合结构域同源。在同工型1c和1d中（分别有29和38个氨基酸的插入），同工型1b原始钙调蛋白结合位点的亚结构域A之后是插入的结构域，然后是原始钙调蛋白结合位点的亚结构域B。选择性剪接序列中第27、28和38位组氨酸残基的正电荷可能是观察到的pH依赖性钙调蛋白与新型“重复”结合位点结合的原因。钙调蛋白对含有富含组氨酸插入片段的同工型1a、1c和1d的C末端结构域的亲和力在pH 5.9时比在pH 7.2时高得多。一个包含31个氨基酸的选择性剪接序列（从第9位到第40位）的合成肽（I31）也以强烈的pH依赖性结合钙调蛋白。C末端结构域的选择性剪接被认为赋予了Ca2+泵同工型活性调节的pH依赖性。
