Domi Teuta, Di Leva Francesca, Fedrizzi Laura, Rimessi Alessandro, Brini Marisa
Department of Biochemistry, University of Padova, Viale G. Colombo 3, 35121 Padova, Italy.
Ann N Y Acad Sci. 2007 Mar;1099:237-46. doi: 10.1196/annals.1387.043.
In mammals, four different genes encode four PMCA isoforms. PMCA1 and PMCA4 are expressed ubiquitously. PMCA2 and PMCA3 are expressed prevalently in the central nervous systems. More than 30 variants are generated by mechanisms of alternative splicing. The physiological meaning of the existence of such elevated number of isoforms is not clear, but it would be plausible to relate it to the cell-specific demands of Ca2+ homeostasis. To characterize functional specificity of PMCA variants we have investigated two aspects: the effects of the overexpression of the different PMCA variants on cellular Ca2+ handling and the existence of possible isoform-specific interactions with partner proteins using a yeast two-hybrid technique. The four basic PMCA isoforms were coexpressed in CHO cells together with the Ca2+-sensitive recombinant photoprotein aequorin. The effects of their overexpression on Ca2+ homeostasis were monitored in the living cells. They had revealed that the ubiquitous isoforms 1 and 4 are less effective in reducing the Ca2+ peaks generated by cell stimulation as compared to the neuron-specific isoforms 2 and 3. To establish whether these differences were related to different and new physiological regulators of the pump, the 90 N-terminal residues of PMCA2 and PMCA4 have been used as baits for the search of molecular partners. Screening of a human brain cDNA library with the PMCA4 bait specified the epsilon-isoform of protein 14-3-3, whereas no 14-3-3 epsilon clone was obtained with the PMCA2 bait. Overexpression of PMCA4/14-3-3 epsilon (but not of PMCA2/14-3-3 epsilon) in HeLa cells together with targeted aequorins showed that the ability of the cells to export Ca2+ was impaired. Thus, the interaction with 14-3-3 epsilon inhibited PMCA4 but not PMCA2. The role of PMCA2 has been further characterized by Ca2+ measurements in cells overexpressing different splicing variants. The results indicated that the combination of alternative splicing at two different sites in the pump structure was responsible for different functional characteristics of the pumps.
在哺乳动物中，四种不同的基因编码四种质膜钙泵（PMCA）亚型。PMCA1和PMCA4在全身广泛表达。PMCA2和PMCA3主要在中枢神经系统中表达。通过可变剪接机制产生了30多种变体。如此众多亚型存在的生理意义尚不清楚，但将其与细胞对钙离子稳态的特定需求联系起来似乎是合理的。为了表征PMCA变体的功能特异性，我们研究了两个方面：不同PMCA变体的过表达对细胞钙离子处理的影响，以及使用酵母双杂交技术研究与伴侣蛋白可能存在的亚型特异性相互作用。四种基本的PMCA亚型与钙离子敏感的重组发光蛋白水母发光蛋白一起在CHO细胞中共表达。在活细胞中监测它们过表达对钙离子稳态的影响。结果表明，与神经元特异性亚型2和3相比，普遍存在的亚型1和4在减少细胞刺激产生的钙离子峰值方面效果较差。为了确定这些差异是否与该泵不同的新生理调节因子有关，PMCA2和PMCA4的90个N端残基已被用作寻找分子伴侣的诱饵。用PMCA4诱饵筛选人脑cDNA文库确定了14-3-3蛋白的ε亚型，而用PMCA2诱饵未获得14-3-3ε克隆。在HeLa细胞中过表达PMCA4/14-3-3ε（而不是PMCA2/14-3-3ε）并结合靶向的水母发光蛋白表明，细胞输出钙离子的能力受损。因此，与14-3-3ε的相互作用抑制了PMCA4而不是PMCA2。通过对过表达不同剪接变体的细胞进行钙离子测量，进一步表征了PMCA2的作用。结果表明，泵结构中两个不同位点的可变剪接组合导致了泵的不同功能特性。
