Structure and Function of Novel Neuron-specific Calcium-binding Proteins

(1)进行了神经元特异性钙结合蛋白（NCaP）新家族成员的分子克隆：我们获得了青蛙视杆细胞S-调制蛋白的克隆;青蛙视锥细胞S26 ;大鼠海马HLP 3和HLP 4。(2)大肠杆菌中的表达系统现在在一些NCaP中可用。它们包括Hippocalcium、S-modulin和s26。(3)分离了海马钙蛋白基因组基因，发现该基因位于人类第2号染色体上。研究了大鼠降钙素基因的转录机制，确定了其增强子和沉默子的位置。(3)发现了S-调制蛋白和神经钙蛋白的靶蛋白，其中S-调制蛋白的靶蛋白为视紫红质激酶。这是通过功能分析以及使用与S-调制蛋白缀合的连接物的结构分析来完成的。发现神经钙蛋白的可能靶点是S100 B和/或微管蛋白B。(4)recoverin的N-末端被肉豆蔻酸酰化。这种脂质修饰的作用进行了检查。发现豆蔻酰化促进恢复素与视紫红质激酶的结合。还证明脂质修饰诱导促进视紫红质激酶靶向盘膜。s26的膜结合比S-调制蛋白弱。发现这种差异的一部分是由于s26中靠近C-末端的氨基酸残基缺乏正电荷。(5)研究了钙调素对毒蕈碱型乙酰胆碱受体磷酸化的可能作用。钙调素的钙结合形式抑制受体磷酸化GRK 2。(6)使用嵌合体钙调蛋白研究钙调蛋白的每个结构域的作用。为了发挥钙调素的功能，需要钙离子与一对钙离子结合位点的协同结合。(7)研究了钙调神经蛋白的可能作用。发现钙调神经蛋白可以抑制长时程增强，这是一种可能的记忆机制。

(1) Molecular cloning of new family members of neuron-specific calcium-binding proteins (NCaP) were performed : we obtained clones of frog rod photoreceptor S-modulin ; frog cone photoreceptor s26 ; rat hippocampal HLP3 and HLP4.(2) Expression systems in E.Coli are now available in some of the NCaPs. They included Hippocalcim, S-modulin and s26.(3) Hippocalcin genome gene was isolated and it was found that this gene is located on the 2nd human chromosome. The transcription mechansism of rat hippocalcin gene was investigated and the sites of enhancer and silencer were identified.(3) The target proteins of S-modulin and neurocalcin were found. In the case of S-modulin, rhodopsin kinase was found to be the target protein. This was done by functional analysis and also structural analysis using a liker conjugated with S-modulin. The possible target of neurocalcin was found to be S100b and/or tublin b.(4) The N-terminus of recoverin is acylated with myristic acid. The role of this lipid modification was examined. It was found that myristoylation facilitates the binding of recoverin to rhodopsin kinase. It was also demonstrated that the lipid modification induces facilitation of the targetting of rhodopsin kinase to disk membranes. The membrane binding is weaker in s26 than S-modulin. It was found that part of this difference is due to a lack of positive charges in s26 at amino acid residues close to C-terminus.(5) Possible role of calmodulin on the phosphorylation of muscarinic acetylcholine receptor was investigated. The calcium-bound form of calmodulin inhibited the receptor phosphorylation by GRK2.(6) The role of each domain of calmodulin was investigated using chimera calmodulin. To exert calmodulin function, co-ordinated calcium binding to a pair of calcium-binding site were required.(7) Possible role of calcineulin were examined. It was found that calcineulin may inhibit long term potentiation, a possible machinery of memory.