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

(1)对神经元特异性钙结合蛋白（NCaP）家族新成员进行了分子克隆：获得了蛙杆光感受器S-modulin的克隆；蛙锥体感光体s26；大鼠海马HLP3和HLP4。(2)大肠杆菌的表达系统现在在一些ncap中可用。它们包括海马钙、s -调节素和s26。(3)分离到Hippocalcin基因组基因，发现该基因位于人类第2染色体上。研究了大鼠海马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s -调节素和神经钙素的靶蛋白。在s -调节蛋白的情况下，发现视紫红质激酶是靶蛋白。这是通过功能分析和结构分析完成的，使用了共轭s -调素。发现神经钙素的可能靶点是S100b和/或tublin b。(4)恢复蛋白的n端被肉豆蔻酸酰化。研究了这种脂质修饰的作用。发现肉豆蔻酰化促进了恢复蛋白与视紫红质激酶的结合。研究还表明，脂质修饰可促进视紫红质激酶靶向盘膜。s26的膜结合弱于s -调节素。发现这种差异的部分原因是由于s26中靠近c端的氨基酸残基缺乏正电荷。(5)探讨了钙调素在毒蕈碱乙酰胆碱受体磷酸化中的可能作用。钙结合形式的钙调素抑制GRK2对受体的磷酸化。(6)利用嵌合体钙调蛋白研究了钙调蛋白各结构域的作用。为了发挥钙调蛋白的功能，需要钙与一对钙结合位点的协调结合。(7)探讨了钙调磷酸酶的可能作用。研究发现钙调磷酸酶可能抑制长时程增强，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.