Signal transduction of Proteoglycan-type protein tyrosine phosphatase, PTPS.

PTPζ是一种受体型蛋白酪氨酸磷酸酶，在发育中的大脑中以硫酸软骨素蛋白聚糖的形式大量表达。PTPζ与多养蛋白和midkine结合具有高亲和力。PTPζ的硫酸软骨素部分在与这些生长因子的结合中起重要作用。通过软骨素酶ABC消化去除PTPζ上的硫酸软骨素链，导致其与多营养因子和midkine的结合亲和力明显降低，信号转导丧失。PTPζ的硫酸软骨素链的精细结构调节其与这些生长因子的结合。PIPζ的硫酸软骨素链含有约1%的d -单位，这似乎是与多营养蛋白和midkine高亲和力结合的必要结构。免疫组化分析结果表明，PTPζ、多营养因子和硫酸软骨素D-unit的表达与小脑浦肯野细胞树突的形成高度相关。因此，我们利用大鼠小脑器官型切片培养系统研究了PTPζ在浦肯野细胞形态发生中的作用。通过添加(1)针对PTPζ的多克隆抗体，(2)钒酸钠（酪氨酸磷酸酶蛋白抑制剂），(3)软骨素酶ABC，(4)硫酸软骨素D，(5)多营养蛋白，具有定向和/或多个初级树突的异常浦肯野细胞显著增加。这些结果表明，多营养蛋白- ptpζ信号通路调节浦肯野细胞树突的形态发生。我们还尝试使用酵母底物捕获系统来鉴定PTPζ的底物分子。该方法基于酵母双杂交系统，有两种调节：有条件地表达v-src以酪氨酸磷酸化猎物蛋白，并使用底物诱捕突变体PTPζ进行筛选。通过该系统，我们确定了GIT1/Cat-1为底物。

PTPζ is a receptor-type protein tyrosine phosphatase, which is abundantly expressed in the developing brain as a chondroitin sulfate proteoglycan. PTPζ binds with pleiotrophin and midkine with high affinity. The chondroitin sulfate portion of PTPζ plays essential roles in the binding to these growth factors. Removal of chondroitin sulfate chains from PTPζ by chondroitinase ABC digestion resulted in the marked decrease of the binding affinity to pleiotrophin and midkine and the loss of signal transduction. The fine structure of chondroitin sulfate chains of PTPζ regulates its binding to these growth factors. Chondroitin sulfate chains of PIPζ contain about 1% D-unit, which seems to be the essential structure for the high affinity binding to pleiotrophin and midkine.Immunohistochemical analysis of PTPζ, pleiotrophin and chondroitin sulfate D-unit demonstrated that the expression of these molecules are highly correlated with the dendrite formation of cerebellar Purkinje cells. So, we examined the roles of PTPζ in the morphogenesis of Purkinje cells using organotypic slice culture system of rat cerebellum. Abnormal Purkinje cells with disoriented and/or multiple primary dendrites markedly increased by the additions of (1)polyclonal antibodies against PTPζ, (2) sodium vanadate, a protein tyrosine phosphatase inhibitor, (3) chondroitinase ABC, (4)chondroitin sulfate D, and (5) pleiotrophin. These results suggest that pleiotrophin-PTPζ signaling regulates the morphogenesis of Purkinje cell dendrites.We also tried to identify the substrate molecules of PTPζ using the yeast substrate-trapping system. This method is based on the yeast two-hybrid system, with two modufucations : conditional expression of v-src to tyrosine phosphorylate the prey proteins and screening by using a substrate-trap mutant of PTPζ. By this system, we identified GIT1/Cat-1 as a substrate.