|Budget Amount *help
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2002: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 2001: ¥2,200,000 (Direct Cost: ¥2,200,000)
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.