Characterization of the Receptor Protein Tyrosine Phosphatase Gene Product, PTPg: Binding and activation by Triphosphorylated Nucleosides.

A full-length cDNA for a novel isoform of the human receptor tyrosine phosphatase gamma gene (PTPRG) was overexpressed in Sf9 insect cells, and the gene product, PTP-gamma, was purified and characterized. The protein was expressed as a M-r apprx 185,000 protein accompanied by a M-r apprx 120,000 putative cleavage product on SDS-PAGE analysis. The protein undergoes N-linked glycosylation and constitutive phosphorylation of serine residues. When assayed for tyrosine-specific phosphatase activity, PTP-gamma dephosphorylated myelin basic protein at a pH optimum of 7.5 and a K-m of 12.6 mu-M; reduced carboxyamidomethylated and maleylated lysozyme (RCM-lysozyme) at a pH optimum of 6.0 and a K-m of 12 mu-M; and p-nitrophenylphosphate with a pH optimum of 5.5 and a K-m of 3.5 mM. Phosphatase activity was inhibited by ZnCl-2 and sodium orthovanadate; Mg-2+, Mn-2+, and Ca-2+ ions were ineffective. The partially purified form of the enzyme was allosterically activated by triphosphorylated nucleosides, with a preference for purines. This activation was prevented by Mg-2+ addition and did not occur when a purified form of the enzyme was utilized, suggesting that its activation depends on specific activating factors or conformational constraints. Interestingly, PTP-gamma protein was specifically bound by an ATP-agarose matrix through its intracellular domain, suggesting a link between binding of nucleotides and activation of the phosphatase.