CHARACTERIZATION OF THE RECEPTOR PROTEIN-TYROSINE-PHOSPHATASE GENE-PRODUCT PTP-GAMMA - 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 p rotein was expressed as a M(r) similar to 185,000 protein accompanied by a M(r) similar to 120,000 putative cleavage product on SDS-PAGE ana lysis. The protein undergoes N-linked glycosylation and constitutive p hosphorylation 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 carboxyamidomet hylated 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 ZnCl2 a nd sodium orthovanadate; Mg2+, Mn2+, and Ca2+ ions were ineffective. T he partially purified form of the enzyme was allosterically activated by triphosphorylated nucleosides, with a preference for purines. This activation was prevented by Mg2+ addition and did not occur when a pur ified 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-agar ose matrix through its intracellular domain, suggesting a link between binding of nucleotides and activation of the phosphatase.