MOLECULAR-CLONING AND EXPRESSION OF A UNIQUE RABBIT OSTEOCLASTIC PHOSPHOTYROSYL PHOSPHATASE

Tyrosyl phosphorylation plays an important regulatory role in osteocla st formation and activity. Phosphotyrosyl phosphatases (PTPs), in addi tion to tyrosyl kinases, are key determinants of intracellular tyrosyl phosphorylation levels. To identify the PTP that might play an import ant regulatory role in osteoclasts, we sought to clone an osteoclast-s pecific PTP. A putative full-length clone encoding a unique PTP (refer red to as PTP-oc) was isolated from a 10-day-old rabbit osteoclastic c DNA library and sequenced. A single open reading frame predicts a prot ein with 405 amino acid residues containing a putative extracellular d omain, a single transmembrane region, and an intracellular portion. PT P-oc is structurally unique in that, unlike most known transmembrane P TPs, it has a short extracellular region (eight residues), lacks a sig nal peptide proximal to the N-terminus, and contains only a single 'PT P catalytic domain'. The PTP catalytic domain shows 45-50% sequence id entity with the catalytic domain of human HPTP beta and with the first catalytic domain of LCA. The PTP-oc gene exists as a single copy in t he rabbit genome. The corresponding mRNA (3.8 kb) is expressed in oste oclasts but not in other bone-derived cells (e.g. osteoblasts and stro mal cells). The 3.8 kb PTP-oc mRNA transcript was also expressed in th e rabbit brain, kidney and spleen. However, the brain and kidney, but not osteoclasts or spleen, also expressed a larger transcript (6.5 kb) . The PTP catalytic domain of PTP-oc was expressed as a GST-cPTP-oc fu sion protein. In vitro phosphatase assays indicated that the purified fusion protein exhibited phosphatase activities at neutral pH values t oward p-nitrophenyl phosphate, phosphotyrosyl Raytide, and phosphotyro syl histone, whereas it had no appreciable activity toward phosphosery l casein. In summary, we have: (a) cloned and sequenced the putative f ull-length cDNA of a unique PTP (PTP-oc) from rabbit osteoclasts; (b) shown that the mature 3.8 kb PTP-oc mRNA was expressed primarily in os teoclasts and the spleen; and (c) shown that the PTP-oc fusion protein exhibited a phosphotyrosine-specific phosphatase activity. In conclus ion, PTP-oc represents a structurally unique subfamily of transmembran e PTPs.