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Molecular and Cellular Biology, September 2000, p. 6496-6507, Vol. 20, No. 17
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Targeted Deletion of Minpp1 Provides New Insight into the Activity of Multiple Inositol Polyphosphate Phosphatase In Vivo

Hongbo Chi,¹ Xiaonian Yang,² Paul D. Kingsley,³ Regis J. O'Keefe,⁴ J. Edward Puzas,⁴ Randy N. Rosier,⁴ Stephen B. Shears,² and Paul R. Reynolds^4,*

Department of Pathology and Laboratory Medicine,¹ Department of Pediatrics,³ and Department of Orthopaedics,⁴ University of Rochester School of Medicine and Dentistry, Rochester, New York 14642, and Inositide Signaling Section, Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709²

Received 18 May 2000/Accepted 23 May 2000

Multiple inositol polyphosphate phosphatase (Minpp1) metabolizes inositol 1,3,4,5,6-pentakisphosphate (InsP₅) and inositol hexakisphosphate (InsP₆) with high affinity in vitro. However, Minpp1 is compartmentalized in the endoplasmic reticulum (ER) lumen, where access of enzyme to these predominantly cytosolic substrates in vivo has not previously been demonstrated. To gain insight into the physiological activity of Minpp1, Minpp1-deficient mice were generated by homologous recombination. Tissue extracts from Minpp1-deficient mice lacked detectable Minpp1 mRNA expression and Minpp1 enzyme activity. Unexpectedly, Minpp1-deficient mice were viable, fertile, and without obvious defects. Although Minpp1 expression is upregulated during chondrocyte hypertrophy, normal chondrocyte differentiation and bone development were observed in Minpp1-deficient mice. Biochemical analyses demonstrate that InsP₅ and InsP₆ are in vivo substrates for ER-based Minpp1, as levels of these polyphosphates in Minpp1-deficient embryonic fibroblasts were 30 to 45% higher than in wild-type cells. This increase was reversed by reintroducing exogenous Minpp1 into the ER. Thus, ER-based Minpp1 plays a significant role in the maintenance of steady-state levels of InsP₅ and InsP₆. These polyphosphates could be reduced below their natural levels by aberrant expression in the cytosol of a truncated Minpp1 lacking its ER-targeting N terminus. This was accompanied by slowed cellular proliferation, indicating that maintenance of cellular InsP₅ and InsP₆ is essential to normal cell growth. Yet, depletion of cellular inositol polyphosphates during erythropoiesis emerges as an additional physiological activity of Minpp1; loss of this enzyme activity in erythrocytes from Minpp1-deficient mice was accompanied by upregulation of a novel, substitutive inositol polyphosphate phosphatase.

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^* Corresponding author. Present address: Center for Oral Biology, Aab Institute of Biomedical Sciences, University of Rochester Medical Center, 601 Elmwood Ave., Box 611, Rochester, NY 14642. Phone: (716) 273-1424. Fax: (716) 473-2679. E-mail: Paul_Reynolds{at}urmc.rochester.edu.

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Molecular and Cellular Biology, September 2000, p. 6496-6507, Vol. 20, No. 17
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

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