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A Carboxyl Leucine-Rich Region of Parathyroid Hormone-Related Protein Is Critical for Nuclear Export

Division of Biological Sciences (J.C.P.), Division of Endocrinology, Department of Medicine (D.W.B., L.J.D.), and Department of Anesthesiology (R.H.H.), University of California, San Diego, California 92161; and Medicine Service (D.W.B., L.J.D.) and Anesthesiology Service (R.H.H.), Veterans Affairs San Diego Healthcare System, San Diego, California 92161

Address all correspondence and requests for reprints to: Randolph H. Hastings, M.D., Ph.D., Veterans Affairs San Diego Healthcare System 125, 3350 La Jolla Village Drive, San Diego, California 92161. E-mail: rhhastings{at}ucsd.edu.

PTHrP is an oncofetal protein with distinct proliferative and antiapoptotic roles that are affected by nucleocytoplasmic shuttling. The protein’s nuclear export is sensitive to leptomycin B, consistent with a chromosome region maintenance protein 1-dependent pathway. We determined that the 109–139 region of PTHrP was involved in its nuclear export by demonstrating that a C-terminal truncation mutant, residues 1–108, exports at a reduced rate, compared with the wild-type 139 amino acid isoform. We searched for potential nuclear export sequences within the 109–139 region, which is leucine rich. Comparisons with established nuclear export sequences identified a putative consensus signal at residues 126–136. Deletion of this region resulted in nuclear export characteristics that closely matched those of the C-terminal truncation mutant. Confocal microscopic analyses of transfected 293, COS-1, and HeLa cells showed that steady-state nuclear levels of the truncated and deletion mutants were significantly greater than levels of wild-type PTHrP and were unaffected by leptomycin B, unlike the wild-type protein. In addition, both mutants demonstrated greatly reduced nuclear export with assays using nuclear preparations and intact cells. Based on these results, we conclude that the 126–136 amino acid sequence closely approximates the structure of a chromosome region maintenance protein 1-dependent leucine-rich nuclear export signal and is critical for nuclear export of PTHrP.

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