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Originally published In Press as doi:10.1074/jbc.M104162200 on July 10, 2001
J. Biol. Chem., Vol. 276, Issue 36, 34175-34181, September 7, 2001
Cloning of a Novel Retinoid-inducible Serine Carboxypeptidase
from Vascular Smooth Muscle Cells*
Jiyuan
Chen,
Jeffrey W.
Streb ,
Kathleen M.
Maltby,
Chad M.
Kitchen, and
Joseph M.
Miano§
From the Center for Cardiovascular Research, University of
Rochester Medical Center, Rochester, New York 14642
Retinoids block smooth muscle cell
(SMC) proliferation and attenuate neointimal formation after vascular
injury, presumably through retinoid receptor-mediated changes in gene
expression. To identify target genes in SMC whose encoded proteins
could contribute to such favorable biological effects, we performed a
subtractive screen for retinoid-inducible genes in cultured SMC. Here,
we report on the cloning and initial characterization of a novel retinoid-inducible serine
carboxypeptidase (RISC). Expression of RISC is low in
cultured SMC but progressively increases over a 5-day time-course
treatment with all-trans-retinoic acid. A near full-length
rat RISC cDNA was cloned and found to have a 452-amino acid open
reading frame containing an amino-terminal signal sequence, followed by
several conserved domains comprising the catalytic triad common to
members of the serine carboxypeptidase family. In vitro
transcription and translation experiments showed that the rat
RISC cDNA generates a ~51-kDa protein. Confocal
immunofluorescence microscopy of COS-7 cells transiently transfected
with a RISC-His tag plasmid revealed cytosolic localization of the
fusion protein. Western blotting studies using conditioned medium from
transfected COS-7 cells suggest that RISC is a secreted protein. Tissue
Northern blotting studies demonstrated robust expression of RISC in rat aorta, bladder, and kidney with much lower levels in all other tissues
analyzed; high level RISC expression was also observed in human kidney.
In situ hybridization verified the localization of RISC to
medial SMC of the adult rat aorta. Interestingly, expression in kidney
was restricted to proximal convoluted tubules; little or no expression
was observed in glomerular cells, distal convoluted and collecting
tubules, or medullary cells. Radiation hybrid mapping studies placed
the rat RISC locus on chromosome 10q. These studies reveal a novel
retinoid-inducible protease whose activity may be involved in vascular
wall and kidney homeostasis.
*
This work was supported in part by Scientist Development
Grant 9730145N (to J. M. M.) and by Postdoctoral Fellowship
Grant 0020233T from the American Heart Association (to J. C.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF330051 (rat RISC) and AF330052 (mouse RISC).
Recipient of National Institutes of Health Cardiovascular Research
Training Grant 1T32HL07949.
§
To whom correspondence should be addressed: Center for
Cardiovascular Research, University of Rochester Medical Center, 601 Elmwood Ave., Rochester, NY 14642. Tel.: 716-273-1664; Fax:
716-273-1497; E-mail: joseph_miano@urmc.rochester.edu.
Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.

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Copyright © 2001 by the American Society for Biochemistry and Molecular Biology.
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