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J. Biol. Chem., Vol. 277, Issue 16, 13488-13493, April 19, 2002
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From the Departments of Medicine and Microbiology-Immunology,
University of California Medical Center,
San Francisco, California 94143-0711
Vasoactive intestinal peptide and its
G-protein-coupled receptors, VPAC-1 and VPAC-2, are highly expressed in
the immune system and modulate diverse T cell functions. The human
VPAC-1 5'-flanking region (1.4 kb) contains four high affinity Ikaros
(IK) consensus sequences. Ikaros native protein from T cell nuclear
extracts and IK-1 and IK-2 recombinant proteins recognized an IK high
affinity binding motif in the VPAC-1 promoter in electrophoretic
mobility shift assays by a sequence-specific mechanism, and anti-IK
antibodies supershifted this complex. Stable NIH-3T3 clones
overexpressing IK-1 or IK-2 isoforms were generated to investigate
Ikaros regulation of endogenous VPAC-1 expression as assessed by
quantifying VPAC-1 mRNA and protein. By traditional and
fluorometric-based kinetic reverse transcription-PCR and
125I-labeled vasoactive intestinal peptide binding,
both IK-1 and IK-2 suppressed endogenous VPAC-1 expression in NIH-3T3
clones by a range of 50-93%. When a series of nested deletions of the VPAC-1 luciferase reporter construct were transiently transfected into
IK-2 clones there was up to a 41% decrease in transcriptional activity
compared with vector control. Two major IK-2 binding domains also were
identified at
Vasoactive Intestinal Peptide Receptor-1 (VPAC-1) Is a Novel Gene
Target of the Hemolymphopoietic Transcription Factor Ikaros*
and
1076 to 623 bp and at 222 to 35 bp,
respectively. As both Ikaros and its novel target VPAC-1 are highly expressed in T cells, this system may be a dominant determinant of the VPAC-1 expression in immune responses.
*
This work was funded by National Institutes of Health Grant
AI 29912 and National Research Service Award F32DK10107-01.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.
To whom correspondence should be addressed: University of
California, UB8B, Box 0711, 533 Parnassus Ave. at 4th, San Francisco, CA 94143-0711. Tel.: 415-476-1531; Fax: 415-471-6915; E-mail: Glenn-Dorsam@excite.com.
Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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