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J. Biol. Chem., Vol. 276, Issue 2, 1516-1522, January 12, 2001
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From the Metabolic Research Unit and Department of Medicine,
University of California, San Francisco, California 94143-0540
The vasorelaxant and anti-mitogenic activities of
the atrial and brain natriuretic peptides depend upon their binding to
the type A natriuretic peptide receptor (NPR-A) expressed on the
surface of vascular cells. Intervention strategies aimed at controlling NPR-A expression are limited by the paucity of studies in this area.
Here we identify a sequence CCAAT between 
141 and 137 of the NPR-A
promoter that, when mutated, reduces promoter activity by 90% in rat
aortic smooth muscle (RASM) cells. Protein/DNA cross-linking and
immunoperturbation of electrophoretically shifted complexes formed
between RASM nuclear extracts and an oligonucleotide surrounding the
CCAAT sequence indicates that the heterotrimeric transcription factor
NF-Y binds specifically to the wild-type, but not mutated, CCAAT
element. Cotransfection of a dominant negative mutant of the NF-YA
subunit results in a concentration-dependent decrease in
the activity of the NPR-A promoter in RASM cells confirming that
endogenous NF-Y is an activator of the promoter. Mutation of the CCAAT
element, in conjunction with mutation of all three Sp1 sites previously
shown to be involved in NPR-A promoter regulation, virtually eliminates
NPR-A promoter activity in RASM cells. Coexpression of all three NF-Y
subunits together with Sp1 in Drosophila cells deficient in
these factors indicates that NF-Y and Sp1 act synergistically to
reconstitute NPR-A promoter activity. A direct physical association between NF-Y and Sp1 can be demonstrated both in vitro by
glutathione S-transferase pull-down assay and in the intact
cell by coimmunoprecipitation and functional studies. Together, these
studies show that NPR-A promoter activity is dominantly regulated
through functional, and possibly physical, interactions of NF-Y and Sp1.
To whom correspondence should be addressed: Metabolic Research
Unit and Dept. of Medicine, 1109 HSW, University of California, San
Francisco, CA 94143-0540. Tel.: 415-476-2729; Fax: 415-476-1660; E-mail: gardner@itsa.ucsf.edu.
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