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A 13-kilobase pair genomic DNA encoding a 78-amino acid
brain-specific calmodulin-binding protein kinase C (PKC) substrate,
neurogranin (Ng/RC3; also known as RC3 or p17), has been sequenced. The
Ng/RC3 gene is composed of four exons and three introns, with the
protein-coding region located in the first and second exons. This gene
was found to have multiple transcriptional start sites clustered within
20 base pairs (bp); it lacks the TATA, GC, and CCAAT boxes in the
proximal upstream region of the start sites. The promoter activity was
characterized by transfection of 293 cells with nested deletion mutants
of the 5`-flanking region fused to the luciferase reporter gene. A
minimal construct containing bp +11 to +256 was nearly as
active as that covering bp -1508 to +256, whereas a shorter
one covering bp +40 to +256 had a greatly reduced activity.
Between bp +11 and +40 lies a 12-nucleotide sequence
(CCCCGCCCACCC) containing overlapping binding sites for AP2
(CCGCCCACCC) and SP1 (CCCGCC); this region may be important for
conferring the basal transcriptional activity of the Ng/RC3 gene. The
expression of a Ng/RC3-luciferase fusion construct
(-1508/+256) in transfected 293 cells was stimulated by
phorbol 12-myristate 13-acetate (PMA), but not by cAMP, arachidonic
acid, vitamin D, retinoic acid, or thyroxines T
Volume 270,
Number 17,
Issue of April 28, pp. 10314-10322, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
and
T
. PMA caused a 2-4-fold stimulation of all the
reporter gene constructs ranging from +11/+256 to
-1508/+256. The stimulatory effects of PMA could be
magnified by cotransfection with both Ca-dependent
and -independent phorbol ester-binding PKC-
, -
,
-, -
, -
, and -
cDNAs, but not by
non-phorbol ester-binding PKC-
cDNA. The Ng/RC3 and PKC-
genes have a similar expression pattern in the brain during
development. These two genes share at least four conserved sequence
segments 1.5 kilobase pair upstream from their transcriptional start
sites and a gross similarity in that they possess several AT-rich
segments within bp -550 to -950. A near homogeneous 20-kDa
DNA-binding protein purified from rat brain was able to bind to these
AT-rich regions of both Ng/RC3 and PKC-
genes with footprints
containing ATTA, ATAA, and AATA sequences.
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