A Novel Mechanism for Cyclic Adenosine 3',5'-Monophosphate Regulation of Gene Expression by CREB-Binding Protein
Kerstin Zanger,
Laurie E. Cohen,
Koshi Hashimoto,
Sally Radovick and
Fredric E. Wondisford
Divisions of Endocrinology Childrens Hospital (K.Z., L.E.C.,
S.R.) and Beth Israel Deaconess Medical Center (K.H., F.E.W.) and
Harvard Medical School Boston, Massachusetts
02215
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ABSTRACT
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The pituitary-specific transcription factor,
Pit-1, is necessary to mediate protein kinase A (PKA) regulation of the
GH, PRL, and TSH-ß subunit genes in the pituitary. Since these target
genes lack classical cAMP DNA response elements (CREs), the mechanism
of this regulation was previously unknown. We show that CREB binding
protein (CBP), through two cysteine-histidine rich domains (C/H1 and
C/H3), specifically and constitutively interacts with Pit-1 in
pituitary cells. Pit-1 and CBP synergistically activate the PRL gene
after PKA stimulation in a mechanism requiring both an intact Pit-1
amino-terminal and DNA-binding domain. A CBP construct containing the
C/H3 domain [amino acids (aa) 16782441], but not one lacking
the C/H3 domain (aa 18912441), is sufficient to mediate this
response. Neither construct augments PKA regulation of CRE-containing
promoters. Fusion of either CBP fragment to the GAL4 DNA-binding domain
transferred complete PKA regulation to a heterologous promoter. These
findings provide a mechanism for CREB-independent regulation of gene
expression by cAMP.
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INTRODUCTION
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Pit-1 is a member of a family (POU) of transcription factors, and
when bound to DNA, activates GH, Pit-1, PRL, and TSH-ß subunit gene
expression (1, 2, 3, 4, 5, 6, 7, 8, 9). In addition to activating basal expression, Pit-1 is
also necessary for cellular regulation of these target genes by
hypothalamic hormones such as GHRH, TRH, and dopamine in a process
requiring Pit-1 (8, 10, 11, 12, 13, 14). In the anterior pituitary, the GHRH
receptor is known to activate (15), while the dopamine receptor is
known to inhibit the cAMP/protein kinase A pathway (16); the TRH
receptor is known to activate phospholipase C, leading to calcium
mobilization and protein kinase C (PKC) activation (17). Interestingly,
GH, PRL, and TSH-ß subunit genes do not contain consensus cAMP
response elements (CREs) or AP-1 sites; instead, Pit-1 DNA response
elements mediate induction by these pathways through a previously
unknown mechanism.
On genes containing CREs, CREB binds as a homodimer and, after
phosphorylation by protein kinase A (PKA), binds to CREB-binding
protein (CBP) and a closely related coactivator (P300) (18, 19, 20, 21, 22, 23). CBP,
via an N-terminal domain, binds to CREB and functions to increase
transcription through a more C-terminal domain, which is proposed to
both activate histone acetyltransferase (24) and displace nucleosomes
as well as recruit RNA polymerase II to the transcription complex
(25, 26, 27). The possibility that CBP could function independently of its
recruitment to the transcription complex was recently suggested
(28).
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RESULTS
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Since CBP is known to integrate a number of diverse cell-signaling
pathways (29), we wanted to determine whether CBP acts as a cofactor
for Pit-1-dependent gene regulation within the anterior pituitary. A
cotransfection assay of the proximal PRL promoter reporter in a
Pit-1-deficient cell line (CV-1) was performed to test this hypothesis.
To activate the PKA pathway, a protein kinase A expression vector (30)
was used; and to activate the PKC pathway, phorbol ester (TPA)
treatment or a reconstituted TRH-signaling pathway was employed (31).
The proximal PRL promoter contains four well defined Pit-1 DNA-binding
sites (10). In this cell line, basal expression of the reporter
construct was low but measurable. CBP or Pit-1 transfection activated
this construct to a small extent after stimulation by these mediators
(Fig. 1A
). However, the combination of
both CBP and Pit-1 expression vectors synergistically activated the
proximal PRL reporter in the presence of PKA, TPA, or TRH
receptor and TRH. Treatment of transfected cells with a cAMP
analog (8-Br-cAMP) elicted similar responses (data not shown). These
responses clearly required Pit-1 DNA-binding, as a Pit-1 DNA-binding
mutant (W261C, Ref. 32) was completely defective in this assay (Fig. 1A
). An intact Pit-1 N terminus was also necessary as the isoform
Pit-1a (also referred to as Pit-1ß or GHF-2), which contains a
26-amino acid (aa) insertion in the N terminus and is defective in
activating pituitary gene expression in vitro (33, 34, 35), was
without stimulatory effect (Fig. 1A
). A mutant PKA expression vector or
TRH treatment in the absence of cotransfected TRH receptor did
not activate this promoter after CBP and Pit-1 cotransfection (data not
shown). These PKA, TPA, and TRH responses with cotransfected Pit-1 and
CBP were not observed on a control promoter (TK, Fig. 1B
). These data
indicate that CBP functions as a Pit-1 cofactor to mediate PKA, TPA,
and TRH induction of the PRL gene.

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Figure 1. Cotransfection Assay of the Proximal PRL and TK
Promoters in CV-1 Cells
CV-1 cells were transfected with a SV-40 expression vector (pSG5)
containing either CBP or Pit-1 (wt, Pit-1a, or W261C) and a RSV
expression vector containing either a wt PKA catalytic subunit cDNA or
a mouse TRH receptor cDNA in the presence of either (A) the bovine PRL
promoter (Prl) or (B) the thymidine kinase promoter (TK) fused upstream
of a luciferase reporter gene. These are representative experiments
(one of four) performed in triplicate, and data are shown as mean
± SE. Fold basal activity is determined relative to
cotransfection of the reporter construct with "empty" pSG5 and RSV
expression vectors.
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Since CBP significantly and specifically activated the proximal PRL
reporter in a Pit-1-dependent manner, the ability of Pit-1 to bind to
CBP was next evaluated. Figure 2A
is a
glutathione S-transferase (GST) pull-down assay using CBP
fragments fused in-frame and C-terminal to GST. In data not shown, the
quality and quantity of each GST-protein was determined on SDS-PAGE to
ensure equivalence of the preparations. As can be seen,
35S-labeled Pit-1 bound specifically to two regions of CBP
(aa 118737 and aa 16772441). As shown in Fig. 2B
, 35S-labeled Pit-1, regardless of PKA treatment, bound to
the C/H1 (aa 362429) and C/H3 (aa 16761844) domains of CBP but not
to either GST alone or GST-CREB-binding domain (aa 463661). In
contrast, 35S-labeled CREB bound only to the CREB domain
and only after in vitro phosphorylation by the catalytic
subunit of PKA. These data indicate that Pit-1 can bind specifically to
CBP in vitro.

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Figure 2. Protein Interactions between CBP and Pit-1
A, GST pull-down assay of radiolabeled Pit-1 and fragments of the CBP
protein. GST-CBP fusions proteins were synthesized, purified, and
exposed to either 35S-labeled Pit-1 or unprogrammed
35S-labeled lysate (C). After extensive washing, proteins
trapped by the resin were resolved on SDS-PAGE and detected by
autoradiography. B, GST pull-down assay of radiolabeled Pit-1 or CREB,
before or after in vitro phosphorylation with the
catalytic subunit of PKA (PKA cat.) and fragments of the CBP protein.
GST-CBP proteins were synthesized as in panel A. C/H1 contains aa
1450 of CBP, CREB contains aa 450720, and C/H3 contains aa
16761891. C, GST pull-down assay of Pit-1 from GH3 cells
using GST-CBP protein. GST alone or GST-CBP, containing aa 118737 of
CBP, was exposed to WCE before (C) or after treatment with 8-Br-cAMP or
TRH. After extensive washing, proteins were eluted from the resin and
resolved by SDS-PAGE, and Western blot for Pit-1 was performed. WCE
without exposure to GST resin demonstrates the 33-kDa Pit-1 protein
(arrow). D, GST pull-down assay of CBP from
GH3 cells using a GST-Pit-1 protein. GST alone or
GST-Pit-1, containing full-length rat Pit-1, was exposed to whole cell
extract before or after treatment with 8-Br-cAMP or TRH. After
extensive washing, proteins were eluted from the resin and resolved by
SDS-PAGE, and Western blot analysis for CBP was performed. WCE without
exposure to GST resin demonstrates the 270-kDa CBP protein
(arrow). E, Coimmunoprecipitation of CBP and Pit-1 from
intact GH3 cells. Whole cell extract was immunoprecipitated
with an anti-CBP or a control (anti-GST) polyclonal rabbit antibody
followed by a Western blot analysis for Pit-1. The arrow
marks the location of Pit-1 coimmunoprecipitated with CBP.
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To prove that Pit-1 and CBP interact in pituitary cells, a GST
pull-down assay using CBP aa 118737 was performed, followed by a
Western blot analysis for Pit-1 immunoreactivity. Figure 2C
demonstrates that GST-CBP, but not GST alone, interacted equally well
with Pit-1 from a rat pituitary cell line (GH3), expressing
both GH and PRL, before or after treatment with either 8-Br-cAMP, or
TRH. The converse experiment followed by a Western blot analysis for
CBP immunoreactivity showed that GST-Pit-1, but not GST alone,
interacted equally well with CBP from the same pituitary cell line,
before or after treatment with either 8-Br-cAMP, or TRH (Fig. 2D
).
Finally, whole-cell extract (WCE) from untreated, 8-Br-cAMP-, or
TRH-treated GH3 cells was immunoprecipitated with a CBP
antibody followed by a Western blot analysis of the immunoprecipitate
for Pit-1 immunoreactivity (Fig. 2E
). CBP antiserum
coimmunoprecipitated Pit-1, indicating that Pit-1 and CBP interact
in vivo in pituitary cells.
Given that CBP and Pit-1 markedly activated the proximal PRL reporter
in response to PKA, TPA, or TRH treatment and that CBP binds to Pit-1,
we next determined what domains of CBP were responsible for this
effect. In Fig. 3A
, CBP deletion mutants
were compared with wild-type (wt) CBP in a cotransfection assay in CV-1
cells. The
81457 construct, which lacks the entire N-terminal
half of CBP, was completely sufficient in mediating PKA stimulation of
the proximal PRL reporter but was unable to mediate a normal TRH
response. In contrast, the 11334 construct was defective in mediating
a PKA effect but sufficient in mediating a TRH effect. The former
result was expected since the known transactivation domain of CBP,
contained within the carboxy terminus of CBP, was deleted in this
construct. The latter result proves that the 11334 construct is
functional and indicates that the TRH signaling pathway utilizes a
different CBP domain, located in the N-terminal half of the molecule,
to mediate transactivation. Finally the 1450 construct, which lacks
the CREB-binding site but contains the C/H1 domain, was fully
sufficient to mediate the TRH response, indicating that the
transactivation domain for this pathway is located in this
amino-terminal region of CBP. In data not shown, treatment with a cAMP
analog (8-Br-cAMP) or TPA produced a response pattern similar to PKA or
TRH stimulation, respectively.

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Figure 3. Determination of the CBP Domains Required for PKA
and TRH Induction of the Proximal PRL Reporter
A, This is a representative transfection experiment (one of three) each
performed in triplicate showing CBP domains required for PKA and TRH
induction. Data are shown as mean ± SD of activity
relative to the wt CBP induction included in each experiment. To the
left of the graph is a schematic
representation of the CBP constructs used. C/H domains are indicated by
black boxes. B, Western blots of CV-1 cells transfected
with the indicated CBP expression vectors: V, pSG5 empty vector; 1, wt
CBP; 2, 81457; 3, 142705; 4, 11334; and 5, 1450.
The antibody used in the Western blot is indicated at the
bottom of the figure; and a molecular mass marker in
kilodaltons is indicated to the left.
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In Fig. 3B
, a Western blot of cellular lysate from CV-1 cells
transfected with these same constructs and probed with an antibody
directed at either the amino or carboxy terminus of CBP. In data not
shown, nuclear localization of each of these proteins was confirmed
using enhanced green fluorescent protein-tagged CBP deletion
constructs. Note that CBP deletion constructs are expressed at similar
levels in transfected CV-1 cells so that differences in protein
expression of these constructs can not explain the results displayed in
Fig. 3A
.
Since the CREB binding domain of CBP is not required for the PKA
response of the PRL gene, we next wanted to determine whether CREB was
required for this response. Transfections were repeated using a human
common glycoprotein
-subunit reporter gene construct, which contains
two well defined CREs (36) (Fig. 4A
).
CBP, but not Pit-1, markedly enhanced the PKA induction of this
reporter vs. transfection of empty vector alone. Since CV-1
cells are relatively deficient in CBP vs. GH3
cells (compare Fig. 3B
with Fig. 2D
) and CBP cotransfection markedly
increased CBP levels in CV-1 cells (compare lanes 1 and 2, Fig. 3B
),
this result was expected. Cotransfection of an expression vector
containing a mutation of the PKA phosphorylation site of CREB (CREBm,
S133A, Ref. 37) completely blocked both the PKA response of the common
glycoprotein
-subunit reporter in the absence (data not shown) and
in the presence of CBP cotransfection (Fig. 4A
). Cotransfection of
CREBm had no significant effect on PKA induction of the PRL reporter
(Fig. 4B
). Cotransfection of wt CREB had no effect on induction of
either reporter (Fig. 4
, A and B). Since CV-1 cells are not deficient
in CREB, it is not surprising that wt CREB cotransfection did not
augment the PKA response. We next tested whether known PKA sites on
Pit-1 mediated the PKA effect seen on the PRL gene. However, mutation
of all three PKA phosphorylation sites on Pit-1 (aa 115, 219, 220, Ref.
38) had no significant effect on PKA induction of the PRL gene (data
not shown).

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Figure 4. PKA Activation of the Proximal PRL or Common
Glycoprotein -Subunit Promoter in CV-1 Cells Using wt and Mutant
CREB (S133A) Expression Vectors
CV-1 cells were transfected with SV-40 expression vectors (pSG5)
containing either wt PKA catalytic subunit, CBP, or Pit-1 cDNAs in the
presence of either a human common glycoprotein -subunit (A) or
proximal PRL (B) promoter fused upstream of a luciferase reporter gene.
In some transfections, a pSG5 expression vector containing either a wt
CREB or CREB mutant cDNA (S133A, CREBm) was also included. Data are
shown as mean ± SE of two independent experiments
each performed in triplicate. Fold basal activity is determined
relative to cotransfection of the reporter construct with empty pSG5
expression vector.
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To determine what C-terminal region of CBP was sufficient for the PKA
response, CBP fragments were fused downstream and in-frame of the GAL4
DNA-binding domain. Figure 5A
demonstrates that GAL4-CBP constructs containing either aa 16772441
or aa 18912441 were sufficient to mediate a robust PKA response when
tested on a UAS-thymidine kinase (TK) reporter, and these
responses were similar to those observed on the PRL gene (Fig. 5B
). The
isolated C/H3 domain (aa 16771891) mediated only a small PKA
response. Mutation of the one consensus PKA site in CBP (S1772A) in the
context of either the 16772441 or 16771891 construct had no effect
on PKA induction, indicating that phosphorylation at this site is not
important for this effect. As shown in Fig. 5B
, only the 16772441
construct was active on the PRL reporter, whereas the 18912441 and
16771891 constructs were not active. Note also that the 16772441
construct alone was inactive on the PRL reporter and required
cotransfection of Pit-1 to mediate a PKA effect. These data indicate
that the C/H3 domain, which binds Pit-1, is required for the
synergistic activation of the PRL reporter (compare aa 16772441 to aa
1891 to 2441) but was not active in isolation (aa 16771891). On a CRE
reporter (four copies of a CRE upstream of the TK promoter), these
constructs were all defective as compared with wt CBP in activating
this reporter after PKA transfection (Fig. 5C
).

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Figure 5. CBP Fusion Protein Mediates PKA Induction of a GAL
or PRL Reporter
A, GAL4-CBP fusion proteins as indicated without and with Pit-1 were
transfected in CV-1 cells, and PKA induction compared with GAL4 DNA-binding domain alone (vector) on
the UAS-TK reporter (A), proximal PRL reporter (B), or a minimal
thymidine kinase promoter containing four consensus CREs (C) is shown.
The GAL4-CBP fusion construct used in shown to the left
of the figure. Fold basal activity is determined relative to
cotransfection of the reporter construct with empty GAL4 DNA-binding
domain expression vector.
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DISCUSSION
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This study suggests a general mechanism for cAMP regulation of
genes without CREs whereby CBP, bound constitutively to a cell-specific
transcription factor directly or via additional cofactor interactions,
stimulates gene expression. A CBP construct containing aa 16772441 is
sufficient to mediate the PKA induction. Pit-1 presumably recruits this
CBP fragment to the transcription complex by binding to aa 16771891,
and subsequently aa 18912441 mediate the PKA induction.
Interestingly, although this fragment does not contain intrinsic
histone acetyltransferase (HAT) activity, it could recruit other
cofactors such as p/CAF or p/CIP with known HAT domains.
Our data do not support a role for CBP phospho-rylation in this
process as mutation of the only consensus PKA site (S1772A) in this
C-terminal fragment did not affect PKA induction; these data are in
contrast to a recent paper by Xu et al. (39). However, it is
possible that another unknown PKA phosphorylation site in the C
terminus of CBP could mediate this effect. Alternatively, PKA may
phosphorylate other cofactors facilitating their recruitment to the C
terminus of CBP. Experiments are in progress to test this
hypothesis.
In contrast to the PKA effect, TRH stimulation of the PRL gene requires
an N-terminal region of CBP (aa 1450). Again, like the PKA effect,
this CBP fragment is without intrinsic HAT activity, but other
cofactors such as p/CAF could also be recruited to this fragment. Xu
et al. also suggested that this same region of CBP could
mediate induction by another growth factor (insulin) on a Pit-1
response element. Thus, CBP acts as a transcriptional cofactor for
Pit-1 to permit regulation of the PRL gene by TRH-, TPA-, and
PKA-signaling pathways. As other genes in the pituitary are regulated
by Pit-1, including GH, TSH-ß subunit, and Pit-1 genes, CBP may
subserve a similar function on these genes.
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MATERIALS AND METHODS
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Transfection Constructs
Luciferase reporter constructs contain either 1000 bp of
5'-flanking DNA of the bovine PRL promoter, 109 bp of 5'-flanking DNA
of the herpes simplex TK promoter (minimal TK construct), 846 bp of
5'-flanking DNA of the human common glycoprotein
-subunit promoter
(glycoprotein
-subunit), five copies of the GAL4 DNA-binding site
upstream of TK (UAS-TK), or four copies of a consensus CRE upstream of
TK. All Pit-1, CBP, and CREB constructs were in the SV40 expression
construct, pSG5. Pit-1a and W261C were modifications of the original wt
Pit-1 cDNA and therefore contained the same translation initiation
site. CBP deletion mutant were made using restriction enzyme digestion
and removal of wt mouse CBP domains as indicated:
81457,
RsrII; 11334, BstXI;
142705,
ApaI; and 1450, EcoRI. The reading frame and
orientation of each mutant were confirmed in pSG5 by DNA
sequencing.
Transfection Assays
In a 24-well format, 0.8 µg of reporter with 1 µg of pSG5
and/or 1 µg of rous sarcoma virus (RSV) expression vectors were
transfected per plate; 16 h after transfection, cultures were
treated with serum-free medium for 8 h. For TRH stimulation, 50
nM TRH was added to the medium. All transfections were
balanced for the same amount of expression vector using empty vector as
needed. For analysis of the expression of transfected CBP constructs,
CBP expression vectors were transfected as above, and a Western blot of
total cellular lysate was performed using a CBP antibody that
recognizes the extreme N terminus (aa 222, Santa Cruz Biotechnology,
Santa Cruz, CA) or C terminus (aa 17362179, Upstate Biotechnology,
Lake Placid, NY) of CBP.
GST and Immunoprecipitation Assays
Regions of the CBP protein were fused in-frame
with GST in the pGEX4T2 vector (Pharmacia Biotech, Piscataway, NJ).
Recombinant proteins were synthesized in JM109 bacteria and purified on
glutathione-Sepharose resin under nondenaturing conditions. GST
proteins were analyzed on SDS-PAGE before use in the assay to ensure
equivalence of preparations. 35S-labeled Pit-1 or CREB was
generated in an in vitro transcription/translation system
(TNT, Promega Biotech, Madison, WI) and exposed to the indicated GST
protein. As a control, an unprogrammed translation with
[35S]methionine was employed. In some experiments the
translated protein was phosphorylated in vitro using the
catalytic subunit of PKA (Boehringer Mannheim, Indianapolis, IN) and 1
mM ATP for 30 min at 37 C before exposure to the GST
protein. After extensive washing with NET (150 mM NaCl, 1
mM EDTA, 0.5% NP40) at 4 C, the proteins trapped by the
resin were resolved on SDS-PAGE and detected by autoradiography.
GST alone or GST-CBP, containing aa 118737 of CBP, was exposed to
whole cell GH3 extract (WCE) made from individual 100-mm
dishes before or after 10 min of treatment with 1 mM
8-Br-cAMP or 50 nM TRH. After extensive washing with NET at
4 C, proteins were eluted from the resin and resolved by SDS-PAGE, and
Western blot analysis was performed with a mouse monoclonal anti-Pit-1
antibody made against the full-length rat Pit-1 molecule (Transduction
Laboratories, Lexington, KY). GST alone or GST-Pit-1, containing
full-length rat Pit-1, was exposed to whole-cell GH3
extract made from individual 100-mm dishes before or after 10 min of
treatment with 1 mM 8-Br-cAMP or 50 nM TRH.
After extensive washing with NET at 4 C, proteins were eluted from the
resin and resolved by SDS-PAGE, and Western blot analysis was performed
with a rabbit polyclonal anti-CBP antibody made against aa 17362179
of mouse CBP (Upstate Biotechnology).
Whole-cell GH3 extracts from 100-mm plates untreated or
treated with either 8-Br-cAMP or TRH were immunoprecipitated with an
anti-CBP or a control GST polyclonal rabbit antibody (anti-GST) and
protein A/G resin. After extensive washing in NET and PBS, a Western
blot analysis for Pit-1 using the monoclonal Pit-1 antibody described
above was performed.
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ACKNOWLEDGMENTS
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We would like to thank M. C. Gershengorn, R. H.
Goodman, J. L. Jameson, R. A. Maurer, and M. Montminy for
plasmids used in this study.
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FOOTNOTES
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Address requests for reprints to: Fredric E. Wondisford, Research North 330C, 99 Brookline Avenue, Boston, Massachusetts 02215. e-mail: fwondisf@bidmc.harvard.edu.
This work was supported by grants from the NIH, March of Dimes, and
Deutsche Forschungsgemeinschaft.
Received for publication October 14, 1998.
Revision received November 9, 1998.
Accepted for publication November 19, 1998.
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