Differential Regulation of Mitogen-Activated Protein/ERK Kinase (MEK)1 and MEK2 and Activation by a Ras-Independent Mechanism
Shuichan Xu,
Shih Khoo1,
Alphonsus Dang,
Sarah Witt,
Vuong Do,
Erzhen Zhen,
Erik M. Schaefer and
Melanie H. Cobb
University of Texas Southwestern Medical Center (S.X., S.K., A.D.,
S.W., V.D., E.Z., M.H.C.), Department of Pharmacology, Dallas,
Texas 75235-9041,
Promega Corporation (E.M.S.), Madison,
Wisconsin 53711
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ABSTRACT
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Mitogen-activated protein (MAP)/ERK kinase (MEK) 1
and MEK2 are the upstream activators of the MAP kinases, ERK1 and ERK2.
MEK1 and MEK2 are
85% identical in sequence but have unique inserts
in their C-terminal domains. MEK isoform-specific antibodies were used
to examine expression and regulation of each enzyme. MEK1 and MEK2 were
expressed in approximately equal amounts in several cell lines; in
some, MEK1 was present in slight excess. Activation of tyrosine
kinase-containing receptors, heterotrimeric G proteins, and protein
kinase C enhanced the activities of both MEK isoforms in 293 and PC12
cells. AlF4- stimulated both
MEK1 and MEK2 in PC12 cells expressing a dominant interfering Ras
mutant that prevents nerve growth factor-dependent activation of the
cascade. Carbachol also stimulated the pathway in these cells. Thus, in
addition to their ability to activate Ras/Raf and the downstream ERK
pathway, heterotrimeric G proteins also appear to trigger a
Ras-independent mechanism to regulate this kinase cascade. In U373,
Chinese hamster ovary (CHO), and INS-1 cells, MEK1 was activated by
regulators of ERKs, while MEK2 was not. These data suggest that, like
the MAP kinases ERK1 and ERK2, in some cell settings the two similar
MEK isoforms are differentially regulated.
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INTRODUCTION
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The mammalian mitogen-activated protein (MAP) kinase or ERK
pathway (1, 2, 3, 4) has been implicated in many hormone-dependent regulatory
events including cell differentiation and proliferation. In addition to
mediating acute events in the cytoplasm and at the membrane, the MAP
kinases ERK1 and ERK2 are thought to alter gene expression, with the
best documented effects on transcription from the serum response
element (5). Tyrosine kinase receptors are believed to activate the ERK
pathway via Ras. Ras·GTP interacts with isoforms of the protein
kinase Raf (6, 7, 8, 9), causing its association with the membrane where it
is activated (6, 7, 8, 10). Raf-1 activates the MAP kinase kinases
(11, 12, 13, 14, 15, 16, 17) also known as MAP kinase/ERK kinases MEK1 and MEK2, which in
turn phosphorylate and activate ERK1 and ERK2. MEK1 and MEK2 are also
activated by other MEK kinases, including the product of the
c-mos protooncogene (18, 19) and MEK kinase 1 (MEKK1) (20, 21), further illustrating the complexity of this signaling cascade.
MEK1 and MEK2 are 85% identical overall and greater than 90%
identical in their catalytic cores. The least similar regions of the
molecules lie near their N termini and in an approximately 40-amino
acid, proline-rich insert between subdomains IX and X. Identity between
MEK1 and MEK2 in this insert region is only approximatley 40%. The
insert is not present in any of the other known MEK family members,
mammalian or yeast, suggesting that it serves functions unique for the
ERK pathway. Although MEK1 and MEK2 are both activated by serum, Weber
and co-workers (22) observed that MEK1, but not MEK2, forms complexes
with Raf-1 (22). Deletion of the insert impaired its ability to form
complexes with Raf (23). Thus, distinct mechanisms, perhaps arising
from the unique catalytic domain inserts, may differentially regulate
these two MEK isoforms.
In this study we examined the regulation of MEK1 and MEK2 in several
cultured cell lines. Earlier studies suggested that these protein
kinases co-chromatograph on ion exchange resins (17), necessitating a
different approach to examine the regulation of each. Thus,
isoform-specific antibodies were used to immunoprecipitate each kinase
individually to measure regulation by activators of the MAP kinase
cascade.
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RESULTS
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Detection of MEK1 and MEK2 by Immunoblotting
MEK isoform-specific antisera were prepared to peptides from the
unique
40-amino acid inserts near the C termini of the kinase
domains of MEK1 and MEK2. We showed previously that each antiserum
immunoblotted only a single MEK isoenzyme without cross-reactivity, and
both antibodies are equally sensitive (21). Using these antisera, we
found that MEK1 and MEK2 were expressed in PC12 and Chinese hamster
ovary (CHO) cells, but MEK1 was in slight excess (Fig. 1A
). The same was true of 3T3 cells (not
shown). In 293, INS-1, and U373 cells, MEK1 and MEK2 were both
expressed approximately equally. Compared with the other cell types,
INS-1 cells expressed much less of either MEK (Fig. 1
, A and B). Both
MEK isoforms were also found in rabbit muscle. As reported earlier for
transfected MEK1 and MEK2, the endogenous rabbit muscle proteins
cochromatographed on MonoQ (Fig. 1
, C and D). The peak of ERK2
phosphorylating activity, in fractions 48, contained all of the MEK1
and MEK2 immunoreactivity.

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Figure 1. Detection of MEK1 and MEK2 in Different Cells
A, Lysates of different cell lines were subjected to SDS-PAGE. MEK1 and
MEK2 were visualized on duplicate blots with the MEK1-specific antibody
A2227 or the MEK2-specific antibody A2228 in the absence or presence of
peptide antigen. B, 293 cell lysates were blotted with A2227 or A2228
in the presence or absence of peptide antigen. C, MEK1 and 2 in rabbit
skeletal muscle cochromatographed on Mono Q. , MBP phosphorylation;
, ERK2 activating activity. D, Immunoblots of the Mono Q fractions
with A2227 and A2228.
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Activities of Both MEK1 and MEK2 Are Increased by Ligands to
Multiple Receptor Types
The regulation of endogenous MEK1 and MEK2 by stimuli that work
through different mechanisms was examined. Activities of MEK1 and MEK2
were measured in immune complexes using isoform-specific antibodies to
immunoprecipitate MEK1 and MEK2 separately from lysates of untreated,
stimulated, and transfected 293 cells. An ERK2 mutant, K52R ERK2, which
has very low intrinsic protein kinase activity, was used as substrate.
Epidermal growth factor (EGF), carbachol,
AlF4-, and phorbol ester enhanced the
activities of MEK1 (21) and MEK2 in serum-deprived 293 cells (Fig. 2
). All of these stimuli increased
activity nearly equally, with EGF being the most effective and phorbol
ester the least.

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Figure 2. MEK2 is Activated by MEKK1 and Ligands for Multiple
Receptor Types in 293 Cells
293 cells transfected with HA-MEKK1 or empty vector were stimulated
with EGF, carbachol, phorbol 12-myristate 13-acetate, and
A1F4- as described. Endogenous MEK2 was
immunoprecipitated with A2228. MEK2 activity was assayed by using
recombinant K52R ERK2 as substrate. Fold activation, compared with MEK2
activity in untransfected cells without stimulation, is shown
under each lane.
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MEKK1 was compared as a stimulus of the endogenous MEK activities. In
MEKK1-transfected cells, both MEK1 (21) and MEK2 were activated even in
serum-deprived cells. MEKK1 overexpression had no significant effect on
the stimulated activities of MEK1 and MEK2; HA-MEKK1 is not activated
by these stimuli (not shown). Expression of MEKK1 in 293 cells also
increased the phosphorylation and activities of cotransfected,
epitope-tagged MEK1 and MEK2 (21). These data indicated that stimuli
activating protein kinase C, tyrosine kinase receptors, and
heterotrimeric G proteins activated both MEK1 and MEK2 in 293
cells.
AlF4- and
Carbachol Enhance MEK Activity in PC12 17N-1 Cells
We measured activation of the enzymes in PC12 cells to examine the
requirement for H-Ras in the regulation of the ERK cascade by
heterotrimeric G proteins. In PC12 cells MEK1 and MEK2 were both
activated by nerve growth factor (NGF) (Fig. 3
) and AlF4-
(24). In NGF-stimulated cells, activity of MEK1 was greater than that
of MEK2. Kinase activity was increased by
AlF4- and to a lesser extent by carbachol (see
Fig. 6
) even in the 17N-1 clone of PC12 cells that overexpresses a
dominant-interfering Ras mutant, S17N H-Ras. In earlier studies we
demonstrated that NGF was unable to activate ERK1, ERK2, or a partially
purified MEK activity in the 17N-1 PC12 line, although these activities
were stimulated by AlF4- (24). These findings
were confirmed below.

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Figure 3. Activation of MEK1 and MEK2 in PC12 and PC12 17N-1
Endogenous MEK1 and MEK2 were immunoprecipitated from NGF-stimulated
PC12 cells (upper panel) or
A1F4--treated PC12 17N-1 cells (lower
panel). MEK activities were assayed as indicated.
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Figure 6. Detection of ERK Activation by an Antibody That
Recognizes the Active Forms
A, Cell lysates (15 µg for 3T3 and 25 µg for all other) were
analyzed by Western blotting. Duplicate blots were probed with Y691, an
ERK antibody (upper panel) and 20291, an antibody that
selectively recognizes active ERK (lower panel). B,
Immunoblots of ERKs (upper) or active ERKs
(lower) in 20 µg of lysate protein from PC12 or PC12
17N-1 cells treated as indicated.
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MEK1 but Not MEK2 Is Activated by Secretagogs in INS-1 Cells
In INS-1 cells the effect of glucose plus forskolin, which
promotes insulin secretion, was examined. As demonstrated previously
(25, 26), ERK1 activity, detected in immune complexes with the
ERK1-specific antibody X837 (27), was strongly increased by glucose
plus forskolin (Fig. 4
). Although the
signaling pathways that link glucose to activation of ERKs are
undefined, glucose metabolism and calcium uptake are required to
control not only secretion but also activation of the protein kinase
cascade (26). Both MEK1 and MEK2 are present in INS-1 cells (Fig. 1A
);
however, glucose plus forskolin stimulate the activity of MEK1 but not
that of MEK2 (Fig. 4
).

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Figure 4. MEK1, not MEK2, Is Activated by Glucose and
Forskolin in INS-1 Cells
INS-1 cell lysates were prepared as described in Materials and
Methods. Endogenous ERK, MEK1, and MEK2 were immunoprecipitated
by antisera X837, A2227, and A2228, respectively. The activities of ERK
and MEK were assayed as described.
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Selective Activation of MEK1 in U373 and CHO Cells
Regulation of MEK1 and MEK2 in several other cell types was
examined to determine whether the selective activation of MEK1 was
unique to INS-1 cells. In CHO, 3T3, and U373 cells, FBS-enhanced MEK1
activity was found, but none was attributable to MEK2 (Fig. 5
). 3T3 (not shown) cells express more
MEK1 than MEK2, perhaps accounting for the difficulty in detecting
activation of MEK2; however, CHO, U373, and INS-1 cells contain
approximately equal amounts of MEK2 as MEK1. Further, U373 cells
express as much immunoreactive MEK2 per mg lysate protein as do 293
cells. Thus, in these cell types, unlike in 293 or PC12 cells,
activation of MEK1 can be detected without a comparable activation of
MEK2.

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Figure 5. Activation of MEK1 and MEK2 in 3T3, CHO, and U373
Cells
3T3, CHO, and U373 cells were stimulated with FBS for 5 min. Endogenous
MEK1 and MEK2 were immunoprecipitated and their activities assayed as
described.
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Activation of MEK1 and MEK2 or MEK1 Alone Results in Activation of
Both ERK1 and ERK2
To compare activation of MEKs to their downstream targets,
activation of ERK1 and ERK2 by a variety of stimuli was also examined.
ERK1 and ERK2 must be phosphorylated on two sites, a tyrosine and a
threonine, to be in the high activity state. Dephosphorylation of
either residue inactivates the enzymes. ERK activation was examined in
lysates from 3T3, CHO, PC12, and U373 cells by immunoblotting with an
antibody raised to a doubly phosphorylated peptide that corresponds to
the active state of ERK1 and ERK2. The antibody selectively detects the
activated forms of these two MAP kinases; once either of the two
activating phosphorylation sites is dephosphorylated, their reactivity
with the anti-active ERK antibody is greatly decreased (28). Equal
amounts of protein were also immunoblotted with antibody 691 that
recognizes ERK1 and ERK2 equally to confirm that equivalent amounts of
ERK1 and ERK2 were analyzed in lysates from stimulated and unstimulated
cells (Fig. 6
).
Both AlF4- and carbachol increased the amounts
of immunoreactive active ERK1 and ERK2 in wild type PC12 cells and the
17N-1 line that overexpressed S17N Ras (Fig. 6
, A and B). The effect of
carbachol was smaller than that of AlF4-, but
the effect was clearly detectable with the anti-active ERK antibody.
Previously, protein kinase assays showed a 5- to 10-fold effect of
AlF4- on total ERK activity in 17N-1 cells,
measured using myelin basic protein as substrate (24). Activation of
ERK1 and ERK2 by both an activator of all heterotrimeric G proteins and
a muscarinic receptor agonist suggests that there is a Ras-independent
mechanism through which heterotrimeric G proteins activate ERKs in PC12
cells.
In 3T3 cells, both ERK1 and ERK2 were activated by insulin, serum, and
EGF, as well as carbachol. Again the effects of carbachol were the
weakest. Serum also potently activated both ERK1 and ERK2 in CHO cells.
U373 cells express more ERK2 than ERK1, but both isoforms were
activated by serum under conditions that cause activation of a single
MEK isoform. Both ERKs were also activated by glucose in INS-1 cells
(26).
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DISCUSSION
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The ERK cascade is activated by tyrosine kinases, certain
heterotrimeric and small G proteins (24, 29), stimulation of protein
kinase C (24, 30, 31, 32), and perhaps by other mechanisms. Both
Ras-dependent and Ras-independent inputs (24, 29, 33, 34, 35) from tyrosine
kinases, heterotrimeric G proteins, and protein kinase C have been
reported. We compared the regulation of MEK1 and MEK2 by all three
types of upstream signals and in various cell types. To measure the
activities of these two MEKs separately, we developed isoform-specific
antibodies for use in immune complex kinase assays. With these
antibodies we found that both MEK1 and MEK2 were activated by all three
types of stimuli.
AlF4-, an activator of all heterotrimeric G
proteins, increased the activity of both MEK1 and MEK2 in wild type
PC12 cells and the 17N-1 line that overexpresses a dominant negative
mutant of Ras. S17N Ras blocks activation of the ERK cascade by NGF,
which acts through a tyrosine kinase receptor (24, 36, 37).
Furthermore, carbachol, a muscarinic agonist, also activated the
cascade in PC12 cells overexpressing S17N Ras. These data support the
conclusion that heterotrimeric G proteins are able to work through a
Ras-independent input to this pathway.
An alternative explanation for our first results with
AlF4--treated PC12 cells could be suggested by
the finding that cAMP synergizes with NGF to activate the ERK pathway
in PC12 cells (38). AlF4- will generate active
species of not only G proteins normally coupled to this pathway
(e.g. Gi, Gq) but also Gs, which will lead to accumulation
of cAMP. Any residual Ras activity (downstream of Gi or Gq) may be
sufficient in the presence of elevated cAMP to elicit increased kinase
activity from MEKs and ERKs. The muscarinic agonist carbachol, however,
does not increase Gs activity and is, therefore, unlikely to work by
the same mechanism. It could potentially activate ERKs through Gi- and
Gq-dependent pathways. Effects on the ERK pathway of phorbol ester, a
protein kinase C activator, and bradykinin, which activates Gq, are
blocked in the 17N-1 cell line, suggesting that the Ras-independent
effect of carbachol is not mediated through Gq or by increasing protein
kinase C activity. Thus, the Gi subfamily may be the most likely to be
able to couple to the ERK cascade without activating Ras.
In 293 and PC12 cells, as well as in rabbit muscle, activation of both
MEK isoforms was detected, supporting the conclusion that in many cell
types MEK1 and MEK2 are coordinately regulated. However, in U373 and
CHO cells, only MEK1 was activated in response to serum, a strong
activator of both MEKs in many other cell types, in spite of ample
expression of both MEK1 and MEK2. Further, in INS-1 cells glucose,
which induces insulin secretion and causes the activation of ERK1 and
ERK2 (25, 26), leads to activation of only MEK1, not MEK2. Thus,
selective activation of one of the two MEK isoforms may occur even in
cell types that express significant quantities of both. This conclusion
is consistent with two other studies in the literature that examined
regulation of endogenous MEK1 and 2. The only study to examine their
activation in multiple cell types identified no differences in their
regulation under the conditions examined (39). However, in mouse
macrophages, which express both MEK isoforms, only MEK1 is responsive
to tumor necrosis factor-
(40).
Activation of a single MEK is not coupled to activation of a single ERK
isoform. Activation of both ERK1 and ERK2 was detected with antibodies
that selectively recognize their active forms in INS-1, CHO, and U373
cells. In all cell types examined here, both ERK1 and 2 were activated
by the agonists tested.
In certain cell settings, such as serum-stimulated U373 or CHO cells or
glucose-treated INS-1 cells, coupling or specificity factors may
distinguish between MEK1 and MEK2, resulting in ligand-selective
activation of a single MEK isoform. Alternately, the specificity may be
entirely attributable to upstream regulators. In this regard, it has
recently been reported by Guan and co-workers that A-Raf phosphorylates
and activates MEK1 but not MEK2 (41). However, in INS-1 cells Raf-1,
which activates both MEKs, is present, suggesting that some other
mechanism must account for the selective activation of MEK1. Future
studies are aimed at determining the mechanisms underlying the
selective activation of MEK1 reported here.
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MATERIALS AND METHODS
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Plasmids and Proteins
A mammalian expression vector encoding hemagglutinin (HA)
epitope-tagged MEKK1 (the C-terminal 682 amino acids) was as described
(21). Recombinant His6-K52R ERK2 was expressed and purified
as described (42).
Cell Culture, Transfection, and Preparation of Extracts
293 (human embryonic kidney), CHO (Chinese hamster ovary), 3T3
(mouse fibroblast), U373 (human astrocyte), and PC12 (rat
pheochromocytoma) cells and the 17N-1 clone PC12 cells (24, 43) were
grown in DMEM containing 10% FBS and 1% L-glutamine. The
rat insulinoma cell line INS-1 was grown in RPMI 1640 containing 11
mM glucose, 10% FBS, 50 µM
ß-mercaptoethanol, and 1 mM sodium pyruvate as described
(44). In some experiments 293 cells at no less than 80% confluence
were transfected with DNA by calcium phosphate coprecipitation (45) and
maintained for 2448 h after transfection. Before stimulation, 293,
CHO, U373, and 3T3 cells were deprived of FBS for 1618 h and then
treated with EGF (50 ng/ml), carbachol (1 mM),
AlF4- (30 mM NaF plus 10
µM AlCl3), phorbol ester TPA (100
nM), or FBS (10%), or left untreated. PC12 cells were
exposed to fresh FBS-containing medium
18 h before treatment with
NGF (50 ng/ml) or FBS, carbachol (1 mM), or
AlF4- (30 mM NaF plus 10
µM AlCl3). INS-1 cells were preincubated in
the absence of glucose in Krebs-Ringer-bicarbonate-HEPES (KRBH) buffer
for 12 h at 37 C and then exposed to 15 mM glucose plus
10 µM forskolin in KRBH without serum at 37 C for 30 min.
Cells were lysed as described (25). After washing with cold PBS, 293
cells were lysed on ice for 10 min in 0.5 ml lysis buffer per 60-mm
dish (20). Lysates were collected and sedimented at 14,000 x
g for 1015 min at 4 C. Other cells were homogenized in ERK
lysis buffer, and supernatants were prepared from them as described
(46). Supernatants were transferred to new tubes and stored at -80 C
or assayed immediately.
Immunoprecipitation and Immunoblotting
The monoclonal antibody to the HA epitope was purchased from
Berkley Antibody Co. (Berkley, CA). Anti-ERK1 antiserum 691, which
recognizes both ERK1 and ERK2, was raised as described (27). Antibodies
to MEK1 (A2227) and MEK2 (A2228) were produced by immunizing rabbits
with peptides from rat MEK1 (MEK11, CQVEGDAAETPPR) and MEK2 (MEK21,
AIFGRPVVDGEEGEPHSIS) (21). These peptides were derived from the unique
inserts in MEK1 and MEK2 not contained in other known MEK family
members. Specificity of immunoprecipitation with antibodies to MEK1 or
MEK2 was confirmed using autophosphorylated recombinant
His6-MEK1 and GST-MEK2 and was comparable to the
specificity of immunoblotting (21). It was shown previously that these
antibodies are approximately equally sensitive; under similar blotting
conditions, amounts of MEK1 or MEK2 detected are comparable. Antibodies
that selectively recognize the activated forms of ERK1 and ERK2 (20291)
were from Promega, and their selective recognition of the high activity
forms of ERK1 and ERK2 was confirmed as described elsewhere (28). To
immunoblot endogenous MEK1 or MEK2, soluble lysates (80 µg protein)
were loaded on gels, transferred to nitrocellulose membranes, and
blotted with A2227 or A2228 in the absence or presence of the
corresponding peptide (MEK11 or MEK21) at 50 µg/ml. For immune
complex kinase assays, supernatants were incubated with the indicated
antibodies and protein A-Sepharose as described (21) with rotation for
2 h at 4 C. The beads were washed with cold 0.25 M
Tris-HCl (pH 7.6) plus 0.1 M NaCl and resuspended for
kinase assay. Immunoblots probed with the above antisera were developed
with the Amersham enhanced chemiluminescence (ECL) kit (Arlington
Heights, IL).
Kinase Assays
Activity of MEK1 or MEK2 was measured in 50 µl of 10
mM HEPES (pH 8.0), 10 mM MgCl2, 1
mM benzamidine, 1 mM dithiothreitol, 100
µM ATP (115 cpm/pmol) containing 50 µg/ml recombinant
K52R ERK2 at 30 C for 60 min using immunoprecipitated kinase associated
with 10 µl protein-A-Sepharose beads. The reactions were stopped by
sedimenting the beads and adding an equal volume of 2x SDS-loading
buffer to the supernatant.
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ACKNOWLEDGMENTS
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We would like to thank Clark Garcia for preparation of bacterial
proteins, Jessie English and Lori Christerson for critical reading of
the manuscript, and Kim McKinney for its preparation.
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FOOTNOTES
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Address requests for reprints to: Melanie H. Cobb, University of Texas Southwestern Medical Center, Department of Pharmacology, 5323 Harry Hines Boulevard, Dallas, Texas 75235-9041.
This work was supported by NIH Research Grant DK-34128 and a fellowship
from the Juvenile Diabetes Foundation (to S.X.).
1 In partial fulfillment of requirements for the Ph.D. 
Received for publication December 23, 1996.
Revision received July 24, 1997.
Accepted for publication July 25, 1997.
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