Contrasting Signaling Pathways of
1A- and
1B-Adrenergic Receptor Subtype Activation of Phosphatidylinositol 3-Kinase and Ras in Transfected NIH3T3 Cells
Zhuo-Wei Hu,
Xiao-You Shi,
Richard Z. Lin1 and
Brian B. Hoffman
Geriatric Research, Education and Clinical Center Veterans
Affairs Palo Alto Health Care System Palo Alto, California
94304
Department of Medicine Stanford University School
of Medicine Stanford, California 94305
 |
ABSTRACT
|
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Activation of protein kinases is an important
intermediate step in signaling pathways of many G protein-coupled
receptors including
1-adrenergic receptors.
The present study was designed to investigate the capacity of the three
cloned subtypes of human
1-receptors,
namely,
1A,
1B
and
1D, to activate phosphatidylinositol
3-kinase (PI 3-kinase) and p21ras in
transfected NIH3T3 cells. Norepinephrine activated PI 3-kinase in cells
expressing human
1A and
1B via pertussis toxin-insensitive G
proteins;
1D-receptors did not detectably
activate this kinase. Transient transfection of NIH 3T3 cells with the
-subunit of the G protein transducin (
t)
a scavenger of ß
-subunits released from activated G proteins,
inhibited
1B-receptor but not
1A-receptor-stimulated PI 3-kinase activity.
Stimulation of both
1A- and
1B-receptors activated
p21ras and stimulated guanine nucleotide
exchange on Ras protein. Overexpression of a dominant negative mutant
of p21ras attenuated
1B-receptor but not
1A-receptor activation of PI 3-kinase.
Overexpression of a dominant negative mutant of PI 3-kinase attenuated
1A- but not
1B-receptor-stimulated mitogen-activated
protein kinase activity. These results demonstrate the capacity
for heterologous signaling of the
1-adrenergic receptor subtypes in promoting
cellular responses in NIH3T3 cells.
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INTRODUCTION
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1-Adrenergic receptors, members of the class of G
protein-coupled receptors, mediate many of the important physiological
effects of catecholamines such as norepinephrine or epinephrine (1, 2).
1-Adrenergic receptors may play a role in many human
diseases, such as atherosclerosis and hypertension (3, 4), restenosis
after coronary dilation (5), myocardial hypertrophy, and cardiac
arrhythmia (6, 7). There are at least three subtypes of
1-receptors expressed in human vascular smooth muscle
cells (VSMCs) and many other cells, namely,
1A-,
1B-, and
1D-receptors (8, 9). It is
generally accepted that activation of all three subtypes of
1-receptors increases hydrolysis of phosphatidylinositol
(PI) 4,5-bisphosphate to inositol (1, 4, 5)-triphosphate and
diacylglycerol via the
-subunit of Gq, a family of pertussis toxin
(PTx)-insensitive G proteins (10, 11). Production of inositol
triphosphate and diacylglycerol can also occur via activation of
phospholipase Cß mediated by ß
-subunits released from G proteins
such as Go and Gi (12). It is becoming increasingly clear that
1-receptors activate other signaling pathways; for
example,
1-receptors activate phospholipase D in brain
and promote the release of arachidonic acid via activation of
phospholipase A2 via PTx-sensitive G proteins (13, 14). Recent studies
indicate that
1-adrenergic receptors may share tyrosine
protein kinase/Ras/mitogen-activated protein (MAP) kinases signaling
pathways with peptide growth factors to stimulate growth responses in
several types of cells including myocytes and VSMCs (Ref. 15 and Z.-W.
Hu, X. Y. Shi, R. Z. Lin, and B. B. Hoffman, submitted for
publication).
Phosphatidylinositol 3-kinase (PI 3-kinase) is a kinase that plays an
important role in mediation of mitogenic actions of many peptide growth
factors and G protein-coupled receptors in various types of cells (for
reviews see Refs. 17, 18). PI-3 kinase has been implicated in the
regulation of cell growth by receptor tyrosine kinases (19, 20),
cytokine receptors (21), and G protein-coupled receptors (22, 23). PI
3-kinase mediates a number of intracellular events including the
PKC-independent serine phosphorylation and activation of a ribosomal S6
kinase family designated p70/p90S6K (24). PI 3-kinases and
ribosomal S6 kinases play a major part in mitogen-stimulated increase
in protein synthesis and changes in cytosolic structure of growing and
dividing cells. Mitogens such as platelet-derived growth factor (PDGF),
insulin, and insulin-like growth factor (IGF) activate PI 3-kinase
leading to rapid phosphorylation of phosphatidylinositol
(4, 5)-diphosphate at the D-3 position of the inositol ring to form
phosphatidylinositol (3, 4, 5)-triphosphate (25). Increasing evidence
suggests that this product is involved in the regulation of the actin
cytoskeleton that plays a critical role in a number of cellular
processes including motility, chemotaxis, and cell division (26, 27).
There are multiple forms of PI 3-kinase with distinct mechanisms of
regulation and different substrate specificities in mammalian cells;
these enzymes are activated both by peptide growth factors and G
protein-coupled receptor agonists. The first identified PI 3-kinase is
a heterodimer consisting of a p85-regulatory subunit with SRC homology
2 (SH2) domains and a p110 catalytic subunit (28). Another PI 3-kinase
has been described; this isoform consisted of a p110 monomeric
subunit that is activated independently of a p85-regulatory subunit
(29). A major mode of activation of the heterodimeric PI 3-kinase by
growth factors likely involves docking of the kinase through SH2
domains on the p85 subunit to phosphorylated tyrosine residues(s) on
receptor tyrosine kinases (30). This PI-3 kinase isoform is also
directly activated by ß
-subunits released from activated G
proteins in platelets (31) and by Gi in cell lines expressing
2A-adrenergic receptors (22). Activation of the
heterodimeric PI 3-kinase has a complex relationship with
p21ras activation. Activation of PI 3-kinase may occur via
Ras-dependent (32) or independent (33) pathways; PI 3-kinase may
function as a target of P21ras (34) in some cells or
function upstream of p21ras in others (22, 35). We recently
demonstrated in human VSMCs, likely expressing the three subtypes of
1-receptors, that
1 receptor-stimulated
mitogenesis is associated with activation of PI 3-kinase (36). In those
cells,
1-receptor activation of PI 3-kinase is mediated
by a PTx-sensitive G protein(s), and PI 3-kinase activity is associated
with activation of p21Ras and increased tyrosine protein
kinase activity. However, there is little specific information about
signaling pathways that mediate
1-receptor subtype
stimulation of PI 3-kinase, particularly in a model system where
potential differences in the signaling pathways used by the three
cloned
1-receptor subtypes could be discerned.
Increasing evidence suggests that each of the subtypes of
1-receptors may activate overlapping or potentially
distinct signaling pathways (2, 13). However, little is known about the
potential biological significance of the various specific
1-receptor subtypes being expressed in same cells.
Availability of expression vectors containing cDNAs encoding each of
the
1-receptor subtypes provides an approach to the
question of which subtype(s) of
1-receptors mediate
activation of PI-3 kinase. NIH3T3 cells stably transfected with each of
three subtypes of
1-receptors provides an interesting
model system as these cells have been extensively characterized in
terms of classical growth factor-stimulated mitogenesis. In the present
study, we have found that stimulation both
1A- and
1B-, but not
1D-receptor subtypes of
human
1-receptors, stably expressed in these cells,
activated PI 3-kinase. The results demonstrate that
1A-
and
1B-receptor subtypes activated PI 3-kinase via
different subunits of G proteins; and there were differentiable
patterns of activation of p21Ras protein and MAP kinase
cascades by
1-receptor subtypes in these cells.
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RESULTS
|
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NIH3T3 cells were used for transfection of control vectors
or expression vectors containing three subtypes (
1A,
1B, and
1D) of human
1-receptors. To confirm functional expression of each of
the three subtypes of
1-receptors in NIH3T3 cells,
1 receptor-stimulated expression of c-fos
mRNA was measured in NIH3T3 cells stably expressing
1A,
1B, and
1D receptors. Induction of
c-fos mRNA was selected as a functional index of
biologically significant expression of
1-receptors (37).
Stimulation of the cells with norepinephrine caused similar increases
in expression of the c-fos gene in the cells transfected
with each of the three subtypes of
1-receptors,
suggesting that these cells expressed functional receptors for each of
the three subtypes (Fig. 1A
).
Norepinephrine did not stimulate c-fos expression in the
wild-type cells (data not shown). Also, norepinephrine-stimulated
increased expression of c-fos mRNA was attenuated by the
1-receptor antagonist prazosin (Fig. 1B
).
Norepinephrine did not activate PI-3 kinase activity in wild-type
NIH3T3 cells or in cells transfected with control vectors, confirming
that these cells do not express endogenous
1-receptors.
As illustrated in Fig. 2
, norepinephrine
(10 µM) stimulated activation of PI 3-kinase in NIH3T3
cells transfected with
1A- or
1B-receptors. However, although cells transfected with
1D-receptors reproducibly had an increased basal
activity of PI 3-kinase compared with cells transfected with control
vectors, norepinephrine did not significantly increase PI 3-kinase
activity in cells transfected with the
1D-receptors.
Interestingly, the increased basal activity of PI 3-kinase in cells
transfected
1D-receptors was inhibited by the
1-receptor antagonists, doxazosin and prazosin (data not
shown). This result suggests that overexpression of
1D-receptors may spontaneously (in the absence of added
agonist), albeit weakly, activate down-stream signaling mechanisms and
that these antagonists are acting as inverse agonists. Norepinephrine
stimulated a concentration-dependent increase in PI 3-kinase activity
in cells expressing
1A- or
1B-receptors,
and theses responses were blocked by
1-receptor
antagonists, doxazosin and prazosin (data not shown), as we reported
previously for VSMCs (36).
To determine whether stimulation of PI 3-kinase in cells expressing
either
1A- or
1B-receptor subtypes
required PTx-sensitive G proteins, cells were incubated with PTx (50
ng/ml) for 16 h. PTx did not block norepinephrine-stimulated PI
3-kinase in cells transfected with either
1A-receptors
(Fig. 3A
, lane 4) or
1B-receptors (Fig. 3B
, lane 4), suggesting that both of
these subtypes of
1-receptors activate PI 3-kinase via
PTx-insensitive G proteins in NIH3T3 cells.
To investigate potential roles of
- and ß
-subunits of G
proteins in activation of PI 3-kinase, cells transfected stably with
1A- or
1B-receptors were transiently
transfected with the
t-construct as
t
functions as a scavenger of free ß
(38). As illustrated in Fig. 3
, overexpression of
t did not inhibit
norepinephrine-stimulated activation of PI 3-kinase in cells
transfected with
1A-receptors. This result suggests that
either an
-subunit or a ß
-subunit not recognized by
t, released from a PTx-insensitive G protein, mediates
this response (Fig. 3A
, lane 5). However, overexpression of
t blocked norepinephrine-stimulated activation of PI
3-kinase in cells transfected with
1B-receptors (Fig. 3B
, lane 5), suggesting that
1B-receptor-mediated
activation of PI 3-kinase may be due to direct effects of
ß
-subunits released from a PTx-insensitive G protein. However,
overexpression of an ß-adrenergic receptor kinase peptide (ß-ARK)
[also called G-protein receptor kinase-2 (GRK2)], which is a
different scavenger of free ß
-subunits (22), did not inhibit
either
1A- or
1B-receptor-stimulated PI
3-kinase activity. Western blotting (Fig. 3C
) demonstrated successful
expression of GRK2 as well
t in these cells. To
demonstrate functional expression of GRK2 in these cells, cells were
cotransfected with the
2A-adrenergic receptor gene and
either
t or GRK2. GRK2 has been shown to block
2A-receptor-stimulated activation of MAP kinase (43).
These cells were then treated with or without norepinephrine, and MAP
kinase activity was measured. Results indicate that both
t and GRK2 attentuated the
2A-receptor-stimulated increase in activity of MAP
kinase, suggesting that these two molecules functioned effectively as
scavengers of the ß
-subunits released by these receptors (Fig. 3D
).
We have previously found that
1-receptor-activated PI 3-kinase activity can be
detected in anti-Ras immunocomplexes, and stimulation of
1-receptors directly increases active Ras-GTP in human
VSMCs (36). Also, our previous study suggested that activation of
Ras-GTP is a downstream target of PI 3-kinase after stimulation of
1-receptors in those cells. NIH3T3 cells provide an
excellent model system by which to pursue the role of the individual
1-receptor subtypes in activating Ras-GTP and PI
3-kinase. We first tested which subtypes of
1-receptors
activated p21Ras protein. The results indicate that
norepinephrine stimulates an increase in active Ras-GTP in cells
expressing either
1A- or
1B-receptors,
but no measurable change was detected in cells expressing
1D-receptors (Fig. 4A
and
B). Both
1A- and
1B-receptor activation
of p21Ras protein was insensitive to PTx (Fig. 4C
).
Overexpression of
-transducin did not inhibit
1A-receptor-activation of Ras-GTP. On the other hand,
t markedly inhibited
1B-receptor-stimulated activation of Ras-GTP, suggesting
that ß
-subunits released from Gq were used by
1A-receptors in signaling to activate
p21Ras.
To obtain additional information about the interactions between
p21Ras protein and PI 3-kinase, overexpression of a
dominant negative mutant of p21Ras
(P21Ala15ras) was used. Transfection of cells with this
dominant negative mutant of p21Ras inhibits agonist-induced
increases in active Ras-GTP (39). If activation of PI 3-kinase were
upstream of p21Ras activation, then transfection of cells
with this mutated gene should not inhibit
1-receptor
activation of PI 3-kinase. On the other hand, if activation of PI
3-kinase by stimulation of
1-receptors were a downstream
target of Ras-GTP, the construct would be expected to inhibit PI
3-kinase activation. As illustrated in Fig. 5
, overexpression of the dominant
negative mutant of p21Ras blocked norepinephrine-stimulated
increases in active Ras-GTP in cells expressing
1A- and
1B-receptors as expected (Fig. 5A
). Expression of this
negative mutant of p21Ras did not inhibit
norepinephrine-stimulated activation of PI 3-kinases in the cells
expressing
1A receptors (Fig. 5B
). However, expression
of the mutant p21ras attenuated activation of PI 3-kinase
in cells expressing
1B-receptors (Fig. 5C
), suggesting
that p21Ras protein functions downstream or independently
of activation of PI 3-kinase after stimulation of
1A-receptors but upstream of PI 3-kinase after
stimulation of
1B-receptors.
Since activation of PI 3-kinase and p21Ras protein induces
activation of a series of growth or differentiation-related protein
kinase cascades in various cells, we determined whether activation of
specific protein kinase cascades was associated with the different
subtypes of
1-receptors. As illustrated in Fig. 6
, norepinephrine activated MAP kinase in
NIH 3T3 cells expressing
1A- and
1B-receptors but not significantly in cells expressing
1D-receptors or in wild-type NIH 3T3
cells. These results were consistent with the results demonstrating
that norepinephrine stimulated activation of p21Ras protein
in cells expressing
1A- and
1B-receptors,
but not detectably in cells expressing
1D-receptors
(Fig. 4A
). Coexpression of the P21A15ras dominant negative
mutant in cells expressing
1A- and
1B-receptors significantly inhibited
norepinephrine-stimulated activation of MAP kinase (Fig. 6C
). These
results demonstrate that stimulation of both
1A- and
1B-receptors activates MAP kinase at least in part via a
p21Ras-dependent signaling path-way.
We were interested in determining the potential role of PI 3-kinase in
MAP kinase signaling pathways used by
1-receptor
subtypes expressed in NIH3T3 cells. Figure 7
illustrates the effects of negative
dominant mutant of PI 3-kinase P85 subunit on MAP kinase activity in
cells expressing
1A- and
1B-receptors.
Coexpression of this mutant of PI 3-kinase p85 significantly attenuated
norepinephrine-stimulated increases in kinase activities in cells
expressing
1A-, but not in cells expressing
1B-receptors (Fig. 7
), suggesting that PI 3-kinase
functions as upstream component in the
1A-receptor-stimulated MAP kinase cascade. On the other
hand, the MAP kinase cascade may not be a target of PI 3-kinase in
1B receptor-signaling pathways.
 |
DISCUSSION
|
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Stimulation of transfected
1A- or
1B-adrenergic receptors in NIH-3T3 cells led to
activation of PI 3-kinase. Cells expressing
1D receptors
did not stimulate PI3-kinase activity above basal values in response to
norepinephrine in these experiments. In parallel experiments we found
that transfection of the
1D-receptors led to
norepinephrine-activation of c-fos in NIH-3T3 cells
demonstrating that the
1D-receptors were being
functionally expressed in the cells. The major result of these studies
is that
1A- and
1B-adrenergic receptors
activated downstream effectors, particularly p21ras and PI
3-kinase, by different signaling mechanisms.
Increased abundance of c-fos mRNA could be readily measured
after stimulation with norepinephrine in the cells expressing in
1D-receptors, but we did not detect changes in PI
3-kinase activity in those cells. While these results clearly
demonstrate functional expression of the
1D-receptors,
they do not exclude the possibility that these receptors might activate
this kinase in different cells or under other conditions. Using
[3H]prazosin, the level of expression of each of the
three
1-receptor subtypes was below the sensitivity of
detection with this ligand (data not shown). Consequently, we can
exclude the possibility of promiscuous coupling due to overexpression
of these receptors. It has been shown previously that a low level of
expression of m1 muscarinic receptors (very difficult to measure
directly with a radioligand) is sufficient to activate MAP kinase
cascades (40).
It is generally accepted that PTx-insensitive G proteins, particularly
members of the Gq family, mediate increases in hydrolysis of PI
4,5-bisphosphate to inositol triphosphate and diacylglycerol by the
three subtypes of
1-receptors (11, 41). Recent evidence
suggests that signal transduction mechanisms of
1-receptors are much more complex than previously
realized. For example, several studies have suggested that
PTx-sensitive G proteins may also be involved in
1-receptor signaling pathways in several types of
cells. Llahi and Fain (42) found that
1-receptors
activate phospholipase D in the brain and promote the release of
arachidonic acid via activation of phospholipase A2 via PTx-sensitive G
protein (13). Stimulation of
1-receptors causes
phosphorylation of c-Jun kinase mediated by a PTx-sensitive G protein
(14). We recently demonstrated that
1-receptor-stimulated mitogenic effects are associated
with activation of PI 3-kinase in human VSMCs and that activation of PI
3-kinase is mediated by a PTx-sensitive G protein in those cells (36).
In the present study, the finding that activation of PI 3-kinase by the
1A- and
1B-receptor subtypes is PTx
insensitive in NIH3T3 cells emphasizes the importance of host cell
factors in modulating receptor function. Our previous study was
performed in human VSMCs that express mainly
1B and
1D mRNA with very low
1A receptor subtype
mRNA. A recent study in COS-7 cells demonstrated that
1B-receptor stimulation of MAP kinase involved a
PTx-insensitive Gq
using a p21ras-independent mechanism,
as it was found that overexpression of the P21N17ras
dominant negative mutant did not inhibit
1B-receptor
stimulation of MAP kinase (43).
Direct interactions between Gß
-subunits and protein kinases have
been implicated in various cells. For example, Gß
-subunits
stimulate PI 3-kinase in platelets and neutrophils (44, 45). Moreover,
transfection studies with COS-7 cells revealed that Gß
-subunits
activate MAP kinase after stimulation of Gs, Gi, and Gq-coupled
receptors (46); these effects of the ß
-subunits are due to
activation of p21ras (46). Gß
-subunits mediate
tyrosine kinase receptor, IGF-I receptor activation of MAP kinase (47).
van Biesen et al. (48) reported that
2-adrenergic receptors activate MAP kinase via
stimulation of G protein ß
-subunits, leading to interactions of
adapter proteins Grb2/SOS1/Shc, indicating that an intact signaling
pathway machinery used by classical receptor tyrosine kinases is shared
by G protein-coupled receptors. Based on these findings,
Gß
-subunits, in G protein-coupled receptor signaling, have been
suggested to be analogous to phosphorylated receptor tyrosine kinases
(49).
In the present study, we obtained evidence that expression of
-transducin attenuated the norepinephrine-induced increase of PI-3
kinase activity in cells expressing
1B-receptors but not
in cells expressing
1A-receptors, suggesting that
ß
-subunits mediate
1B-receptor activation of PI-3
kinase. However, we found that overexpression of GRK2,
ß-adrenergic receptor kinase peptide, used successfully to
sequester ß
-subunits in other studies (47), did not inhibit
1B receptor-stimulated PI-3 kinase activity in our
experiments (Fig. 3
, A and B). This result raises the possibility that
the ß
-subunits involved in
1B receptor-stimulated
PI-3 kinase in NIH 3T3 cells have differing affinities for
tvs. GRK2. Since there are multiple forms of
ß
-subunits, this possibility is tantalizing but requires further
experimental testing.
A number of studies have suggested that there is a complex interaction
between activation of PI 3-kinase and activation of the
P21ras/MAP kinase cascade (17). For instance, several
studies have demonstrated that activation of PI 3-kinase by growth
factors or by G protein-coupled receptor agonists is associated with
activation of p21ras. In PC12 cells, Downward et
al. (50) demonstrated that PDGF-stimulated p21ras
targets PI3-kinase. On the other hand, activation of PI 3-kinase may
also function upstream of p21ras/MAP kinase cascade. A
recent study suggests that the sequential activation of PI 3-kinase,
Ras protein, and MAP kinase is involved in the insulin-signaling
pathways during differentiation of adipocytes by hormones and
phosphodiesterase inhibitors; inhibition of PI 3-kinase by wortmannin
inactivated the Ras/MAP kinase pathway, leading to suppression of
adipocyte differentiation (51). Other studies have demonstrated that PI
3-kinase activity is an important intermediate step in G
protein-coupled receptor agonist activation of the
p21ras-signaling cascade (22, 36). Hawes et al.
(22) reported that stimulation of
2A-ARs or
lysophosphatidic acid receptors activates p21ras and
MAP kinase cascade via Gß
-subunits. Treatment of cells with
specific inhibitors of PI 3-kinase, wortmannin or LY294002, or
overexpression of a dominant negative mutant of the p85 subunit of PI
3-kinase, attenuated activation of the Ras/MAP kinase cascade.
Moreover, a Gß
-specific PI 3-kinase
has recently been
identified to mediate G protein-coupled receptor activation of MAP
kinase cascades (52). Given the fact that
1B-receptor
mediates MAP kinase activation via Gß
-subunits, it will be
important to determine which subtype of PI 3-kinases (p85/p110 or p110
) plays a signaling role in
1B-receptor signal
transduction.
We recently found that
1-receptor stimulation of
PI 3-kinase activity is associated with activation of Ras protein, and
Ras may function as a target of PI 3-kinase in smooth muscle cells
(36). Our present study provides evidence demonstrating that activation
of PI 3-kinase is greatly involved in
1-receptor
subtype-signaling pathways. Overexpression of the dominant negative
mutant of p21A15Ras markedly inhibited the capacity of
1A- and
1B-receptor subtypes to activate
p21ras (Fig. 4
). This construct attenuated
1B-receptor-stimulated, but did not attenuate
1A receptor-stimulated, PI 3-kinase activity, suggesting
that p21Ras may act either as downstream component of
1A receptor-stimulated PI 3-kinase or upstream component
of
1B receptor-stimulated PI 3-kinase. However,
overexpression of a dominant negative mutant of the PI 3-kinase p85
subunit, namely
p85, blocked
1A- but not
1B-receptor subtype-stimulated MAP kinase activity. This
result demonstrates that PI 3-kinase is critical for activation of MAP
kinase by
1A-receptors. However, the results suggest
that
1B-receptor subtype activates PI 3-kinase and MAP
kinase in parallel. There is other evidence indicating that
1- receptors may utilize other intracellular signal
pathways such as protein kinase C/Raf-1 to activate MAP kinase
independently of p21Ras (43). These complex interactions
among the
1-receptors, subunits of G proteins, and
downstream protein kinase cascades are presented schematically in Fig. 8
.
In summary, the results indicate that
1A- and
1B-receptors activate PI 3-kinase, likely via different
subunits of PTx-insensitive G proteins. Moreover, the
1A-receptor subtype may use Gq
and
1B
receptor subtype may use ß
-subunits of Gq to stimulate PI 3-kinase
activity in NIH3T3 cells. We also provide evidence demonstrating that
increases in PI 3-kinase activity by stimulation of
1A-receptor subtype turn on p21ras
activation, which in turn activates the MAP kinase cascade. However,
1B-receptor subtype activation of MAP kinase cascade is
likely independent of activation of PI 3-kinase. These experiments
demonstrate the heterogeneity of the signaling pathways activated by
the various receptor subtypes. Further experiments aimed at
understanding structure-function relationships for
1-receptor-coupling mechanisms should provide new
insights into the functions of the physiologically very important
receptor systems.
 |
MATERIALS AND METHODS
|
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Materials
Norepinephrine and myelin basic protein (MBP) were purchased
from Sigma Chemical Co. (St Louis, MA); [
32P]ATP (2000
Ci/mmol), [
32P]dCTP, and enhanced chemiluminescence
(ECL) Western detection system were purchased from Amersham
Corp.(Arlington, IL); expression vectors of human
1A-,
1B-, and
1D-adrenergic receptor and
control vectors were a gift of Dr. Johnston and colleagues of Central
Research (Pfizer Inc., Sandwich, England); expression vectors of
-subunit of transducin (
t-pcDNA-I) and control vector (pcDNA-I)
were kindly provided by Dr. Henry Bourne of University of California at
San Francisco; expression vectors of a minigene (cDNA 495689) of GRK2
(ß-ARK1) was a gift from Dr. R. J. Lefkowitzs laboratory of
Duke University (Durham, NC); the p21Ala15ras dominant
negative mutant and control vectors were kindly provided by Joan H.
Brown of University of California at San Diego; phosphatidylinositol
was obtained from Avanti Polar Lipids Inc. (Alabaster, AL); human
recombinant IGF-I, cell culture medium, and FBS were purchased from
GIBCO/BRL (Grand Island, NY); wortmannin was from Worthington
Biochemical Co. (Freehold, NJ); and antibodies against PI-3 kinase
p85-
, p110, tyrosine protein kinases, H-ras, p44ERK1/2,
GRK2, and
t, as well as protein A/G-agarose, were from
Santa Cruz Biotechnology (Santa Cruz, CA). All other chemicals were
reagent or molecular biology grade and were obtained from standard
commercial sources.
Cell Culture
NIH3T3 mouse fibroblasts were purchased from the American Type
Culture Collection (Manassas, VA). Cells were grown in DMEM with 5%
FBS at 37 C in a humidified atmosphere of 5% CO2-95% air.
The cells were harvested for passaging at confluence with trypsin-EDTA
and plated in 100-mm dishes at a density about 5 x
105, with a 8090% confluence being reached about 5 days
later. The medium was replaced every 2 days. To examine effects of
agonist-stimulated changes, cells were incubated with DMEM without
serum for indicated times after achieving confluence. The cells were
treated with agonists or vehicle solution (as control) starting from
the longest time point, and the cells were harvested at the same
time.
Stable Transfection of NIH3T3 Cells with
1-Adrenergic Receptors
NIH3T3 cells (3 x 105) were seeded in
100-mm2 culture dishes with DMEM with 10% FBS. The
following day, each adrenergic receptor subtype expression vector
(
1A and
1B, 10 µg;
1D,
50 µg) was transfected using 50 µg of lipofectamine (GIBCO-BRL) in
serum-free medium after the instruction of manufacture. Five hours
later, an equal volume of DMEM with 20% FBS was added. Twenty four
hours after the start of transfection, the cells were washed and placed
in fresh medium; 48 h later, the transfected cells were reseeded
into ten 100-mm2 dishes, and culture medium was changed to
DMEM/10% FBS with 500 µg/ml of Geneticin (GIBCO-BRL). Ten to 14 days
later, the surviving cell colony was isolated and grown in medium
containing Geneticin. Cells expressing a similar number of receptors
were used for the further experiments. For various assays, the cells
were made quiescent by serum starving for 24 h before treatment
and harvest.
Transient Transfection of NIH3T3 Cells with Expression Vectors
Containing
-Transducin, ß1-Adrenergic
Receptor Kinase, p21Ala15ras, or
p85
PI3K
Transfection of NIH3T3 cells with control or expression vectors
containing
-transducin, ß1-adrenergic receptor kinase
(now termed GRK2), the p21Ala15ras dominant negative
mutant, or a dominant negative mutant of PI 3-kinase p85
(
P85)
was performed as described above. NIH3T3 cells that stably expressed
each of the three subtypes of
1-adrenergic receptors
were cultured in DMEM with 10% FBS in the presence of 500 µg/ml of
geneticin as described above. The cells were seeded into
100-mm2 dishes and transfected at
80% confluence.
Transfection was performed in 3.0 ml of Optim-MEM (GIBCO-BRL)
containing 50 µg of lipofectamine and 10 µg of control vectors or
expression vectors containing
-transducin,
ß1-adrenergic receptor kinase, p21Ala15ras,
or
P85. Five hours later, 3 ml of DMEM with 20% FBS were added.
Twenty four hours from the start of transfection, the cells were
washed, and fresh DMEM with 10% FBS was replaced. Next day, the cells
were made quiescent by serum starving for 18 h before treatment
and harvest.
1-Adrenergic receptor subtype-stimulated
activation of PI 3-kinase, Ras-GTP, and MAP kinase was then determined
as described below.
RNA Preparation and Northern Blotting Analysis
A single-step method of RNA isolation using acid guanidinium
thiocyanate-phenol-chloroform extraction as described previously (53)
was used to isolate total RNA from NIH3T3 cells. Briefly, cells were
rinsed with cold calcium-magnesium-free PBS, and then the cells were
homogenized with a Polytron in 10 vol of denaturing buffer containing 4
M guanidinium thiocyanate, 25 mM sodium citrate
(pH 7), 0.5% sarcosyl, 0.1 M 2-mercaptoethanol. One volume
of 2 M sodium acetate (pH 4.0), 10 vol of water-saturated
phenol, and 2 vol of chloroform-isoamyl alcohol (49:1) were
sequentially added to the homogenate with thorough mixing after
addition of each reagent. The homogenate was incubated on ice for 20
min and then centrifuged at 12,000 x g for 20 min. The
aqueous phase was taken and RNA was precipitated with isopropanol (1:1
volume). The resulting RNA pellet was dissolved in the denaturing
buffer and again precipitated with isopropanol by cooling and
centrifugation. The RNA pellet was washed with 75% ethanol,
sedimented, vacuum dried, and dissolved in Tris-EDTA buffer. For
Northern blotting analysis of c-fos, 10 µg of total RNA
were heated at 65 C for 10 min, cooled rapidly on ice, and denatured
with 6% formaldehyde. The RNA was fractionated by 1% agarose gel
electrophoresis and transferred to a nylon filter by capillary
blotting. The blot was prehybridized in 50% formamide, 5 x
SSPE buffer (1 x SSPE = 0.18 M NaCl, 10
mM sodium phosphate, pH 7.7, and 1 mM EDTA),
5 x Denhardts solution, 0.5% SDS, at 42 C for 4 h, and
hybridized at 42 C for 1216 h to the rat c-fos or
ß-actin cDNA probes that were labeled by [32P]dCTP
using Amershams random priming labeling kit. After hybridization the
filter was washed twice in 2 x SSPE, 0.1% SDS at 65 C for 15 min
and once in 0.1 x SSPE, 0.1% SDS at 56 C for 30 min. The filter
was exposed to Kodak XAR-5 film at -70 C with an intensifying screen
for 1624 h. The autoradiograms were scanned using a laser
densitometer. The amount of c-fos mRNA was quantified
relative to the amount of 18s tRNA on the same filter.
Immunoprecipitation
After treatment, cultures on 100-mm2 plates were
rinsed with ice-cold PBS containing 1 mM sodium
orthovanadate. Cells were incubated with lysis buffer (1% Nonidet
P-40, 25 mM HEPES (pH 7.5), 50 mM NaCl, 50
mM NaF, 5 mM EDTA, 10 nM okadaic
acid, 1 mM sodium orthovanadate, 1 mM
phenylmethylsulfonyl fluoride, and 10 µg/ml of antipain, aprotinin,
and leupeptin) for 1015 min on the ice. Insoluble material was
removed by centrifugation at 12,100 x g for 20 min.
The amount of cell lysate was normalized by protein content in each
experiment using a kit from Bio-Rad (Richmond, CA). The lysate was
incubated with an appropriate amount of antibody and agitated for
2 h. The lysate was further incubated with 2030 µl of protein
A/G plus-agarose with agitation for 1 h. The beads containing the
immunoprecipitates were washed three times with lysis buffer, once with
distilled water, and once with washing buffer (0.1 M NaCl,
1 mM EDTA, 20 mM Tris-HCl, pH 7.5), and
subjected to PI 3-kinase and MAP kinase assays or analysis of Ras-bound
GTP.
Analysis of Ras-Bound GTP and GDP
Agonist-stimulated change in Ras-bound GTP was performed
following a method described previously (36). Quiescent cells were
labeled with 0.1 mCi/ml of [32P]orthophosphate in
phosphate-free DMEM for 12 h. After stimulation with agonists for
the indicated times, cells were washed with cold PBS for three times
and cells were lysed as described above. Ras proteins were recovered by
immunoprecipitation with an anti-Ras polyclonal antibody. After
extensively washing as described above, the immunoprecipitates were
suspended in 20 µl of reaction mixture containing 20 mM
HEPES, pH 7.5, 20 mM EDTA, 2% SDS, 0.5 mM GDP,
and 0.5 mM GTP. The suspension was heated at 90 C for 3 min
and centrifuged for 5 min. The bound nucleotides were separated by TLC
on polyethyleneimine-cellulose plate with 0.75 M
KH2PO4 for development. GDP and GTP were
visualized using unlabeled standards. Ras-associated GTP was calculated
from the ratio of GTP/(GDP+GTP). The radioactivity was quantitated with
a PhosphorImager system (Molecular Dynamics, Sunnyvale, CA).
In Vitro Assays of PI 3-Kinase
For measurement of PI 3-kinase activity, cell lysates (1 mg
protein) were incubated with antibody against the p85-
subunit of PI
3-kinase (2 µg/mg protein) as described above. Assay of PI 3-kinase
activity was conducted as described previously (36). Briefly, the
washed pellets were resuspended in 50 µl of kinase reaction buffer
(20 mM Tris-HCl, pH 7.5, 100 mM NaCl, and 0.5
mM EGTA) and incubated at 25 C for 10 min after addition of
0.5 µl of 20 mg/ml phosphatidylinositol dissolved in chloroform to
make micelles of PI. Assays were initiated with addition of 5 µl of
ATP solution (0.4 M ATP, 0.1 M
MgCl2, and 1 µCi/ml [
32P]ATP) and
incubated at room temperature for 30 min. During this period of time
the formation of phosphatidylinositol phosphate was linear (data not
shown). The reaction was stopped upon addition of 100 µl of
chloroform-methanol-11.6 N HCl (100:200:2). After
centrifugation, the lower organic phase was taken for TLC on silica gel
plates from J. T. Baker Inc. (Phillipsburg, NJ) and developed in
chloroform-methanol-25% ammonium hydroxide-water (43:38:5:7). The
plates were exposed to Kodak XAR-5 film at -70 C with an intensifying
screen for 1624 h or were visualized after development with a
PhosphorImager system.
In Vitro Assay of MAP Kinase Activity
Assay of MAP kinase activity was performed following a method
described previously (36). Confluent cells were incubated in the
absence of serum overnight and treated with norepinephrine or other
agonists for various times as indicated. The cells were lysed in 0.4 ml
lysis buffer as described above. For MAP kinase activity assay, cell
lysates (400 µg protein) were incubated with antibody against ERK1 (2
µg/mg protein) and washed as above. The washed immunocomplexes were
resuspended in 50 µl of kinase buffer (25 mM HEPES, pH
7.5, 10 mM MgCl2, 1 mM
dithiothreitol, 0.5 mM EGTA, 40 µM ATP, 1
µCi of [
32P]ATP), and MBP (1 mg/ml) as a
substrate. The reaction mixture was incubated for 10 min at 30 C
because preliminary experiments indicated that MBP-induced
phosphorylation is linear for 2030 min. The reaction was stopped by
spotting 10 µl of reaction mixture onto p-81 phosphocellulose paper
(Whatman LabSales, Hillsboro, OR), which was then washed in 75
mM phosphoric acid with constant stirring for 1 h and
transferred to another washing overnight. The papers were washed with
acetone for 5 min and dried. 32P, which represented the
phosphorylation of MBP by MAP kinase, was quantitated by scintillation
spectrophotometry. Alternatively, reaction mixtures were loaded and
separated on 14% SDS-PAGE, and the dried gels were exposed to Kodak
XAR-5 film at -70 C with an intensifying screen for 1624 h for
visualization.
Data Analysis
Data are presented as mean ± SEM, and
treatment effects were compared by one-way ANOVA or Students paired
t test (two tailed). P < 0.05 was taken as
level of significance.
 |
ACKNOWLEDGMENTS
|
---|
We thank Geoffrey Johnston for providing expression vectors
encoding human
1A-,
1B-, and
1D-adrenergic receptors and control vectors; Joan H.
Brown for providing the dominant negative mutant of p21Ras
expression and control vectors; Henry R. Bourne for providing the
t
and control constructs; and Wataru Ogawa for the dominant negative
mutant of PI 3-kinase p85
and control vector.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Zhuo-Wei Hu, M.D., Ph.D., Veterans Affairs Palo Alto Health Care System, GRECC 182B, 3801 Miranda Avenue, Palo Alto, California 94304. E-mail: huzhwei{at}leland.stanford.edu
This work was supported by NIH Grant HL-41315 and by a preclinical
grant from Pfizer Inc.
1 Supported by a Pharmaceutical Research and Manufacturers of America
Foundation Fellowship for Careers in Clinical Pharmacology. 
Received for publication December 4, 1997.
Revision received August 17, 1998.
Accepted for publication September 24, 1998.
 |
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