(Received for publication, October 21, 1994; and in revised form, December 27, 1994)
From the
Epinephrine stimulation of rat ,
, and
adrenergic receptor
subtypes, expressed stably in Chinese hamster ovary (CHO) cells, caused
a rapid, transient activation of mitogen-activated protein kinase
(MAPK), with subtype-specific different efficiencies. The order of
activation was CHO-2B
CHO-2D
CHO-2C. Pertussis toxin blocked
the stimulation of MAPK enzymatic activity and the parallel MAPK
phosphorylation, demonstrating that these responses are mediated by
pertussis toxin-sensitive G
proteins.
Contrary to what
has been reported for the subtype expressed in rat-1
fibroblasts, epinephrine did not cause any detectable activation of
p21
in the CHO transfectants.
Furthermore,
combined application of epinephrine and phorbol myristate acetate had a
potent cooperative but not additive effect in clones CHO-2D and CHO-2B
but not in CHO-2C, suggesting that protein kinase C is probably
differently involved in the signaling by the three receptor subtypes.
These results show that in CHO cells, the
different adrenergic receptor subtypes utilize
differential pathways to activate MAPK in a
p21
-independent way.
At least three distinct adrenergic receptor
subtypes,
,
, and
, mediate the cellular effects of epinephrine and
norepinephrine in the
rat(1, 2, 3, 4) . They are expressed
in a tissue-specific manner and differ in their pharmacological,
biochemical-molecular biological, and probably signal transduction
properties(5, 6, 7, 8) .
adrenergic receptors mediate the action of
epinephrine by regulating a variety of effectors including among others
inhibition/stimulation of adenylyl cyclase through pertussis
toxin-sensitive G proteins (9, 10) and stimulation of
cytosolic phospholipase A
(11) . However, the
function(s) and the physiological differences of the subtypes are
virtually unknown, in part because their differential coupling to
different intracellular signaling mechanisms has not been
characterized. Studies on the coupling of
adrenergic
subtypes to adenylyl cyclase have shown that the rat
subtypes couple differently to adenylyl cyclase in certain cells (12) and point to the possibility of further differences and
increased plasticity in the coupling of subtypes to other effectors,
including mitogen-activated protein kinase (MAPK). (
)
MAPK, also known as extracellular signal-regulated kinase, is a central component of an evolutionarily conserved kinase cascade, which integrates various extracellular signals converging to it(13) . The cascade consists of three consecutive kinases, which relay and at the same time switch tyrosine phosphorylation signals, initiated from cell surface receptors, to serine/threonine phosphorylation of downstream serine/threonine protein kinases and other effectors that regulate mitogenesis, differentiation, and cellular metabolism.
Thus, upon activation,
p74, a serine/threonine kinase, acts as MAPK
kinase kinase (MEKK) phosphorylating and activating MAPK kinase, a dual
specificity protein kinase, that activates MAPK by dual phosphorylation
at closely spaced residues Thr-183 and Tyr-185. A parallel
p74
-independent pathway, including MEKK
(instead of p74
) that can phosphorylate and
activate MAPK kinase, is thought to operate in the transmission of
signals initiated by certain G protein receptors(14) . Although
in most systems different isoforms of MAPK, mainly p42 and p44 MAPK,
are coactivated by a variety of stimuli, p42 MAPK is selectively
activated independently of p44 MAPK in some cells(15) . The
activated MAPK activates, by phosphorylating consensus sites,
downstream effectors, including p90
and
phospholipase A
in the cytosol and transcription factors
(c-jun, c-myc, c-fos,
p62
) in the nucleus, where MAPK translocates
when activated (16) .
Ligand activation of tyrosine kinase
receptors (like the EGF receptor) stimulates MAPK via a pathway that
involves accumulation of active GTP-bound p21.
However, EGF activates also, in rat-1 and NIH 3T3 fibroblasts, a
Ca
-dependent signaling pathway capable of
phosphorylating p42 MAPK in a p21
-independent
way(17) . In a similar way some G protein-coupled receptors
(like the endogenous thrombin receptor and the lysophosphatidic acid
receptor in hamster lung fibroblasts and rat-1 cells) use activated
p21
in signaling to
MAPK(18, 19) . Yet other G protein-linked receptors
(like the platelet-activating factor receptor in CHO cells) utilize a
p21
-independent pathway(20) .
Recently it has been reported that the agonist-stimulated
adrenergic receptor activates MAP kinase activity in
rat-1 fibroblasts through the activation of the p21
pathway (21) . However, it is not known if the
activation of MAPK by
adrenergic receptor stimulation
is subtype-specific and, furthermore, if there are cell type-related
differences in the signaling pathway employed by
subtypes.
To characterize potential differences of the
biochemical signals induced upon stimulation of cloned adrenergic receptor subtypes, we have expressed the rat
,
, and
subtypes
in CHO cells and studied their coupling to MAPK.
We have found that
in CHO cells adrenergic receptor subtypes couple to
this effector with different efficacy and that they transmit their
signal to MAPK activation through a
p21
-independent pathway probably involving
different protein kinase C.
The blots were incubated with the anti-MAPK antibody and then with peroxidase-conjugated rabbit anti-mouse antibody (1:600). Immunostained proteins were visualized using the ECL detection system.
For the Western blotting analysis of G proteins, membrane proteins were prepared as described(27) . Membrane proteins (50 µg) were separated by SDS-PAGE, electroblotted to nitrocellulose, and the filters were incubated with anti-G protein antibodies (diluted 1:1000) and processed as described above.
Guanine nucleotides bound to
p21 were eluted from the immunoprecipitate and analyzed
by ascending thin layer chromatography on a polyethyleneimine cellulose
plate as described elsewhere(28) . After autoradiography the
spots corresponding to GTP and GDP were scraped off the TLC plates, and
the radioactivity was determined by liquid scintillation.
In initial experiments we analyzed the CHO transfectants by
ligand binding and determined that they express comparable levels of
adrenergic receptors in the range of 3-10
pmol/mg of membrane protein (see ``Experimental
Procedures''). This was necessary as receptor density such as in
our clones (pmol/mg protein) has been found to permit consistent and
comparable analysis of
adrenergic receptor-mediated
responses(29) .
Measurement of the cAMP in
agonist-stimulated CHO clones verified that the expressed subtypes are
functionally linked, but in a different way, to adenylyl cyclase in CHO
cells. Epinephrine activation of and
subtypes inhibited forskolin-stimulated elevation of cAMP,
whereas activation of
receptor produced a biphasic
effect with a maximum inhibition at 100 nM, followed by
stimulation at doses of 1-100 µM epinephrine
(results not shown).
Kinetic analysis of MAPK activation in
response to epinephrine (10 µM) showed almost identical
time courses in the three clones with a rapid, transient increase in
the MAPK activity. MAPK increase was measurable at 1 min after
stimulation, reached a maximum at 5 min, and decreased rapidly
thereafter, with continued epinephrine treatment, so that at 30 min MAP
kinase activity was to nearly prestimulated levels. The time course of
the activation was paralleled by an identical time course in the
phosphorylation and subsequent mobility shift of p42 MAP kinase
(results not shown). This uniform time course of activation of MAPK by
the subtypes is similar to what has been found with
other G-protein-coupled receptors (
-thrombin, lysophosphatidic
acid, platelet-activating factor receptors) (18, 19, 20) in a variety of cells, as well
as receptor tyrosine kinases in certain cell types (like EGF receptor
in PC12 cells)(30) .
As shown in Fig. 1, epinephrine
applied at 10 µM for 5 min activates MAPK in all three
clones but with substantially different efficiencies. The order of
activation is CHO-2B CHO-2D
CHO-2C. No activation was
observed in non-transfected CHO cells. The activation of MAPK by
epinephrine is 80% of the effect of serum on MAPK activity in clone
CHO-2D, 75% in clone CHO-2B, and 25% in CHO-2C. Yohimbine (10
µM) caused partial inhibition of activation of MAP kinase,
whereas pretreatment of cells with pertussis toxin (100 ng/ml) for 4 h
inhibited the epinephrine-induced activation completely in CHO-2C and
almost completely in CHO-2D and CHO-2B cells (85 and 67% inhibition,
respectively), without affecting basal MAPK activity (not shown). This
proves that the activating effect of epinephrine through
adrenergic receptor subtypes is mediated by pertussis
toxin-sensitive G
proteins. Pertussis toxin had only a
moderate inhibitory effect on the serum activation of MAP kinase, an
expected result of many of the growth factors present in the serum
signal to MAP kinase through tyrosine kinase receptors.
Figure 1:
Analysis of MAP
kinase activation. Cultures of parental CHO cells and transfectants,
expressing the (CHO-2D),
(CHO-2B), and
(CHO-2C) adrenergic receptor
subtypes, were serum-deprived for 24 h. Then the cells were treated for
5 min with epinephrine (Epi, 10 µM) in the
absence or presence of yohimbine (Yoh, 10 µM) or
pertussis toxin (PTX, 100 ng/ml), or alternatively they were
treated for 5 min with dialyzed fetal calf serum (FCS, 10%) in
the absence or presence of pertussis toxin (100 ng/ml). Cell extracts
were tested for MAPK activity as described under ``Experimental
Procedures.'' The values of MAPK activity obtained under different
conditions were normalized with respect to control cells, that is cells
non-stimulated with any agonist. The results shown are the average of
duplicate determinations from two separate
experiments.
The changes in enzymatic activity of MAP kinase were paralleled by modifications of MAP kinase phosphorylation. Fig. 2shows that epinephrine induced a different degree of shift in the mobility of p42 MAP kinase in the different clones: minor shift in CHO-2C, moderate in CHO-2D, and pronounced in CHO-2B cells. In this last clone specifically, retarded migration of an additional electrophoretic zone can be observed, most likely representing the 44-kDa (p44) form of MAP kinase. The slower migrating forms of p42/p44 on anti-MAPK immunoblots represent the threonine and tyrosine phosphorylated and activated form of MAPK(31) . Incubation of the same filter with anti-phosphotyrosine antibody showed that the shifted electrophoretic bands are tyrosine-phosphorylated (not shown). Consistent with the enzymatic activity of MAPK, the retardation in the electrophoretic mobility of p42/p44 MAPK is prevented partially by yohimbine and completely by pertussis toxin treatment.
Figure 2: Phosphorylation of MAP kinase in CHO transfectants. Cell extracts were prepared from cells treated under the conditions described in the legend of Fig. 1, and proteins were resolved by SDS-PAGE followed by immunoblotting with the anti-p42 MAPK antibody (see ``Experimental Procedures''). The position of the inactive unphosphorylated MAPK is indicated by p42, whereas the phosphorylated activated form of p42 MAPK displays reduced electrophoretic mobility and is indicated by pp42. Abbreviations are the same as listed in the legend to Fig. 1.
The substantial
quantitative differences in the MAPK activation observed among the CHO
clones in this study cannot be attributed to differences in receptor
number, since the three CHO clones express comparable levels of
receptor protein. They also express similar levels of G protein, as shown by immunoblot analysis of G
subunits (Fig. 3). In this context it should be noted
that, in the course of these experiments, while preparing the cells by
serum deprivation for the MAP kinase assays, we have observed a
reproducible induction of the levels of G
proteins. The
immunoblots of Fig. 3show clearly the induction of both
G
and G
proteins (the G
isoforms
expressed in CHO cells) (32) when parental or transfected CHO
cells were serum-depleted for 24 h.
Figure 3:
Immunoblot analysis of Gi proteins.
Parental non-transfected CHO cells and transfectants were grown in
medium supplemented with 10% fetal calf serum. Membrane proteins were
prepared from the cells under these conditions (+) or after serum
deprivation for 24 hours(-). The proteins were separated by
SDS-PAGE, transferred onto nitrocellulose membrane, and revealed by
antibody AS7 recognizing the G
i
and G
i
subunits and antibody EC2 specific for the G
i
and G
subunits.
The functional significance of
these changes in conditioning the signal transduction apparatus is
currently under investigation. We report these preliminary findings
here in order to show that the G levels change under serum
deprivation in the same direction (increase) in all clones studied. We
reason therefore that the found differences in MAPK activation rather
reflect different coupling efficiency of different subtypes to the same
signaling pathway to MAPK or reflect the employment by the subtypes of
different signaling pathways regulating MAPK activation such as
p21
activation, protein kinase C activation, or
Ca
mobilization(17) .
Figure 4:
Activity of p21 in
parental CHO cells and CHO cells expressing
adrenergic receptor subtypes. Confluent
P-labeled
CHO-R cells were stimulated with 1 µM insulin for 5 min.
P-Labeled CHO-2D and CHO-2B were stimulated with 10
µM epinephrine (Epi) for 5 min. Cell lysates were
immunoprecipitated with anti-p21
monoclonal
antibodies. Guanine nucleotides bound to p21
were eluted from the immunoprecipitates and separated by
thin layer chromatography as described under ``Experimental
Procedures.'' The positions of origin (Ori) and GTP and
GDP standards are indicated. The percentage of GTP bound to
p21
is calculated from the ratio of GTP/GDP
+ GTP. In CHO-R, the percent of GTP bound to p21
was stimulated by insulin to 185% of the control
value.
p21-independent activation of MAPK is not without
precedent. It has been described among others for platelet-activating
factor receptor in CHO cells(20) .
However, the finding of
stimulation of MAPK activity by all adrenergic
receptor subtypes without activation of p21
is to our
knowledge the first concerning the
receptor. It is in
contrast to the p21
-mediated activation of MAPK by the
human
and the m
muscarinic receptor
subtype in rat-1 cells (21, 34) or by the
fluoroaluminate AlF
in PC 12 cells(35) , and it
shows that activated p21
, like other signaling effectors,
can be employed by the same receptors in an alternative way depending
on the cell type(36) .
Alternatively the inability to detect
p21 activation by epinephrine in our study may mean that
a very slight, undetectable increase in the GTP-p21
is
sufficient for the activation of MAP kinase by
adrenergic subtypes in CHO cells.
We examined the possibility
of protein kinase C as a signaling pathway in the activation of p42
MAPK by epinephrine. Fig. 5demonstrates that stimulation of
protein kinase C by PMA activated MAPK in the three clones.
Furthermore, combined application of epinephrine (10 µM)
and PMA had a potent cooperative, but not additive, effect in clones
CHO-2D and CHO-2B but not in CHO-2C. These results suggest that protein
kinase C is probably involved in a different fashion in the signaling
by the three adrenergic receptor subtypes.
Figure 5: Effect of PMA pretreatment on epinephrine-induced activation of MAPK. MAPK activity was determined as described in Fig. 1in cells treated with 10 µM epinephrine (Epi) for 5 min, 100 nM PMA for 10 min, or a combination of 10 µM epinephrine and 100 nM PMA for 10 min.
In
conclusion, our results show that adrenergic receptor
subtypes expressed in CHO cells utilize differently biochemical
pathways signaling to MAPK in a p21
-independent way.