From the
The coupling of the
G protein-mediated signal transduction pathways are involved in
a wide range of biological processes, including metabolism, neuronal
activities, some of the sensory processes and hematopoietic functions,
etc. The signal transducing cascades are initiated with interactions of
ligands with receptors, followed by specific interactions between
ligand-bound receptors and G proteins and subsequent activation of
effectors by activated G proteins(1, 2) . Molecular
cloning has revealed a great deal of diversity in the molecular natures
of receptors, G proteins and effectors(3) . The intriguing
question is how the signal transduction pathways are organized so that
they can function properly and smoothly in order to meet different
needs in a variety of cellular systems. The tissue- or cell
type-specific expression of the receptors, G proteins, and effectors
and selectivity in their interactions are the key factors in
organization of the signal transduction networks. For instance, as many
as five G
Norepinephrine and epinephrine
transduce their biological information through a group of cell surface
receptors, the adrenergic receptors (AR). These receptors can be
divided into three classes,
In the present study we investigated whether
We acknowledge Dr. Allen Smrcka and Mark Betz for
reading the manuscript.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-adrenergic receptor (AR)
to the
subunits of the Gq class of G proteins was investigated in
a cotransfection system. COS-7 cells cotransfected with the
-AR cDNA and the G
or G
cDNA showed marked norepinephrine-induced increases in
accumulation of inositol phosphates in a concentration-dependent
manner. However, cells cotransfected with the cDNA encoding G
q,
G
, or G
instead of G
gave no ligand-dependent activation of phospholipase C (PLC). The
facts that the
-AR agonist isoprenaline can also induce activation
of PLC in cells coexpressing
-AR and G
and that the
-AR-specific antagonist propranolol
can block norepinephrine-induced activation of PLC in these
cotransfected cells further indicate that it is the
-AR that mediates the activation of phospholipase C in
these cotransfected cells. To test the possibility of involvement of
G
, a G
antagonist, G
3 mutant with substitution
of a Ser residue for the C-terminal Cys residue, was used because this
protein, when expressed in COS-7 cells, can inhibit only
G
-mediated but not G
-mediated activation of PLC. The
result that the G
3 mutant could not inhibit
-adrenergic receptor-mediated activation of PLC in
cells cotransfected with the G
cDNA suggests that
G
is unlikely to be a major mediator of
-adrenergic receptor-induced activation of PLC. Thus,
we conclude that the
-adrenergic receptor can
specifically couple to G
and G
, but
not to G
q, G
, or G
to activate
PLC.
subunits, including G
q, G
,
G
, G
, and G
, have
been identified as the members of the Gq class(3) . G
q and
G
were found in almost all the cells and tissues that
have been examined(4, 5) . However, expression of
G
, G
, and G
is
restrictive: G
was found most abundant in the kidney
and lung(6) , whereas G
(6) and
G
(6) were only found in hematopoietic cells.
Although all the G
subunits of the Gq class can activate
phospholipase C
(PLC)
(
)(7, 8, 9, 10, 11, 12, 13, 14) ,
they show different specificity in coupling to receptors. Although
-AR can couple to all the G
subunits of the Gq
class to activate PLC(11) , the interleukin-8 receptor can
couple only to G
, G
, and
G
, but not to G
q or
G
(15) . The similar results were also observed
for the C5a and formylmethionylleucylphenylalanine receptors: these two
receptors can couple to G
, but not to G
q and
G
(16) .
,
, and
, based on the pharmacological and molecular cloning studies, and
multiple subtypes exist in each class(17) . Most of the studies
that used the in vivo systems or transfected cells indicated
that the
-ARs couple to the G
subunits of the Gq
class to activate PLC(11) ; the
-ARs couple to
the Gi proteins to inhibit adenylyl cyclase, and the
-ARs couple
to the Gs proteins to activate adenylyl
cyclase(17, 18) . However, a few other studies suggested
that the selectivity in the interactions of these ARs with different G
proteins is rather promiscuous in certain systems. For example, two
reports revealed that
-AR could stimulate PLC activity in turkey
erythrocytes in a cAMP-independent pathway(19, 20) .
-AR
can couple to any of the G
subunits of the Gq class. We found that
-AR can specifically couple to G
and
G
, but not G
q, G
, or
G
in the cotransfection assay system.
Cell Culture, Transfection, and Enzyme
Assays
COS-7 cells were cultured in Dulbecco's modified
Eagle's medium containing 10% fetal calf serum under 5% CO at 37 °C. For transfection, COS-7 cells were seeded into
12-well plates at a density of 1
10
cells/well the
day before transfection. The media were removed the next day, and 0.5
ml of Opti-MEM (Life Technology, Inc.), containing 3.3 µg of
lipofectamine (Life Technology, Inc.) and 1 µg of plasmid DNA, was
added to each well. Five hours later the transfection media were
replaced by the culture media. Then the cells were labeled with 10
µCi/ml of myo-[2-
H]inositol or
[
H]adenosine the following day, and the levels of
inositol phosphates or cAMP were determined 1 day later as described
previously(10) . All cDNAs used in this study were constructed
in the pCMV expression vector(10) .
Binding Assays
COS-7 cells in 12-well plates were
cotransfected with the cDNAs encoding the -AR and one
of the
subunits of the Gq class. After 48 h, the cells were
washed with phosphate-buffered saline and incubated with varying
amounts of [
H]propranolol (30 Ci/mmol, DuPont
NEN) in phosphate-buffered saline for 40 min at 4 °C. Then the
cells were washed three times with ice-cold phosphate-buffered saline
and lysed in 0.5 ml of 0.2 N NaOH. Aliquots of 0.1 ml were
taken for counting in a scintillation counter. The nonspecific binding
was determined by measuring binding of
[
H]propranolol to nontransfected cells. The
number of specific binding sites were determined by the Scatchard
analysis.
-AR was found to stimulate the release of
inositol phosphates from the turkey erythrocyte membranes through
cAMP-independent and pertussis toxin-insensitive
pathways(19, 20) . To determine the signal transduction
pathway for this phenomenon, we tested whether
-AR can
couple to any of the G
subunits of the Gq class of G proteins in
the cotransfection assay. We found that cells transfected with the
-AR cDNA showed little ligand-induced accumulation of
IPs (Fig. 1), suggesting that
-AR cannot
activate endogenous Gq/11 (COS-7 cells contain endogenous G
q and
G
, but not G
, G
,
or G
(10, 11) . Cotransfection of the
cells with the G
q, G
(Fig. 1) or
G
(data not shown) cDNA did not lead to
ligand-induced activation of PLC either. This is consistent with the
previous results that
-AR was a poor activator of
G
q, and its specific agonists hardly stimulated the release of IPs
in most systems (17, 21) where G
q and G
are usually expressed. However, when cells were cotransfected
with cDNAs encoding
-AR and G
,
marked increases in ligand-induced accumulation of IPs were found. The
concentration-dependent curve indicates that the EC
is
about 2 µM of norepinephrine (Fig. 1). In addition,
cotransfection with the G
cDNA gave the same result,
suggesting
-AR can also couple to G
(data not shown). We have demonstrated previously that in this
cotransfection system recombinant proteins are usually expressed at
levels 3-10-fold higher than endogenous proteins, if there are
any(10, 11, 15, 22, 23) , and
that cells transfected with the G
cDNA expressed
G
at a level about 3-fold higher than endogenous
Gq/11(15, 22) . Furthermore, the numbers of
-AR on cells transfected with different cDNAs also
remain constant (230 fmol/10
cells). Therefore, all the
results indicated that
-AR may specifically couple to
G
and G
, but not to G
q,
G
, or G
.
Figure 1:
Coupling of -AR to
G
subunits of the Gq class. COS-7 cells were cotransfected with
the
-AR cDNA and the cDNA encoding
G
, G
, G
q, or the control
-galactosidase (LacZ). The levels of inositol phosphates were
determined 20 min after addition of norepinephrine.
To confirm that
norepinephrine-induced activation of G is mediated by
-AR, we used the
-AR-specific agonist
isoprenaline. As shown in Fig. 2, 2 µM isoprenaline
stimulated accumulation of IPs in cells coexpressing
-AR and G
. The same amount of
isoprenaline showed little effect on cells expressing
-AR or G
alone, nor did it show any
effect on cells cotransfected with the G
q or G
cDNA and the
-AR cDNA (data not shown).
Furthermore, the
-AR-specific antagonist propranolol
(10 µM) could inhibit norepinephrine-induced activation of
PLC in the cells coexpressing G
and
-AR. Since
-AR is known to activate
adenylyl cyclases through the Gs proteins, increases in the cAMP levels
may indirectly activate the recombinant G
protein. To
test this possibility, we added forsklin to the cells transfected with
the cDNA encoding G
. Forsklin showed little effect on
the levels of IPs (data not shown), suggesting that
-AR-induced activation of G
is not
mediated by adenylyl cyclases. We also excluded the possibility that
coexpression of G
may change G protein-coupling
specificity of
-AR, because
-AR can
still stimulate accumulation of cAMP in the presence of G
(data not shown). Therefore, norepinephrine-dependent activation
of PLC in cells coexpressing G
and
-AR is
-AR-specific and cAMP-independent.
Figure 2:
Coupling
between -AR and G
. COS-7 cells were
transfected or cotransfected with cDNAs encoding G
,
LacZ, and/or
-AR as indicated in the figure.
Ligand-induced accumulation of inositol phosphates was determined 20
min after addition of norepinephrine (Nor, 2.5
µM), isoprenaline (Iso, 2 µM),
and/or propranolol (Pro, 10 µM), as indicated.
We
have observed previously that COS-7 cells cotransfected with
G and G
showed a synergistic increase in
the level of IPs over those transfected with G
or
G
alone(22) . Thus, it is possible that
-AR-mediated activation of PLC in cells cotransfected
with the G
cDNA may result from the synergistic
effect between G
and the G
subunits that
are released from
-AR-activated Gs proteins. To test
this possibility, we used a dominant negative inhibitor of G
,
G
3CS, with substitution of a Ser residue for the C-terminal Cys
residue. We have previously demonstrated that G
3CS could inhibit
C5a- or formylmethionylleucylphenylalanine-induced activation of PLC
, which is mediated by G
; but G
3CS did
not have any effect on m1-muscarinic receptor-mediated activation of
PLC, which is mediated by the Gq proteins.
(
)In
this report we demonstrated that G
3CS was also able to inhibit
interleukin-8-induced activation of PLC
(Fig. 3A), which is mediated by G
(15) without affecting m1-muscarinic receptor-mediated effect (Fig. 3B). These findings indicate that G
3CS can
serve as a specific antagonist for G
-mediated effects. To
test whether G
is directly involved in
-AR-mediated activation of PLC, COS-7 cells were
cotransfected with the G
and
-AR
cDNAs and the G
3CS cDNA. As shown in Fig. 3C,
coexpression of G
3CS had no effect on norepinephrine-induced
activation of PLC in cells coexpressing
-AR and
G
, suggesting that the G
subunits released
from Gs do not play a significant role in
-AR-mediated
activation of PLC and that
-AR directly couples to
G
.
Figure 3:
Effects of
G on
-AR-mediated activation of PLC. COS-7
cells were cotransfected with various cDNAs, including
-galactosidase (LacZ), G
3CS, G
, and
-AR, as indicated in the figure.
Norepinephrine-induced accumulation of IPs was determined 20 min after
addition of ligand (2 µM of norepinephrine).
We have provided clear evidence by the
cotransfection assay for the notion that -AR couples
to G
. The next question is whether the coupling
exists in natural systems. Thus, we tested whether isoprenaline can
induce accumulation of IPs in THP-1 cells. THP-1 is a human
promonocytic leukemia cell line, and
-AR on these
cells has been well characterized(24, 25) . This cell
line also contains G
(6) . We found that
isoprenaline (10 µM) clearly stimulated accumulation of
inositol phosphates in THP-1 cells (Fig. 4), thus providing
evidence for the natural occurrence of the coupling between
-AR and G
. In addition,
-AR was previously found to stimulate PLC activity in
the erythrocytes(19, 20) . Since both G
(6) and
-AR (19, 20) were found
in the erythrocytes, this result may provide another example for
-AR-mediated activation of PLC in natural
systems(19, 20) .
Figure 4:
-AR-induced activation of
PLC in THP-1 cells. THP-1 cells were labeled with
[
H]inositol for 1 day and then the levels of
inositol phosphates were determined 30 min after addition of
isoprenaline (10 µM). The dark bars represent
cells that were treated with pertussis toxin (500 µg/ml) for 4 h
immediately before addition of the ligand. The asterisks represent p < 0.01.
G,
G
, and
-AR were all found in a
variety of hematopoietic cells. G
was detected in
many erythroids, T cell and myeloid lineage(6) , whereas
G
was detected in a number of B cell as well as
myeloid lineages(26) .
-AR was also found in
many T and B cells as well as myeloid
cells(27, 28, 29, 30, 31, 32, 33) .
The specific coupling of
-AR to G
and G
suggests that norepinephrine and
epinephrine may not only activate adenylyl cyclases, but also activate
PLC in many of the hematopoietic systems. In addition, our findings may
also explain some of the
-AR-mediated effects in certain
hematopoietic systems that previously could not be completely
interpreted by increases in the levels of cAMP(34) . The
specific expression of G
and G
and
the inability for
-AR to couple to G
q,
G
, and G
prevent the
-AR ligands from activating PLC in most systems. This
may serve as an example for how the signal transduction pathways are
organized by both tissue-specific expression and selectivity in
receptor-G protein interactions.
-AR appears to
play important roles in the hematopoietic systems, and it provides a
link between the neuroendocrine system and the immune
system(35, 36, 37, 38) . The expression
levels of
-AR in leukocytes vary in some pathological
conditions, such as asthma(39) , rheumatoid arthritis, and
Crohn' s disease(40) . Most previous research was directed
at cAMP, which is certainly one of the messengers downstream of the
receptor. However, activation of PLC by
-AR through
G
should also be investigated as a possible signal
transduction pathway for
-AR-mediated effects in
hematopoietic systems. It would be interesting to understand why
-AR couples to two signal transduction pathways in the
hematopoietic system and what the precise physiological function of
-AR-mediated activation of PLC is.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.