From the
The murine G-protein
A wide variety of hormones and neurotransmitters regulate
cellular functions by binding to transmembranous receptors which couple
to and activate heterotrimeric guanine nucleotide binding proteins
(G-proteins).
The G
In order to study the interaction of different receptors and
G-proteins of the G
In this paper, we demonstrate that G
The receptors shown to activate
G
The chemokine receptors for C5a and
interleukin 8 have been shown to activate G
Since a variety of receptors
can activate G
The fact that G
We thank Drs. Michael J. Brownstein, Marc G. Caron,
Shaun R. Coughlin, Masakazu Hirata, Robert J. Lefkowitz, Ernest G.
Peralta, Peter R. Schofield, and Lei Yu for providing expression
plasmids of receptors.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-subunit G
and its
human counterpart G
are expressed in a subset of
hematopoietic cells, and they have been shown to regulate
-isoforms of inositide-specific phospholipase C. We studied the
ability of a variety of receptors to interact with G
and G
by cotransfecting receptors and G-protein
-subunits in COS-7 cells. Activation of
adrenergic and muscarinic M
receptors in cells
expressing the receptors alone or together with G
,
G
, or G
led to a very small
stimulation of endogenous phospholipase C. However, when the receptors
were coexpressed with G
and G
,
addition of appropriate ligands caused a severalfold increase in
inositol phosphate production which was time- and dose-dependent. A
similar activation of phospholipase C was observed when several other
receptors which were previously shown to couple to members of the
G
and G
family were coexpressed with
G
. In addition, stimulation of inositol phosphate
formation via receptors naturally coupled to phospholipase C was
enhanced by cotransfection of G
and
G
. These data demonstrate that G
and G
are unique in that they can be activated
by a wide variety of G-protein-coupled receptors. The ability of
G
and G
to bypass the selectivity
of receptor G-protein interaction can be a useful tool to understand
the mechanism of receptor-induced G-protein activation. In addition,
the promiscuous behavior of G
and G
toward receptors may be helpful in finding ligands corresponding
to orphan receptors whose signaling properties are unknown.
(
)Receptor-activated G-proteins
then regulate different cellular effectors, such as specific enzymes
and ion channels(1, 2, 3, 4) . Sixteen
mammalian genes encoding G-protein
-subunits, which define the
individual G-proteins, have been identified, and they have been grouped
into four families, G
, G
,
G
, and G
, according to sequence
homologies(5) . Many of the factors which determine the
specificity of G-protein-mediated signal transduction are still
unknown. Nonetheless, the selective coupling of an activated receptor
to a distinct pattern of G-proteins is regarded as an important
requirement to achieve accurate signal transduction. For example,
receptors which upon activation lead to stimulation of adenylyl cyclase
primarily couple to G
, whereas the receptor-mediated
pertussis toxin-insensitive activation of phospholipase C is due to the
coupling of receptors to members of the G
family(6, 7, 8) .
family consists of five members whose
-subunits show
different expression patterns. Whereas G
and
G
, which are 88% identical, seem to be almost
ubiquitously expressed and are primarily responsible for coupling
receptors in a pertussis toxin-insensitive manner to phospholipase C
-isoforms(7, 8, 9) , the expression of
G
, which is 81% identical with G
, is
more restricted(10) . The human G
and its
murine counterpart G
are only expressed in a subset
of hematopoietic cells(10, 11) . G
and
G
, which are 85% identical, have been placed into the
G
family since their sequences show the highest similarity
toward G
(57%). All five members of the G
family share functional properties, i.e. they can
regulate the
-isoforms of phospholipase C (12-14). Purified
G
, G
, and G
indistinguishably activate different isoforms of phospholipase
C-
in a reconstituted system(15, 16) . Recent data,
however, demonstrate that receptors for interleukin 8 and C5a interact
selectively with G
but not with G
and
G
(17, 18, 19) , demonstrating that
there are differences among members of the G
family with
regard to receptor interaction. In the present study, we show that a
wide variety of structurally and functionally different receptors
couple to G
and G
but not to other members of
the G
family, indicating that G
and G
are unique, i.e. they possess the ability to
nonselectively couple a large variety of receptors to phospholipase C.
Materials
Carbachol, isoproterenol, dopamine,
8-hydroxy-2-(di-n-propylamino)tetralin, thrombin, serotonin,
[Arg]vasopressin,
[D-Ala
,N-MePhe
,Gly
-ol]enkephalin,
11
,9
-epoxymethano-PGH
(U46619), and N-formyl-Met-Leu-Phe were from Sigma. CGS-21680 was from RBI
(Natick, MA) and 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (Ro
20-1724) was from Life Technologies, Inc.
Transient Transfection and Labeling of COS-7
Cells
COS-7 cells were cultured as described(13) . For
transfection experiments, cells were seeded in 24-well plates at a
density of 4 10
cells per well and grown overnight.
Cells were then washed with phosphate-buffered saline, and 0.4 µg
of DNA mixed with 2 µl of lipofectamine (Life Technologies, Inc.)
in 0.25 ml of Opti-MEM was added to each well. In cotransfection
experiments with two different plasmids, 0.2 µg of each plasmid was
added. In control experiments, the total amount of DNA was maintained
constant by adding DNA from a vector encoding
-galactosidase.
After 5 h at 37 °C, 0.25 ml of DMEM containing 20% (v/v) fetal
bovine serum was added to each well. About 24 h after transfection,
cells were labeled for 20-24 h with 120 pmol of myo-[2-
H]inositol (758.5 GBq/mmol; Du
Pont NEN) per well as described(13) .
Determination of Inositol Phosphate Levels
Labeled
cells were washed with phosphate-buffered saline and then incubated for
10 min at 37 °C with 0.25 ml of inositol-free DMEM containing 10
mM LiCl. Thereafter, medium was aspirated, and the indicated
agents were added in DMEM containing 10 mM LiCl. Inositol
phosphate formation was stopped after 20 min by removing the medium and
adding 0.2 ml of 10 mM ice-cold formic acid. After keeping the
samples on ice for 20 min, 0.45 ml of 10 mM NHOH
was added, and the whole sample was loaded onto a column containing
0.75 ml of anion exchange resin (AG 1-X8; Bio-Rad) equilibrated with 5
mM borax and 60 mM sodium formate. Total inositol
phosphates were then separated and measured as described(20) .
If not stated otherwise, measurements were done in triplicate
representing three independently transfected wells.
Determination of Cellular cAMP Levels
For
determination of cAMP levels, cells were grown and transfected in
24-well plates as described. 48 h after transfection, cells were
preincubated for 15 min with DMEM containing 300 µM
3-isobutyl-1-methylxanthine and 20 µM
4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (Ro 20-1724).
Thereafter, medium was replaced by DMEM containing both
phosphodiesterase inhibitors and the indicated concentrations of
ligands. At the end of this treatment (20- min incubation time), the
reaction was stopped by aspiration of the medium and addition of 150
µl of ice-cold 10% (w/v) trichloroacetic acid. Samples were kept
for 10 min on ice, and 90 µl of 1 M Tris (pH 9.8) was
added to neutralize the sample. cAMP was determined by the competitive
binding assay(21, 22) . Briefly, 100 µl of the
sample were incubated for 2 h with 2 pmol of
[8-H]cAMP (925 Gbq/mmol; Amersham) and 62.5
µg of cAMP-dependent protein kinase purified from porcine heart
(Sigma) in a final volume of 200 µl at 4 °C. Thereafter, 100
µl of 4% (w/v) charcoal in 5 mM EDTA and 50 mM Tris-HCl (pH 7.5) was added, and samples were immediately
centrifuged for 2 min at 12,000
g. To determine the
amount of [8-
H]cAMP bound to the binding protein,
supernatants were counted in a liquid scintillation counter. Each
experiment was calibrated by running a set of cAMP standards along with
the unknown test sample. For the standard samples, the log of total
counts/min bound was plotted versus the log of total cAMP per
sample (labeled plus unlabeled), and the amount of cAMP in the test
sample was calculated from the resulting standard curve(21) .
Assays were done in triplicate representing three independently
transfected wells.
COS Cell Expression Vectors
cDNAs corresponding to
G-protein -subunits G
, G
,
G
, G
, and G
were
carried by the cytomegalovirus vector
pCIS(12, 13, 17, 19, 20) . A
-galactosidase construct inserted into pCIS was used as a
transfection control. cDNAs encoding the human muscarinic
M
, the human
adrenergic, the murine
5-HT
, and the human thrombin receptors were in the
vector pCIS. The human dopamine D
construct was in pCMV5
(23), the rat µ opioid receptor and the human adenosine A
receptor encoding cDNAs were in pRc/CMV (Invitrogen), the human
vasopressin V
receptor and the human fMLP receptor cDNAs
were in the vector pcDNAI/Amp (Invitrogen), the human 5-HT
receptor construct was in pSVL (Pharmacia Biotech Inc.), the cDNA
encoding the human vasopressin V
receptor was carried by
the pcD3 vector(24), and the human thromboxane A
receptor
cDNA was in pCDM8 (Invitrogen).
family, cDNA clones encoding receptors
and G-protein
-subunits were transiently cotransfected into COS-7
cells, and inositol phosphate production in response to receptor
ligands was measured. First, we tested the
adrenergic
and the M
muscarinic receptor, which have been shown to
couple primarily to G
and G
, respectively
(25-28), to determine if they can mediate ligand-dependent
inositol phosphate production in COS-7 cells (Fig. 1). When both
receptors were expressed alone, there was a slight increase in the
inositol phosphate production in response to the respective ligands. In
the case of the muscarinic M
receptor, this increase could
be blocked by pretreatment of cells with pertussis toxin and is
presumably mediated by
-subunits of
G
(29) . Cotransfection of G-protein
-subunits
G
, G
, and G
, all
of which have been shown to be expressed at high levels and to mediate
receptor-dependent phospholipase C activation in COS cells (12, 13, 17,
19, 20), slightly increased the basal inositol phosphate production,
but had no effect on the ligand-dependent increase in inositol
phosphate formation. In contrast, ligand-dependent inositol phosphate
production was severalfold enhanced when both receptors were
cotransfected with G
and G
(Fig. 1).
Figure 1:
Accumulation of inositol phosphates in
COS-7 cells that coexpress the adrenergic or the
M
muscarinic receptor and G
subunits. COS-7 cells were
cotransfected with cDNAs encoding the the
adrenergic (left panel) or the M
muscarinic receptor (right panel) and cDNAs encoding
-galactosidase (lz) or
-subunits of the G
family,
G
(
), G
(
), G
(
),
G
(
), and G
(
) as described under ``Experimental
Procedures.'' After 48 h,
[
H]inositol-labeled cells were incubated in the
absence (-; open bars) or presence (+) of 10
µM isoproterenol (closed bars, left
panel) and 10 µM carbachol (carb.; closed bars, right panel) for 20 min, and levels of
inositol phosphates were determined as described. Cells which were
cotransfected with the M
muscarinic receptor cDNA and
G
subunit cDNAs were processed as described (-PT)
or were pretreated with 100 ng/ml pertussis toxin (+PT)
for 18 h prior to incubation with ligand. Shown are mean values of
triplicates ± S.D.
Fig. 2shows that the adrenergic or the M
muscarinic receptor-mediated
increase in inositol phosphate production in cells cotransfected with
G
or G
was linear with time for at
least 30 min. Isoproterenol- and carbachol-induced inositol phosphate
formation in cells transiently expressing G
or
G
and the corresponding receptors was
concentration-dependent (Fig. 3). Half-maximal and maximal
effects of carbachol were observed at concentrations of 0.1 and
1-10 µM, whereas isoproterenol-stimulated inositol
phosphate production was half-maximal and maximal at concentrations of
0.3 and 10 µM, respectively. We then tested the ability of
isoproterenol and carbachol to stimulate inositol phosphate formation
in the presence of G
and G
in order
to test if the ligand concentration dependence was in the same range as
the dosage required to regulate the natural effector target of their
respective receptors. Therefore, in a set of parallel experiments we
measured the effect of increasing concentrations of both ligands on the
cellular cAMP content in COS-7 cells transfected with the
or the M
receptor cDNAs. Isoproterenol induced an
increase in the intracellular cAMP content in cells expressing the
receptor, and the extent of this effect was slightly
increased in cells coexpressing G
. In order to study
the M
receptor-mediated decrease in intracellular cAMP,
cells were cotransfected with the muscarinic M
receptor and
the
adrenergic receptor. When cAMP levels were
increased through the stimulation of the
receptor, a
carbachol-dependent decrease of the cAMP content could be observed. In
both cases, effects mediated by the
and the M
receptor were dose-dependent and occurred with a very similar
dose-response relationship as the
G
/G
-dependent stimulation of
phospholipase C via both receptors (Fig. 3). This shows that the
activated
and the M
receptor regulated
the intracellular cAMP content in COS-7 cells with a very similar
efficacy as they induced inositol phosphate formation when
cotransfected with G
and G
into
COS-7 cells.
Figure 2:
Time course of the
adrenergic and M
muscarinic receptor-mediated inositol
phosphate formation in COS-7 cells. COS-7 cells were cotransfected with
cDNAs encoding the
adrenergic (left panel)
or the M
muscarinic receptor (right panel) and
cDNAs encoding
-galactosidase (
, lz) or G
(
,
), G
(
,
), and G
(
,
) as described under ``Experimental
Procedures.'' Cells were incubated in the absence or presence of
10 µM isoproterenol (left panel) and 10
µM carbachol (right panel) for the indicated time
periods (abscissa), and released inositol phosphates were
measured as described. Shown is the ligand-dependent inositol phosphate
release, and data points represent mean values of triplicates ±
S.D.
Figure 3:
Accumulation of inositol phosphates and
cAMP in COS-7 cells transfected with the adrenergic
or M
muscarinic receptor cDNAs. A and C, COS-7 cells were cotransfected with cDNAs encoding the
adrenergic (A) or the M
muscarinic receptor (C) and cDNAs encoding
-galactosidase (
, lz) or
-subunits of the G
family, G
(
,
), G
(
,
), G
(
,
),
G
(
,
), and G
(
,
) as described under
``Experimental Procedures.'' Cells were then incubated with
the indicated concentrations of isoproterenol (A) and
carbachol (C), and the ligand-dependent inositol phosphate
formation was determined as described. B, COS-7 cells were
cotransfected with cDNAs encoding the
adrenergic
receptor and
-galactosidase (
, lz) or G
(
,
). Cells were incubated at increasing
concentrations of isoproterenol for 20 min, and cellular cAMP content
was determined as described under ``Experimental
Procedures.'' D, COS-7 cells were cotransfected with
cDNAs encoding the the
adrenergic and M
muscarinic receptor (0.2 µg of each per well). Transfected
cells were incubated with 2 µM isoproterenol and the
indicated concentrations of carbachol (abscissa) for 20 min,
and the carbachol-induced decrease in cellular cAMP content was
measured as described. Basal cAMP levels (in the absence of any ligand)
were 1.2 ± 0.2 pmol per well. Values are mean values ±
S.D.
To further determine the spectrum of receptors able to
activate G and G
, we cotransfected COS-7
cells with cDNAs of a variety of different receptors alone or together
with G
, G
, G
,
G
, or G
. We then measured the
effect of increasing agonist concentrations on inositol phosphate
formation. Fig. 4shows the results obtained with three receptors
which are naturally coupled to the stimulation of adenylyl cyclase, the
vasopressin V
, the dopamine D
, and the
adenosine A
receptor(23, 30, 31, 32) . When
these receptors were expressed alone, a small dose-dependent increase
in the inositol phosphate formation could be observed. In cells
coexpressing the receptors and G
, G
,
and G
, the ligand-dependent inositol phosphate
production was the same as in cells transfected with the receptors
alone. However, when the vasopressin V
, the dopamine
D
, and the adenosine A
receptors were
coexpressed with G
or G
, the
ligand-dependent inositol phosphate formation increased severalfold.
Figure 4:
Formation of inositol phosphates in COS-7
cells coexpressing vasopressin V, dopamine D
,
or adenosine A
receptors and G-protein
-subunits.
COS-7 cells were cotransfected with cDNAs encoding the dopamine D
receptor (A), the vasopressin V
(B), or the adenosine A
receptor (C) and cDNAs encoding
-galactosidase (
, lz) or
-subunits of the G
family, G
(
,
), G
(
,
), G
(
,
),
G
(
,
) and G
(
,
) as described under
``Experimental Procedures.'' Cells were then incubated with
the indicated concentrations of dopamine (A),
[Arg
]vasopressin (AVP; B), and
CGS-21680 (C), and the ligand-dependent inositol phosphate
formation was determined as described. Shown are mean values of
triplicates ± S.D.
We then tested the ability of G and G
to couple receptors, which activate G
and G
proteins, to the production of inositol phosphates (Fig. 5). The µ opioid receptor, the 5-HT
receptor, and the fMLP receptor, which have been shown to
activate G-proteins of the G
family(33, 34, 35) , also mediated a small
increase in the formation of inositol phosphates when expressed alone.
No significant increase in the ligand-dependent inositol phosphate
formation was observed when the receptors were coexpressed with
G
or G
. Coexpression of the fMLP or
the 5-HT
receptor but not the µ opioid receptor and
G
slightly increased the ligand-dependent inositol
phosphate production. Ligand-induced release of inositol phosphates was
again markedly increased when the receptors were coexpressed with
G
and G
, indicating that the
receptors can couple to G
and G
,
but not to G
or G
.
Figure 5:
Accumulation of inositol phosphates in
COS-7 cells cotransfected with cDNAs encoding the 5-HT,
the fMLP or the µ opioid receptor, and G-protein
-subunits.
COS-7 cells were cotransfected with cDNAs encoding the 5-HT
(A), the fMLP (B), or the µ opioid receptor (C) and cDNAs encoding
-galactosidase (
, lz) or
-subunits of the G
family, G
(
,
), G
(
,
), G
(
,
),
G
(
,
), and G
(
,
) as described under
``Experimental Procedures.'' Cells were then incubated with
increasing concentrations of
8-hydroxy-2-(di-n-propylamino)tetralin (A), fMLP (B), and
[D-Ala
,N-MePhe
,Gly
-ol]enkephalin (C), and the ligand-dependent inositol phosphate formation was
determined as described. Shown are mean values of triplicates ±
S.D.
To prove that
receptors which activate phospholipase C in a pertussis
toxin-insensitive manner by coupling to G can also
activate G
and G
, we expressed the
thrombin, the thromboxane A
, the vasopressin
V
, and the 5-HT
receptors(24, 36, 37, 38) together with G
, G
,
G
, G
, and G
in
COS-7 cells (Fig. 6). Since COS cells express G
and G
(13) , the effects of cotransfected
G
family members on the inositol phosphate formation
mediated by phospholipase C-linked receptors is much weaker, as the
receptors can interact with endogenous G
and
G
. Nevertheless, the tested phospholipase C-coupled
receptors mediated the stimulation of inositol phosphate production,
and the ligand-dependent portion could be significantly increased when
the receptors were coexpressed with G
family members,
including G
and G
.
Figure 6:
Formation of inositol phosphates in COS-7
cells coexpressing thromboxane A, 5-HT
,
vasopressin V
or thrombin receptors, and G-protein
-subunits. COS-7 cells were cotransfected with cDNAs encoding the
thromboxane A
receptor (A), the 5-HT
(B), the vasopressin V
receptor (C), or the thrombin receptor (D) and cDNAs encoding
-galactosidase (lz) or
-subunits of the G
family, G
(
), G
(
), G
(
),
G
(
), and G
(
) as described under ``Experimental
Procedures.'' Cells were then incubated in the absence or presence
of 3 µM U46619 (A), 10 µM serotonin (B), 1 µM [Arg
]vasopressin (C), or 3 units/ml thrombin (D), and the
ligand-dependent inositol phosphate formation was determined as
described. Shown are mean values of triplicates ±
S.D.
Thus,
different receptors which couple to G, G
, and
G
family members can be functionally linked to endogenous
phospholipase C when coexpressed with G
and
G
in COS cells, indicating that they all activate
G
and G
.
and
G
are capable of coupling a variety of receptors to
the stimulation of inositol phosphate formation when coexpressed with
the receptor in COS-7 cells. Receptors which are not linked to a
pertussis toxin-insensitive regulation of phospholipase C under
physiological conditions mediated little increase in inositol phosphate
formation when expressed alone. This increase was not altered by
coexpression of G
and G
. In some
cases, coexpression of G
led to a slight increase of
the basal ligand-dependent inositol phosphate formation (Fig. 5).
All receptors tested gained the ability to mediate a severalfold
increase in the inositol phosphate production when they were
coexpressed with G
and G
,
indicating that they functionally interacted with G
and G
. In addition, the inositol phosphate
formation mediated by several receptors which are physiologically
linked to phospholipase C in a pertussis toxin-insensitive manner,
could be increased by coexpression of G
,
G
, G
, G
, and
G
. Thus, these receptors were able to act through all
the transfected G
subunits.
and G
represent a wide spectrum
of structurally and functionally different ligand binding proteins,
demonstrating that G
and G
can be
activated by a wide variety of receptors which serve very different
functions under physiological conditions. This appears to be a unique
feature of G
; other G-proteins usually are
selectively activated by a defined spectrum of receptors. A certain
degree of specificity in the receptor G-protein interaction is regarded
as a prerequisite for proper signal transduction. Thus, a G-protein
which can nonselectively link functionally different receptors in a
given cell to the same effector would be thought to produce
inappropriate signaling and block specific intracellular information
processing pathways. However, G
and G
exhibit very restricted expression patterns. Expression has only
been shown in a subset of hematopoietic cells, and especially at
premature stages in different cell lineages(11, 39) .
Therefore, coupling of most of the tested receptors to G
and G
in the COS cell system is presumably
without direct physiological significance, since many of these
receptors and G
and G
may not be
coexpressed in vivo.
, but not
G
and
G
(17, 18, 19) , and they are
known to be expressed in mature cells of the immune system, especially
in leukocytes. Therefore, they have been implicated in the
physiological regulation of G
activity. Whereas
chemokine receptors for interleukin 8, C5a, and fMLP can undoubtedly
couple to G
and G
(17-19, Fig. 5), our current work indicates that this ability is not
restricted to this receptor class, but rather due to the unique
properties of G
. In addition, expression of
chemokine receptors and G
seems to be regulated in
a reciprocal manner during leukocyte development. For example, in
undifferentiated HL-60 cells which express high levels of
G
, chemokine receptors are absent or at low levels,
whereas chemokine receptor expression increases and the expression of
G
dramatically decreases during differentiation of
HL-60 cells(11, 40, 41, 42) . The
effects of chemokine receptors in myeloid cells are mainly pertussis
toxin-sensitive(43, 44, 45) , indicating that
they are mediated by G
-type G-proteins. Recently it has
been shown that the fMLP receptor primarily functions through pertussis
toxin-sensitive G-proteins even when stably expressed in
undifferentiated HL-60 cells where G
is
present(46) . Chemokine receptors probably interact with
G
and G
under some conditions in vivo; it is, however, tempting to speculate that the main
receptors physiologically coupled to G
and
G
are still to be identified. They might be involved
in the regulation of growth and differentiation of hematopoietic cells,
and G
may allow the coupling of diverse receptors
to the stimulation of phospholipase C.
and G
while being
unable to activate G
and G
, the
question arises of which structural determinants of G
and G
are responsible for their ability to
become activated by many different receptors. The carboxyl-terminal 55
amino acids of G
and G
are most
divergent from G
, G
, and
G
(9, 10, 11) . This region
includes the very carboxyl terminus of the
-subunit which has been
shown to affect receptor specificity(47, 48) .
Interestingly, G
and G
possess a
unique insert of several amino acids including different charged
residues between helix
4 and helix
5 which are just adjacent
to a region homologous to residues 311-329 of
transducin(10, 11, 49) . This region of
transducin has also been implicated in the interaction with
receptor(50) . However, the situation appears to be more
complicated as shown by a recent study in which different chimeras
between G
and G
were examined for
their ability to interact with the C5a receptor, which couples to
G
but not to G
(51) . This
study suggests that the carboxyl-terminal 133 amino acids of
G
do not alone account for its ability to interact
with the C5a receptor, and that multiple regions of G
are responsible for the functional difference between
G
and G
, including a segment which
comprises residues 220-240 of G
. Thus, there
are obviously many domains of G
and G
which directly or indirectly may affect the structure of
G-protein and in this way may modulate the specificity of receptor
G-protein interaction.
and
G
interact with a wide variety of receptors can be
useful for understanding the molecular details of the receptor
G-protein interaction, once more structural data pertaining to
receptors and different G-protein
-subunits are available. In
addition, G
promiscuity may facilitate the
examination of orphan receptors whose ligands and signal transduction
properties are unknown. Cotransfection of orphan receptors and
G
or G
into COS cells and
subsequent determination of phospholipase C activity can be a way to
search for ligands independent of the physiological signaling
properties of the receptor.
,9
-epoxymethano-PGH
.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.