(Received for publication, November 28, 1994; and in revised form, January 11, 1995)
From the
Differentiated HL-60 cells acquire responsiveness to
fMet-Leu-Phe (fMLP), which activates phospholipase C and
O generation in a pertussis
toxin-sensitive manner. Addition of retinoic acid (RA) for the last 24
h during dimethyl sulfoxide (Me
SO)-induced differentiation
enhanced fMLP-dependent signals and interaction between fMLP receptor
and G
. RA modifies both the function and subunit
composition of G
, the predominant G
of HL-60
membranes, as shown by comparing purified G
from membranes
of Me
SO-treated cells (D-G
) to G
from membranes of cells treated with both Me
SO and RA
(DR-G
). As compared to D-G
, DR-G
induced more fMLP binding when added to membranes of pertussis
toxin-treated HL-60 cells and, in the presence of GTP
S, stimulated
-sensitive phospholipase C in extracts of HL-60 cells to a
much greater extent and at lower concentrations. Immunoblots revealed
that RA induced expression of the
subunit, which was
otherwise undetectable in G
purified from HL-60 cells or
in HL-60 membranes. Possibly by inducing expression of
, RA alters two functions of the G
subunit, modulation of fMLP receptor-G
coupling and activation of the effector, phospholipase C.
Heterotrimeric () G proteins are GTP-dependent
molecular switches that relay signals from cell surface receptors to
effector enzymes and ion channels(1) . G proteins consist of
two functional subunits,
and
. Historically
subunits were assumed to transmit primary signals, while
subunits were thought to regulate or terminate signals and to be
interchangeable among G proteins(2) . Recently, however,
accumulating evidence has shown that
subunits can directly
regulate activities of many effectors, including adenylyl
cyclases(3, 4) , phospholipase C
(PLC
)(
)(5, 6) , certain K
channels(7, 8) , and PI3 kinase(9) , and
that
can mediate hormonal stimulation of the
mitogen-activated protein kinase pathway (10) . Moreover,
discovery of multiple
(
) and
(
) subunits (11, 12, 13, 14, 15) suggested
that combinations of different
and
gene products might
perform different specific functions. Indeed, experiments with
antisense oligonucleotides suggest that specific
and
subunits may determine the specificity of interactions between G
proteins and receptors(16, 17) .
One of the
signaling systems in which , rather than
, stimulates
the effector is the G
-mediated activation of PLC
by
fMet-Leu-Phe (fMLP) in differentiated HL-60 cells(18) . Several
agents, including Me
SO and RA (19) induce HL-60
cells to differentiate into neutrophil-like cells. One result of this
differentiation program in HL-60 cells is that PLC
and the
machinery for generating O
become
responsive to stimulation by fMLP; both responses can be blocked by
pertussis toxin (PTX) (20, 21) and are mediated by
G
(22) , possibly by both G
and
G
, although the former predominates in HL-60
cells(23, 24) .
, rather than
, is thought to activate PLC
, based on the
observations that
can activate PLC
isoenzymes (5, 6) and that no known PLC
can be stimulated by
(25) .
The mechanism by which
differentiation factors allow fMLP-dependent activation of PLC is not
known. Possible targets of these agents include the fMLP receptor,
G, and PLC
. Differentiation of HL-60 cells induced by
Me
SO or dibutyryl cAMP (Bt
cAMP) is associated
with increases in the number of fMLP-binding sites (26, 27, 28, 29, 30) and in
the G
content of
membranes(23, 28, 31) . In surprising
contrast, differentiation induced by RA alone is not associated with
detectable increases in either fMLP-binding sites, fMLP receptor
transcripts(26, 27, 29, 32) , or
G
content, (
)although it clearly increases
responses to fMLP. Thus, the mode of action of RA differs from that of
Me
SO.
Here we report studies of the potentiation by RA
of the MeSO-induced fMLP-dependent activation of PLC
in HL-60 cells. This potentiation occurs, at least in part, via an
effect on G
. RA enhances both the interaction of G
with fMLP receptors and its ability to activate
-sensitive PLC
. RA treatment increases expression of a
specific
polypeptide,
, which may be responsible
for both changes in the function of G
.
For the reconstitution of GTPS-sensitive fMLP
binding in HL-60 cell membranes by addition of purified G
,
the membranes were prepared from differentiated HL-60 cells which had
been cultured in the presence of 50 ng/ml PTX for 24 h. The membranes
from PTX-treated cells were incubated on ice for 15 min with indicated
amounts of purified G
and then assayed for fMLP binding
with 80 nM [
H]fMLP, as
described(34) .
Phospholipase C activity was measured using sonicated
micelles of 50 µM [inositol-2-H]phosphatidylinositol-4,5-bisphosphate
(20,000 counts/min/tube) and 450 µM phosphatidylethanolamine in a solution containing 50 mM MES-NaOH, pH 7.0, 1.5 mM MgCl
, 3 mM EGTA, and 1.2 mM CaCl
(to give 0.3 µM free Ca
)(37) .The final concentration of
sodium cholate was 0.08%. Reactions were terminated and released IP3
was quantitated as described(38) . Before the assay, G
was activated by incubation with 10 µM GTP
S in
a reaction mixture containing 10 mM Tris-HCl, pH 7.5, 10%
glycerol, 0.5% cholate, and 10 mM MgCl
at 30
°C for 30 min.
Figure 1:
Effects of
RA or MeSO on fMLP-dependent O
generation or PLC activation in intact HL-60 cells. A,
cells were exposed to RA alone (1 µM), Me
SO
alone (1.4%), or Me
SO (DMSO) (1.4%) for the
indicated number of days plus RA (1 µM) during the last 24
h of treatment. Cells (4
10
) were then incubated
with 1 µM fMLP for 10 min at 37 °C, and
O
generation was measured as described
under ``Experimental Procedures.'' Bars in panel
A represent the mean ± S.E. of four determinations. B, cells were treated with Me
SO alone (1.4%) for 5
days (D) or with Me
SO (1.4%) for 5 days plus RA (1
µM) during the last 24 h of treatment (DR). Cells
(5
10
) were then incubated with 1 µM fMLP for 1 min at 37 °C, and production of IP
was
measured as described under ``Experimental Procedures.'' Bars represent the mean ± S.E. of three
determinations.
Like the binding of many agonists to
G-coupled receptors, fMLP binding is diminished in the
presence of GTP
S(35, 44) ; presumably, this is
because receptor-G
complexes have a higher binding affinity
for agonists, and binding of GTP
S to
promotes
dissociation of G
from receptors. D and DR membranes
contained apparently equal numbers of fMLP-binding sites, as measured
in the presence of GTP
S, while the number of sites sensitive to
GTP
S was greater in DR than in D membranes (Fig. 2, A and B). The fMLP-binding sites that disappear in the
presence of GTP
S are assumed to represent fMLP receptors that
would be coupled to G
in the absence of the guanine
nucleotide, while the GTP
S-insensitive-binding sites are thought
to represent receptors uncoupled from G
. Following this
interpretation, the data in Fig. 2, A and B,
show that treatment with Me
SO plus RA enhanced coupling of
fMLP receptors to G
more than did treatment with
Me
SO alone.
Figure 2:
fMLP binding (A and B)
and fMLP-stimulated GTP hydrolysis (C) in membranes of
MeSO (D) or Me
SO plus RA (DR)-treated HL-60 cells. A, D (circles) and
DR (triangles) membranes (20 µg) were incubated at 25
°C for 20 min in 50 µl of a reaction mixture containing the
indicated concentration of [
H]fMLP. The specific
binding was measured in the presence (open symbols) or in the
absence (closed symbols) of 10 µM GTP
S, as
described under ``Experimental Procedures.'' B,
GTP
S-sensitive fMLP binding, estimated as the difference between
the two specific binding curves performed in the presence and absence
of GTP
S, depicted in panel A. C, D (circles) and DR (triangles) membranes (10 µg)
were incubated at 30 °C for 5 min in the presence of the indicated
concentration of fMLP, and GTPase activity was measured as described
under ``Experimental
Procedures.''
Although GTP hydrolysis stimulated by
maximally effective concentrations of fMLP was almost equal in D and DR
membranes, fMLP stimulated GTPase activity with an EC that
was almost 10-fold lower in DR, as opposed to D membranes (Fig. 2C). By this criterion also, RA enhanced the
interaction between fMLP receptors and G
.
Membranes from
HL-60 cells treated with RA alone (for 1 or 2 days) showed no
detectable fMLP-binding or fMLP-stimulated GTPase activity. In the
presence of MeSO, exposure of the cells to RA for more than
24 h inhibited the induction of fMLP binding and the G
increase induced by Me
SO (data not shown).
To
assess interactions of pure G with fMLP receptors, we
prepared acceptor membranes from PTX-treated cells; in these membranes,
fMLP binding was completely unaffected by the presence of GTP
S,
indicating that PTX treatment had completely uncoupled endogenous
G
from the receptors. In order to test specifically the
additional differentiating effect of RA, the acceptor membranes were
prepared from cells differentiated in the presence of Me
SO
alone. Addition of pure G
to these membranes increased the
binding of fMLP to its receptors, and this increased binding was
blocked by GTP
S. G
purified from DR cells
(DR-G
) was much more effective in enhancing
GTP
S-sensitive fMLP binding than was pure G
from D
cells (D-G
) (Fig. 3A), in
agreement with parallel observations (Fig. 2, A and B) of the effects of GTP
S on fMLP binding to sites in DR versus D membranes, in experiments in which the receptors were
coupled to endogenous G
. We compared the abilities of
three purified G
preparations to enhance fMLP binding in
PTX-treated acceptor membranes; at a concentration of 5 nM,
these pure G
proteins enhanced fMLP binding with a rank
order of DR-G
> G
purified from bovine
brain
D-G
(Fig. 3B).
Figure 3:
Effects of D-G or DR-G
on GTP
S sensitive fMLP binding. A, the indicated
concentrations of pure D-G
(circles) or pure
DR-G
(triangles) were added to PTX-treated D
membranes, and specific fMLP binding was assayed in the absence (closed symbols) or in the presence (open symbols) of
10 µM GTP
S as described under ``Experimental
Procedures.'' B, fMLP binding was assayed in the absence (open circle) or in the presence of 5 nM D-G
(closed circles), bovine brain G
(open
squares), or DR-G
(closed triangles);
symbols represent duplicate determinations.
In addition
to increasing the ability of G to interact with fMLP
receptors, RA treatment greatly enhanced the ability of
GTP
S-activated G
to stimulate PLC activity partially
purified from HL-60 cytosol. Cytosol, obtained from DR HL-60 cells, was
fractionated on a Hi-Trap Heparin column in an NaCl gradient (see
``Experimental Procedures''), and PLC activities of
individual fractions were assayed without an activator (control) or in
the presence of either 30 nM
purified from bovine
brain or 10 nM GTP
S-activated DR-G
(Fig. 4A). PLC activity was eluted from the
column in two peaks; the second peak of PLC activity was similarly
activated by both GTP
S-activated DR-G
and bovine
brain
, in keeping with previous evidence (18, 25, 46, 47) that G
in neutrophils (and HL-60 cells) activates PLC via its
subunit, while activated
cannot stimulate PLC in
neutrophils or other cells rather than via
-GTP.
Figure 4:
G and
dependent
activation of PLC activity in HL-60 cytosol extract fractionated on a
Hi-Trap Heparin column. A, the cytosol fraction of DR HL-60
cells (see ``Experimental Procedures'') was applied to a
Hi-Trap Heparin column, and PLC activities in fractions eluted on an
NaCl gradient were assayed in the absence of any stimulus (open
circles) or in the presence of GTP
S-activated DR-G
(10 nM; filled triangles) or 30 nM
(filled circles), as described under
``Experimental Procedures.'' B, the indicated
concentrations of GTP
S-activated D-G
,
DR-G
, bovine brain
(open
squares), or bovine brain
subunit were added to
aliquots of fraction 26 from the Hi-Trap Heparin column, and PLC
activities were assayed.
Column fraction 26, which showed maximal fold stimulation by both
activated DR-G and
, was used to test the molar
potency of pure
, DR-G
, and D-G
as
stimulators of
-sensitive PLC activity. On a molar basis,
GTP
S-activated DR-G
appeared slightly more potent as
a stimulator of PLC than did pure
from bovine brain. Both
were much more effective stimulators than was GTP
S-activated
D-G
(Fig. 4B). In accord with work by
others (18, 25, 46, 47) ,
GTPgS-activated
from bovine brain did not stimulate
this PLC activity.
Figure 5:
Immunoblot analysis of in
D-G
, DR-G
, and membranes of HL-60 cells. A, D-G
(lane 2), DR-G
(lane 3), or bovine brain
(lane 1)
were resolved on a 11% SDS-polyacrylamide gel, and then immunoblotted
with a
1 specific antibody (
-636), a
2 specific antibody
(
-637), or a
specific antibody as described under
``Experimental Procedures.'' B, D-G
(lane 1) or DR-G
(lane 2) were
resolved on a 15% SDS-polyacrylamide gel, and then immunoblotted with a
-specific antibody (B-17), a
-specific antibody (D-9), or a
-specific antibody (A-67) as described under
``Experimental Procedures.'' C. cholate extracts of
membrane fractions (100 µg) from undifferentiated cells or cells
treated with RA (2 days), Me
SO (5 days), or
Me
SO (DMSO) (5 days) plus RA (during the last 24 h
of treatment) were resolved on a 15% SDS-polyacrylamide gel and then
immunoblotted with a
-specific antibody (B-17). The
standard represents 4 µg of purified bovine brain G proteins
containing a mixture of
and
subunits.
After
ADP-ribosylation by PTX and radioactive NAD, D- and
DR-G
preparations were subjected to SDS-polyacrylamide gel
electrophoresis and isoelectric focusing. Apparent molecular weights
and isoelectric points of
in the two preparations
were identical (results not shown), suggesting that differentiation in
the presence of RA did not produce a change in size or charge of
.
In this report we show that RA potentiates MeSO
as an inducer of fMLP-dependent PLC activation in HL-60 cells and that
this potentiation results, at least in part, from a qualitative change
in the
subunit of G
. RA enhances the abilities
of G
both to interact with fMLP receptor and also to
activate a
-sensitive PLC in the cytosol of differentiated
HL-60 cells. RA induces expression of a specific
subunit,
2,
which is otherwise not found in either G
or membrane
fractions of HL-60 cells. This is the first demonstration that a
differentia-tion factor regulates both the function and the polypeptide
composition of a G protein
subunit. Moreover, if expression
of
is responsible for the enhanced signaling ability
of G
, this is the first demonstration that the specific
composition of a
subunit physiologically accounts for the
ability of a G protein to activate a specific effector.
In this
section, we shall first discuss the three novel observations in this
report. Then we shall return to the question of whether expression of
2 accounts for the qualitative change in G
function
we observed.
Indeed, our second new observation is
that G, more specifically, its
subunit, is a
regulatory target of RA in HL-60 cells. RA induced changes in the
ability of G
to interact with fMLP receptors, as shown by
measurements of fMLP binding and stimulation of GTP hydrolysis in HL-60
membranes (Fig. 2) and also by reconstitution into PTX-treated
membranes of pure G
preparations from cells treated with
Me
SO plus RA or Me
SO alone (DR-G
versusD-G
; Fig. 3). In addition, reconstitution of a partially purified
PLC
with GTP
S-activated G
indicated that
DR-G
was a more effective and more potent activator of
PLC, on a molar basis, than was D-G
(Fig. 4). The
latter result clearly implicated the
subunit of DR-G
as the key element that accounts for the difference between
DR-G
and D-G
, because (a) the
PLC
preparation was quite sensitive to stimulation by
but not by
-GTP
S (both purified from bovine
brain); (b) PLC preparations from HL-60 and other cells (6, 18, 25) do not respond to activated
subunits; (c) although both D-G
and DR-G
contained equal amounts of
subunit, the latter produced a very large stimulation of PLC in
response to GTP
S, whereas the former had hardly any effect (Fig. 4B). Taken together, the enhanced abilities of
DR-G
to interact with the fMLP receptor and to activate
PLC
explain, at least in part, the synergistic effect of RA on
Me
SO-induced responsiveness of HL-60 cells to fMLP.
The
third new finding is that RA induces expression of a specific
subunit,
, which is not present in HL-60 cells unless
RA is added. Several reports have shown differentiationrelated changes
in the amount of different G
subunits in various cell types (23, 31, 50, 51) . To our knowledge,
however, differentiation factor-regulated expression of a specific
G
subunit has not previously been reported, although different
cells and tissues of mammals do contain different complements of
and
subunits.
Thus, while and
are expressed in a variety of tissues,
and
are preferentially expressed in the
brain(14) .
subunits are widely distributed in different
tissues, with the exception of
5, which is selectively expressed
in brain(13) . Immunocytochemistry techniques have shown more
precise and specific localizations. In the retina,
1 and
1
are found in the outer segments of rod cells, while
3 and
2
are found in cone cells(52) .
has been
reported to co-localize with vinculin and actin filaments in cultured
cells (53) .
Clear
precedents indicate that specific complexes can influence
the specificity of interactions between receptors and
subunits,
as is likely to be the case for
in
HL-60 cells. Antisense experiments have shown that a somatostatin and
an m4-muscarinic receptor in a single cell require
subunits
composed of different, specific
and
polypeptides to trigger
G
-dependent inhibition of Ca
currents(16, 17) .
complexes of
different composition differ in their ability to facilitate
rhodopsin-catalyzed binding of GTP
S to
(54, 55, 56) . Moreover, a
farnesylated peptide mimicking the C terminus of
can
directly stabilize metarhodopsin II, the active form of
rhodopsin(57) ; this shows that structurally specific segments
of a
subunit can interact directly with a receptor and, by
implication, help to determine receptor-G protein specificity.
Our
data suggest that a specific
(
) activates PLC in HL-60 cells with
greater efficiency than other
/
combinations. In other
tissues, what precedents indicate the specificity of different
subunits as regulators of effectors? First, a strong general
argument: if all
complexes were equivalent, all G proteins
should regulate
-sensitive effectors in the same way; for
example, stimulation of G
by
-adrenoreceptors should
stimulate PLC activity and open K
channels just as
well as does stimulation of G
. This is demonstrably not the
case, suggesting that different
complexes are not
equivalent.
In addition to this general argument, several studies
have used purified subunits of different composition to
search for specific regulation of effectors, including K
channels (7) and PLC
(58, 59) . In
general these studies indicate that
,
the
of retinal rod outer segments, regulates these effectors
less effectively than do other
complexes, but that the other
complexes are more or less indistinguishable in potency or
efficacy. In a study using PLC
3 purified from bovine brain, a
recombinant
containing
appeared to be a
somewhat more potent stimulator than other
complexes (see Fig. 5of (58) ), although the authors concluded that all
complexes were equivalent.
If is
indeed a superior stimulator of PLC
, how can this fact have
escaped previous investigators? One possibility is that the right
combination of
and
subunits has not been tested. If
is the key activator of PLC
in
RA/Me
SO-treated HL-60 cells,
could be
1,
2, or
4; the last of these has not been tested as a
stimulator of PLC in combination with
. (
)It is also possible that differentiated HL-60 cells
contain a PLC
that is specifically more sensitive to
, but which has not yet been tested
in experiments with recombinant proteins. HL-60 cells have been
reported to contain PLC
2 (which is found only in HL-60 cells;
Refs. 5, 60) and PLC
3, both of which are sensitive to activation
by
(5, 6) ; PLC
3 is more sensitive to
than is PLC
2(5, 6) . We have not
established the identity of the PLC used in our experiments, which was
partially purified from HL-60 cytosol; the active fractions contained
material that was detected by anti-PLC
2 antibody, but did not
react with an antiserum to the carboxyl terminus of PLC
3 (results
not shown). Thus we cannot rule out the presence of a previously
unknown
-responsive PLC in our experiments. (
)
In summary, we have shown that RA, a differentiation
factor, enhances fMLP signals in HL-60 cells and that the enhanced
stimulation of PLC depends on a qualitative change in the
subunit of G
. In parallel with these changes, RA induces
expression of a specific
polypeptide,
. From
this circumstantial evidence, we infer that RA-induced
accounts both for enhanced interactions of G
with
fMLP receptors and for its more effective stimulation of PLC. Critical
tests of this inference will require further experiments. It will be
necessary to show that (a) pure
does activate a PLC in these cells more effectively than do other
complexes, and (b) removal or specific inactivation
of
blocks the effect of RA on fMLP-stimulated PLC
activity.