(Received for publication, March 3, 1997, and in revised form, March 31, 1997)
From the Department of Cell Biology, Institute for Molecular and
Cellular Regulation, Gunma University, Maebashi 371, Japan and the
Department of Pharmacology, University of Virginia Health
Science Center, Charlottesville, Virginia 22908
When the calcium-permeable cation channel CD20 is
expressed in Balb/c 3T3 cells, it is activated by insulin-like
growth factor-I (IGF-I) via the IGF-I receptor (Kanzaki, M., Nie,
L., Shibata, H., and Kojima, I. (1997) J. Biol. Chem.
272, 4964-4969). The present study was conducted to investigate the
role of G proteins in the regulation of the CD20 channel. In the
excised patch clamp mode, activation of the CD20 channel by IGF-I
required GTP, Mg2+, and ATP in the bath solution, and
removal of either GTP or ATP attenuated the activation.
Non-hydrolyzable ATP could substitute for ATP, and guanyl-5-yl
thiophosphate blocked the activation of the channel by IGF-I. The CD20
channel was also activated by guanosine
5
-3-O-(thio)triphosphate, and ATP was not required for the
activation. Addition of a preparation of Gi/Go
holoprotein purified from bovine brain activated the CD20, and the
-adrenergic receptor kinase peptide did not affect the number of
channel openings induced by the G protein. The CD20 channel was
stimulated by the GTP-bound form of recombinant Gi2
subunit purified from Sf9 cells. The Gi3
subunit was
less effective, and the Gi1
subunit had no effect.
Purified recombinant
1
2 subunits did not
affect the activity of the channel. Finally, IGF-I-induced activation of CD20 was inhibited by an antibody against Gi2
subunit. These findings indicate that the CD20 channel expressed in
Balb/c 3T3 cells is activated by the IGF-I receptor via the
subunits of heterotrimeric G proteins.
CD20 is a transmembrane protein with molecular mass of 35 kDa that
is expressed in B lymphocytes (1). This protein has been considered to
be involved in growth regulation since monoclonal antibodies against
CD20 modify the growth rate of the cells (2-4). Molecular cloning of
the CD20 protein has provided information on the putative structure,
which resembles that of ion channels and transporters (5, 6). Bubien
et al. (7) showed that the CD20 channel expressed in
fibroblasts functions as a calcium-permeable cation channel. Using
whole cell recordings, they showed that CD20 is a
voltage-independent cation channel that permeates calcium. Since
calcium entry is a prerequisite for the progression through G1 phase (8), we expressed the CD20 channel in Balb/c 3T3
cells and studied the changes in the growth characteristics of the
cells (9). Indeed, expression of the CD20 channel resulted in three major alterations in G1 progression induced by insulin-like
growth factor-I (IGF-I).1 First, expression
of the CD20 channel shortened the period required for the entrance to
the S phase by accelerating the G1 progression induced by
IGF-I. Second, expression of the CD20 channel reduced the dependence of
the G1 progression on extracellular calcium, and
CD20-expressing cells could progress toward S phase in medium containing lower concentrations of calcium. Third, expression of the
CD20 channel enabled IGF-I alone to induce progression in cells
arrested in the G0 phase. In Balb/c 3T3 cells, IGF-I is not
capable of stimulating DNA synthesis in G0-arrested cells (10). IGF-I promotes G1 progression only when
G0-arrested cells are treated sequentially with
platelet-derived growth factor and epidermal growth factor (EGF)
(10-12). Therefore, expression of the CD20 channel at least partly
reproduces the effect of platelet-derived growth factor and EGF. These
results indicate that the expression of CD20 modulates the action of
IGF-I. Our recent study revealed that the calcium-permeable channel
activity of CD20 is activated by IGF-I in Balb/c 3T3 cells expressing
CD20 (13). When IGF-I is added to quiescent cells expressing CD20, the
opening probability of the CD20 is markedly augmented, and calcium
entry via the CD20 channel is greatly increased. Interestingly, the
effect of IGF-I on the CD20 channel is blocked by pertussis toxin (PTX)
(13). Conversely, mastoparan, an activator of
Gi/Go (14), activated the CD20 channel, and
transfection of gip2, a gene encoding constitutive active
form of the subunit of Gi2 (15), also activated the CD20 channel (13). Therefore, it is possible that IGF-I activates the
CD20 channel by a mechanism involving a PTX-sensitive G protein. The
present study was conducted to address this issue, and the data suggest
that the CD20 channel is activated principally by the
subunit of
Gi2.
Recombinant human IGF-I was supplied by Fujisawa
Pharmaceutical Co. Ltd. (Osaka, Japan). [32P]dCTP and
125I-labeled protein A were obtained from DuPont NEN.
Na[125I] was purchased from ICN Pharmaceuticals, Inc.
(Irvine, CA). Anti-CD20 antibodies were obtained from Cymbus Bioscience
Ltd. (CBL 456; Southampton, UK) and Coulter Immunology (Coulter clone B1; Hialeah, FL). The anti-Gi2 subunit antibody was
obtained from Calbiochem (anti-Gi
1 and
Gi
2 subunits C-terminal-(345-354) rabbit IgG).
Mastoparan was purchased from Peptide Institute, Inc. (Osaka, Japan).
Purified Gi/Go was prepared as described previously (16, 17). The
subunits of the G proteins
Gi1, Gi2, and Gi3 were expressed
using the baculovirus/Sf9 insect cell system and purified to
homogeneity using DEAE, hydroxyapatite, and Mono P chromatography as
described previously (18, 19). The recombinant
1
2 dimers were also expressed using the
baculovirus Sf9 cell system and purified as described previously (20).
The C-terminal peptide of the
-adrenergic receptor kinase (
ARK) (21) was synthesized by using peptide synthesizer (Applied Biosystems, Foster City, CA). The crude peptide was purified by preparative high
performance liquid chromatography to better than 98% homogeneity as
judged by analytical high performance liquid chromatography. All the
chemicals were of reagent grade and obtained from commercial sources.
Balb/c 3T3 cells (clone A31) were provided by the RIKEN cell bank (Tsukuba, Japan). Balb/c 3T3 cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal calf serum (Life Technologies, Inc.). These cells were cultured under humidified conditions of 95% air and 5% CO2 at 37 °C.
Transfection of cDNAThe inducible CD20 expression vector (CD20-pMEP4) was transfected into Balb/c 3T3 cells by electroporation as described previously (9). CD20 expressing quiescent Balb/c 3T3 cells were obtained by incubating confluent cells in Dulbecco's modified Eagle's medium containing 0.5% platelet-poor plasma and 40 µM ZnCl2 for 24 h. The constitutively active Gi2 mutant (Gip2) expression vector Gip2-pcDNA I was generously provided by Dr. H. Bourne of UCSF. Balb/c 3T3 cells were co-transfected with CD20-pMEP4 and Gip2-pcDNA I using a transfection reagent N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate (DOTAP, Boehringer Mannheim GmbH, Germany). Hygromycin (Wako Pure Chemicals, Osaka, Japan)-resistant colonies were independently picked up and screened by Northern blotting for high expression of Gip2 and CD20 as described previously (13).
We performed a binding study using a mAb against CD20 and
immunostaining with anti-CD20 antibody to show that the
CD20-transfected cells express CD20. For the binding assay, the IgG was
iodinated by the chloramine-T method (9) to a specific activity of
approximately 80 mCi/mg. The confluent cells grown in 6-well plates
were incubated for 1 h in 0.2% bovine serum albumin/Dulbecco's
modified Eagle's medium containing 125I-labeled IgG
(2 × 105 cpm/0.4 ml) at 32 °C. Nonspecific binding
was determined in the presence of excess unlabeled antibody. For
immunostaining, cells were grown on coverslips, fixed for 10 min in
10% formalin/PBS, washed with PBS, blocked with PBS containing 3%
bovine serum albumin, and incubated with the anti-CD20 antibody. After
washing in PBS, coverslips were incubated with tetramethylrhodamine
isothiocyanine-conjugated rabbit anti-mouse IgG antibody (Kappel,
dilution 1:100) at room temperature for 1 h. After a final wash
with PBS, samples were mounted in glycerol/PBS. Fluorescence was
observed by microscopy (Olympus, Japan). To confirm the expression of
Gip2, we performed the immunoblotting and immunostaining of
Gi2 subunit. For immunoblotting, cells were solubilized
in 20 mM Tris-HCl (pH 7.4) containing 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 1 mM EGTA, 5 mM iodoacetamide, 10 µg/ml pepstatin A, and 1000 units/ml
trypsin inhibitor. Lysates were centrifuged at 12,000 rpm at 4 °C
for 10 min to remove unsolubilized materials, and the supernatants were
mixed with equal volume of 2-fold concentrated Laemmli electrophoresis sample buffer in the presence of 50 mM mercaptoethanol.
Protein concentration was determined by using the BCA protein assay kit (Pierce). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was
carried out using 12% gels, and the protein was blotted to an
Immobilon membrane (Millipore, Yonezawa, Japan). The antibody against
the
i2 subunit was used as the primary antibody, and detection was achieved with 125I-labeled protein A (0.2 µCi/ml). The blots were subjected to autoradiography, analyzed using
a FUJIX BAS2000, and photographed by a FUJIX Pictrography 3000 (Fuji
Photo Film, Japan). Immunostaining was performed as described above
except PBS containing 0.1% saponin was used to permeabilize the plasma
membrane, and tetramethylrhodamine isothiocyanine-conjugated goat
anti-rabbit IgG antibody was used as the second antibody.
The cell-attached patch
clamp and the inside-out patch clamp techniques were used for the
voltage clamp studies (13). Micropipettes were pulled from borosilicate
glass capillaries and heat-polished at the tip. They had resistance
values between 5 and 8 M after being filled with a pipette solution.
High resolution membrane currents were recorded using an EPC-9
patch-clamp amplifier (HEKA, Lambrecht, Germany) controlled by
"Pulse" (HEKA) software on a Macintosh computer. All voltages were
corrected for a liquid junction potential between the bath and pipette
solutions. Single channel currents were recorded as described by Hamill
et al. (22). The signal was stored on video tape after
analogue/digital conversion (Sony PCM 501 ES, modified by Shoshin EM
Corp., Okazaki, Japan). For studies using the cell-attached
configuration, the micropipettes were filled with a solution containing
110 mM BaCl2 or CaCl2, 200 nM tetrodotoxin (Seikagaku-Kogyo, Japan), and 10 mM HEPES (pH 7.4, adjusted by adding Ba(OH)2 or
Ca(OH)2). In some experiments, Cl
was
replaced with aspartate. The bath solution contained 137 mM
NaCl, 5 mM KCl, 1 mM MgCl2, 1.25 mM CaCl2, 5 mM glucose, and 10 mM HEPES (pH 7.4, adjusted with NaOH). Single channel
recordings were analyzed by using the "EP ANALYSIS" (HEKA) and Igor
Pro (Wave Metrics, Lake Oswego, OR). The total number of functional
channels (N) in the patch were estimated by observing the
number of peaks detected on the amplitude histogram. As an index of
channel activity, NPo (number of channels
multiplied by the open probability) was calculated as shown in Equation 1.
![]() |
(Eq. 1) |
Statistical Analysis
The data are expressed as means ± S.E., and differences between them were analyzed using Student's t test and analysis of variance. Results were considered to be significantly different when p < 0.05.
Previous experiments have shown that the
activity of the CD20 channel in a cell-attached patch is observed when
IGF-I is added to the pipette solution and that IGF-I-induced CD20
channel activation is abolished by pretreatment with PTX (13). These
findings suggest that IGF-I activates CD20 via a PTX-sensitive G
protein. To investigate whether or not IGF-I-induced CD20 channel
activation is mediated by a G protein, we performed experiments using
inside-out patches. Fig. 1A shows the results
of a representative experiment obtained in an excised patch. The
pipette solution contained 110 mM BaCl2 and 1 nM IGF-I. After the patch excision, the single channel
currents disappeared even in the presence of 0.4 mM GTP and
4 mM Mg2+ in the bath solution. However, the
channel activity appeared again by the subsequent application of 1 mM ATP to the bath. To quantitate the effects of GTP and
ATP, we calculated the mean open probability. Fig. 1B shows
the mean open probability of the CD20 channel. As depicted, the CD20
channel openings were rarely detected in the bath solution containing
GTP and Mg2+, but the channel openings were observed when
ATP was added to the bath solution. Similarly, channel activity was not
observed in the excised patch when only ATP and Mg2+ were
in the bath, and activity was restored when GTP was added to the bath
(Fig. 1C). Fig. 1D shows the open probability of
the CD20 channel. Similar Ba2+-permeable channels
responsive to the internal solution containing ATP, GTP, and
Mg2+ were observed in 17/31 inside-out experiments. In
experiments without IGF-I in the pipette solution, the current was not
observed even if the bath solution contained GTP, Mg2+, and
ATP. We assumed that these channels were CD20 since the channel events
in either cell-attached or inside-out patches were blocked when a
monoclonal antibody against CD20 (CBL456; 2 µg/ml) was present in the
pipette solution (see Table I). Table I shows the number
of cells in which CD20 was activated by each treatment. In the presence
of ATP, GTP, and Mg2+ in the internal side of the membrane,
CD20 channel was activated in about 50% of the cells. The open
probability also decreased in inside-out patches compared with
cell-attached patches. NPo values were 0.44 ± 0.11 and 0.21 ± 0.07 in cell-attached and inside-out patches,
respectively. The single channel conductance was about 7 pS in excised
patches, and this size was similar to those in cell-attached patches
(13). None of these channel events in inside-out patches were observed
in the absence of Mg2+ in the bath solution. Channel
activation was not detected by adding GDP instead of GTP (data not
shown). Furthermore, pretreatment of the cells with PTX (100 ng/ml for
4 h) abolished the channel activation both in cell-attached and in
inside-out patches.
|
Next, we examined the effect of a
non-hydrolyzable GTP analogue, GTPS, on CD20 activation in
inside-out patches. The pipette solution did not contain IGF-I. Fig.
2 shows the effect of GTP
S added to the internal side
of the plasma membrane on CD20 channel activity. The channel events
were not observed in the cell-attached patch because of the absence of
IGF-I (Fig. 2, top trace). After the patch excision, the
activity of the channel was markedly stimulated by adding 0.1 mM GTP
S alone but not by GDP
S (data not shown) in the
presence of 4 mM Mg2+. The effect of GTP
S
was dose-dependent and, at concentrations less than 1 µM, GTP
S did not activate the CD20 channel (data not
shown). The channels activated by GTP
S had identical conductance to
the CD20 channel stimulated by IGF-I (pipette), GTP, and ATP (bath) in
inside-out patches. Channel events induced by GTP
S were abolished by
the mAb against CD20 (data not shown). ATP in the internal side of the
membrane was required for IGF-I-induced CD20 activation. As shown in
Table II, however, ATP was not required and had no
stimulatory effect on GTP
S-activated CD20 channel events.
|
As shown in the above sections,
ATP on the internal side of the plasma membrane was required for
IGF-I-induced but not for GTPS-induced CD20 activation in inside-out
patches. These results suggest that ATP is essential for IGF-I-induced
CD20 activation but is not necessary for CD20 activation when G protein
is activated directly. To evaluate this possibility, we examined
whether or not ATP is necessary for CD20 activation induced by
mastoparan. As shown in Fig. 3A, GTP and
Mg2+ on the internal side of the membrane were required and
sufficient for CD20 channel activation with mastoparan in the pipette
(21/22 patches). Likewise, when Gip2, a constitutively active mutant of
Gi2
, was co-expressed with CD20, ATP was not necessary
to activate CD20 (19/19 patches). These channel openings were not augmented by ATP in each condition (Table II). On the other hand, Mg2+ was necessary for CD20 activation, and these channel
events disappeared when Mg2+ was removed by adding EDTA
(data not shown). Furthermore, in cells activated by mastoparan, CD20
channel events disappeared when GDP
S was added to the internal side
of the membrane (Fig. 3B). Similarly, the CD20 channel
activity in Gip2-transfected cells was blocked by EDTA (Fig.
3C).
Effect of ATP Analogues on CD20 Activation in Inside-out Patches
As indicated above, ATP was necessary for IGF-I to
activate the CD20 channel in addition to GTP and Mg2+ in
excised patch. Since the IGF-I receptor has intrinsic tyrosine kinase
activity in the intracellular domain of the subunit, it is an
interesting question whether or not protein phosphorylation is involved
in the IGF-I-induced CD20 activation. To address this question, we
examined the effect of non-hydrolyzable analogues of ATP, ATP
S and
AMP-PNP, on IGF-I-induced CD20 activation in inside-out patches. The
pipette solution contained 1 nM IGF-I. As shown in Fig.
4A, the channel activity was detected upon
application of either 1 mM AMP-PNP or 1 mM
ATP
S in the presence of 0.4 mM GTP and 4 mM
Mg2+. NPo activated by either
ATP
S or AMP-PNP was slightly higher than that by ATP (Fig.
4B). In the absence of GTP and Mg2+, neither
ATP
S nor AMP-PNP alone had a stimulatory effect. These results
suggest that ATP binding rather than hydrolysis of ATP is necessary for
IGF-I-induced activation of the G protein.
Effect of Purified G Protein on CD20 Activation
The present
findings as well as those of our previous study (13) indicate that the
CD20 channel is regulated by G protein(s). To further assess the
involvement of G protein in the regulation of the CD20 channel, we
investigated the effect of reconstituting purified G proteins obtained
from bovine brain into the excised patches. The preparation used in
these experiments contains heterotrimeric Gi and
Go proteins. The pipette solution did not contain IGF-I. The purified G protein was pretreated with an equimolar concentration of GTPS. As shown in Fig. 5, the buffer solution
without G protein did not activate the channel, but the
GTP
S-activated brain G protein (20 nM) applied to the
internal side of CD20-expressing cell membrane patches induced
persistent openings of the Ca2+-permeable channel. The
channels activated by purified G protein had identical conductance as
the CD20 channel stimulated by IGF-I (pipette), GTP and ATP (bath), or
GTP
S (bath) alone. These single channel currents were abolished by
the mAb against CD20 (data not shown). The G protein-induced activation
of the CD20 channel was observed in all patches tested (16/16 patches
in three different experiments). Neither GTP
S alone added at 20 nM nor the GDP-bound form of the G proteins activated the
channel.
The observation that the expression of Gip2 results in
CD20 channel activation (13) suggests the importance of the subunit of the G protein to activate the CD20 channel. Since both
-GTP and
can activate multiple effectors (23), we examined the effect of
the C-terminal peptide of the
ARK peptide to assess the possibility
that the
subunit might be activating the CD20 channel in the
excised patches. The C-terminal domain of
ARK would be expected to
interact with the
subunit of the activated G protein and
neutralize its action (21). As shown in Fig. 5, the G protein-activated
CD20 channel event was not affected by 10 µM
ARK
peptide (a 500-fold excess of peptide). The
ARK peptide had no
significant effects on channel events in various conditions using
IGF-I, mastoparan, or GTP
S to activate the channel or using patches
activated by co-expression of Gip2 (data not shown). These results
suggest that the
subunits and not the
dimers have a
stimulatory effect on the CD20 channel.
To confirm the involvement of the subunit of
Gi on CD20 activation and to clarify the subtype of
i subunit that activates the CD20 channel, we
investigated the stimulatory action of recombinant
subunits of
Gi1, Gi2, and Gi3 on CD20 channel
activity in inside-out patches. As shown in Fig. 6, the
CD20 channel was activated by reconstitution of 1 nM
recombinant
i2 and
i3 (GTP
S-activated) but not by
i1. Activation of the CD20 channel by
recombinant
i subunits was normalized to the activity
induced by 5 nM
i2 subunit. As shown in Fig.
6B,
i2 was the most potent. The minimal concentration of
i2 required to activate the channel was
approximately 250 pM, and the EC50 was about 6 nM. The
i3 subunit was less effective than
the
i2 subunit and
i1 had essentially no
effect. As expected from the data shown in Fig. 5, 20 nM
recombinant
1
2, which could activate
PLC
2 (24), had no significant effect on CD20 channel
activity (Fig. 6C). These channel events activated by
recombinant
i2 subunits were not affected by recombinant
1
2. Neither GTP
S alone (concentrations
up to 50 nM) nor the detergent solution used as a vehicle
for the recombinant proteins affected the channel activity. Exposing
the cytoplasmic side of the membrane patches to exogenously applied ATP
did not modify the channel events (data not shown).
Finally, to confirm the involvement
of i2 subunit in IGF-I-induced CD20 activation, we
investigated the effect of an antibody against
i2 in
inside-out patches. As shown in Fig. 7, channel events
activated by IGF-I in the bath solution containing ATP, GTP, and
Mg2+ were diminished by adding an anti-
i2
subunit antibody (5 µg/ml).
The CD20 protein functions as a calcium-permeable cation channel
(7, 9) and, when expressed in Balb/c 3T3 cells, alters the growth
characteristics of the cells (9). In particular, expression of the CD20
channel modifies the ability of IGF-I to stimulate progression (9).
This alteration is due at least in part to the activation of the CD20
channel by IGF-I via the IGF-I receptor (13). Interestingly, although
the IGF-I receptor is very similar to the insulin receptor (25), the
IGF-I-mediated activation of CD20 is blocked by PTX. The present study
was conducted to examine which G proteins might be involved in the
IGF-I-mediated activation of the CD20 channel. We studied the activity
of CD20 in excised patches in which the condition of the cytoplasmic
face of the plasma membrane could be manipulated by changing the bath solution. Activation of the channel by IGF-I in the cell-attached mode
was examined first, and the existence of the ligand, receptor, channel,
and transducers in the patch was confirmed. The following observations
combined with our previous results (13) indicate that IGF-I activates
the CD20 channel by a G protein-dependent mechanism. First,
IGF-I-induced activation of the CD20 required GTP as well as
Mg2+ and removal of either GTP or Mg2+
attenuated the activation. Second, GDPS, which inactivates G proteins, inhibited the IGF-I action on CD20. Third, G protein-mediated activation of the CD20 channel was directly demonstrated by adding purified G protein (Gi/Go class) purified from
the brain. Like the calcium-permeable cation channel activated by
erythropoietin (26), calcium-permeable voltage-independent cation
channel CD20 is regulated by Gi proteins.
There are abundant data to show that ion channels are regulated by
activation of G protein-coupled receptors. The signaling pathways used
include second messengers, phosphorylation, and direct regulation by G
proteins (27, 28). Currently, K+, Ca2+, and
Cl channels are thought to be regulated by either the
or
subunits of G proteins (27, 29). For example, it is now clear
that the muscarinic K+ channel in the atrium is regulated
by G protein
subunits (27, 30), and there is growing evidence
that the N-type Ca2+ channel is also regulated
by these subunits (27, 28, 31, 32). In contrast, the Cl
channels in renal epithelial cells may be regulated by a Gi
subunit (27). The data in this report clearly suggest that the CD20
channel is also regulated by members of the Gi
family
of proteins. Interestingly, only the Gi2
subunit is
fully effective in activating this channel, whereas Gi1
protein is essentially inactive. The particular preparations of
Gi1, Gi2, and Gi3
subunits used
in this study are fully active as judged by their efficacy in other
assays. For example, all three Gi
s couple equally well to recombinant A1 adenosine receptors (33). When a blocking antibody against
i1 and
i2 was added, the
IGF-I-induced activity was markedly inhibited. Therefore, the CD20
channel is principally regulated by
i2 in the cell. This
notion is in agreement with the report by Okamoto et al.
(34) that IGF-I activates Gi2 in Balb/c 3T3 cells.
Considered together, these results suggest that the interaction between
the CD20 Ca2+-permeable channel and the different
Gi
subunits is very selective.
While the multiple variations of the mitogen-activated protein kinase
pathway are thought to be the major signaling mechanisms used by
receptors with tyrosine kinase activity (35, 36), the finding that
receptors in this family can also couple to G protein subunits is
not unique. A number of investigators have shown that responses to both
insulin and EGF receptors may be elicited via G protein-coupled
mechanisms. In rat hepatocytes, the ability of EGF to stimulate
inositol lipid breakdown in intact cells or to stimulate the binding of
GTP
S to isolated membrane preparations is blocked by pertussis
toxin, suggesting that the receptor couples to the Gi
subunit in these cells (37, 38). Moreover, in rat cardiomyocytes, EGF
stimulates adenyl cyclase by coupling to the Gs protein
(39). In BC3H-1 myocytes, the ability of insulin to generate
diacylglycerol can be blocked by pertussis toxin (40).
The IGF-I receptor resembles the insulin receptor and has intrinsic
tyrosine kinase activity in its subunit (25). Binding of the ligand
to the
subunit leads to the activation of the receptor kinase,
which phosphorylates its own subunit, and autophosphorylation of the
receptor up-regulates the activity of receptor kinase, thus
phosphorylating other substrates including insulin receptor substrate-1
(see Ref. 41 for review). An interesting question is whether or not
receptor kinase activity is necessary for the channel activation. The
present results show that non-hydrolyzable analogues of ATP reproduced
the effect of ATP. Thus, ATP binding rather than hydrolysis may be
necessary for the receptor-mediated activation of the G protein. It
should be mentioned that, in our experiments, the cell was first
activated by IGF-I added inside the patch in the cell-attached mode. It
is likely that autophosphorylation of the receptor took place in the
beginning of the experiment. However, it is not certain whether or not
the receptor remained autophosphorylated in the excised patch. In any
event, based on the results obtained with AMP-PNP, ATP binding rather
than hydrolysis is necessary for the channel activation. In this
regard, we recently found that insulin activates calcium-permeable
cation channel in Chinese hamster ovary cells expressing human insulin
receptor.2 Interestingly, insulin-induced
activation of the channel requires GTP, ATP, and Mg2+ in
the excised mode of the patch clamp, and non-hydrolyzable ATP analogues
can be substituted for ATP. In the case of the insulin receptor, ATP
binding is known to induce alterations of the conformation of the
active domain of the receptor (42). This raises the possibility that
receptor-mediated activation of the G protein requires a conformational
change induced by ATP binding. Given the structural similarity between
the insulin receptor and the IGF-I receptor, ATP binding may induce a
structural change in the
subunit of the receptor, which is
necessary for the activation of the Gi2
subunit. This
possibility should be examined experimentally in the future.
CD20 is expressed in B lymphocytes (1, 2). The present results provide
insights into the regulation of CD20 in lymphocytes and regulation of
the IGF-operated channel in Balb/c 3T3 cells. Since the CD20 channel
can be activated by i2, other receptors coupled to the
Gi2 heterotrimer should be expected to activate the channel
in B lymphocytes. Besides the function as a calcium-permeable channel,
CD20 also functions in the phosphorylation cascade (43). It is an
interesting question whether or not the latter function is also
modulated by the Gi2 protein. In Balb/c 3T3 cells, IGF-I stimulates calcium entry by activating the IGF-operated
calcium-permeable channel (44), and regulation of the IGF-operated
channel resembles in many respects that of CD20. The IGF-induced
calcium entry is blocked by PTX (45) and is augmented by GTP
S (46).
Since IGF-I activates Gi2 (34), it seems likely that the
regulatory mechanism of the IGF-operated channel is similar to that of
CD20 expressed in Balb/c 3T3 cells.
In summary, the CD20 channel expressed in Balb/c 3T3 cells is activated
by IGF-I. Activation of the CD20 channel is caused principally by the
subunit of Gi2.
We are grateful to Dr. Henry Bourne for providing us with Gip2-pcDNA I, to Julia E. Fletcher for help with protein purification, and to Mayumi Odagiri for secretarial assistance.