The Netherlands Cancer Institute, Division of Cellular Biochemistry, 1066 CX Amsterdam, The Netherlands
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Gap junctions mediate cell-cell communication in almost all tissues, but little is known about their regulation by physiological stimuli. Using a novel single-electrode technique, together with dye coupling studies, we show that in cells expressing gap junction protein connexin43, cell-cell communication is rapidly disrupted by G protein-coupled receptor agonists, notably lysophosphatidic acid, thrombin, and neuropeptides. In the continuous presence of agonist, junctional communication fully recovers within 1-2 h of receptor stimulation. In contrast, a desensitization-defective G protein-coupled receptor mediates prolonged uncoupling, indicating that recovery of communication is controlled, at least in part, by receptor desensitization. Agonist-induced gap junction closure consistently follows inositol lipid breakdown and membrane depolarization and coincides with Rho-mediated cytoskeletal remodeling. However, we find that gap junction closure is independent of Ca2+, protein kinase C, mitogen-activated protein kinase, or membrane potential, and requires neither Rho nor Ras activation. Gap junction closure is prevented by tyrphostins, by dominant-negative c-Src, and in Src-deficient cells. Thus, G protein-coupled receptors use a Src tyrosine kinase pathway to transiently inhibit connexin43-based cell-cell communication.
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
COMMUNICATION between adjacent cells through gap
junction channels occurs in almost all tissues and is
fundamental to coordinated cell behavior. In excitable tissues, such as cardiac muscle and neurons, gap
junctions allow rapid and synchronous propagation of action potentials. More in general, junctional communication has been implicated in the control of cell proliferation,
embryonic development, and tumor suppression (for reviews see Bennett et al., 1991; Beyer, 1993
; Hotz-Wagenblatt and Shalloway, 1993
; Paul, 1995
; Mesnil et al., 1995
;
Goodenough et al., 1996
). Gap junction channels are permeable to small (<1 kD) molecules, including second messengers such as Ca2+, inositol phosphates, and cyclic nucleotides. The integral membrane proteins forming these
channels are termed connexins, which are encoded by a
multigene family (Bennett et al., 1991
; Goodenough et al.,
1996
; Kumar and Gilula, 1996
). Connexin43 (Cx43)1 is the
most widespread and abundant member of this family.
While much has been learned in recent years about the
cellular and molecular biology of gap junction channels
(Bennett et al., 1991; Musil and Goodenough, 1991
; Goodenough et al., 1996
; Kumar and Gilula, 1996
), it is still not
known how junctional communication is regulated under
physiological conditions. Regulation of cell-cell communication has often been evaluated by using nonphysiological effectors such as ionophores, phorbol esters, or cell-permeable cAMP analogues. There is evidence that an increase in cytoplasmic Ca2+ or H+ levels can disrupt cell-cell
coupling (Spray, 1994
), whereas a rise in cAMP frequently
upregulates junctional communication (Godwin et al., 1993
;
Burghardt et al., 1995
; Goodenough et al., 1997; but see
Lasater, 1987
, for an opposite effect). Furthermore, membrane potential (transjunctional voltage) has been implicated in the gating of gap junction channels (for references
see Beyer, 1993
). Of particular relevance is the finding
that several protein kinases can influence junctional permeability (for reviews see Hotz-Wagenblatt and Shalloway, 1993
; Goodenough et al., 1996
). For example, persistent activation of protein kinase C (PKC) or expression of
the active v-Src tyrosine kinase abrogates cell-cell communication, which correlates with enhanced phosphorylation of Cx43 on serine or tyrosine residues, respectively
(Crow et al., 1990
; Filson et al., 1990
). EGF inhibits junctional communication in some cell types (Maldonado et
al., 1988
) and stimulates serine phosphorylation of Cx43,
which is thought to be mediated by mitogen-activated protein (MAP) kinase (Kanemitsu and Lau, 1993
; Hii et al.,
1994
). Consistent with this, MAP kinase can directly phosphorylate Cx43 in vitro (Warn-Cramer et al., 1996
). However, no specific receptor-linked signaling pathway involved in modulating junctional communication has been
identified to date.
Lysophosphatidic acid (LPA) is a platelet-derived serum mitogen that acts on its cognate G protein-coupled
receptor present in numerous cell types (Moolenaar et al.,
1997). The LPA receptor couples to stimulation of phospholipase C, inhibition of adenylyl cyclase, and activation
of the Ras and Rho GTPases (Moolenaar et al., 1997
). In a
recent patch-clamp study on confluent Rat-1 fibroblasts,
we found that LPA evokes a long-lasting membrane depolarization due to activation of a Cl
conductance (Postma
et al., 1996
). While analyzing Cl
channel opening in response to LPA, we made the unexpected observation that
the cell under study rapidly isolates itself from adjacent cells. This prompted us to analyze G protein regulation of
cell-cell communication in more detail. Rat-1 cells are ideally suited for these studies because (a) they express Cx43
as the sole gap junction protein (Goldberg and Lau, 1993
)
and (b) G protein signaling is well characterized in these
cells (van Corven et al. 1989, 1993; Hordijk et al., 1994a
;
Postma et al., 1996
; van Biesen et al., 1996; Kranenburg
et al., 1997
).
Using a newly developed single patch-clamp electrode technique together with dye coupling studies, we show here that Cx43-based junctional communication is rapidly but transiently disrupted upon activation of various G protein-coupled receptors, including those for LPA, thrombin, and neuropeptides. We show that agonist-induced gap junction closure is independent of Ca2+, PKC, MAP kinase, membrane potential, and Ras/Rho signaling. Instead, we find that Cx43-based gap junctions are closed through a novel G protein-mediated signaling pathway involving c-Src. In addition, we show that recovery of communication is controlled, at least in part, by homologous receptor desensitization.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Materials
Staurosporine, tyrphostins 25 and 47, sodium orthovanadate, 8-Br-cAMP,
and 8-Br-cGMP were from Calbiochem-Novabiochem (La Jolla, CA). Lucifer yellow (LY), Indo-1, and rhodamine-conjugated palloidin were from
Molecular Probes (Eugene, OR). EGF was from Collaborative Research
Inc. (Waltham, MA). LPA (1-oleoyl), thrombin, endothelin, neurokinin
A, isoproterenol, phorbol ester, ionomycin, and thapsigargin were from
Sigma Chemcial Co. (St. Louis, MO). TRP (sequence: SFLLRNPNDKYEPF) was synthesized as described (Jalink and Moolenaar, 1992). C3
and pertussis toxin were from List Laboratories. Antibodies to Cx43 and
phosphotyrosine (PY20) were from Transduction Laboratories (Lexington, KY) and anti-Src monoclonal 327 from Oncogene Science (Manhasset, NY). [
-32P]ATP was from Amersham Corp. (Arlington, Heights, IL).
Cell Culture
Rat-1 cells, v-Src-transformed Rat-1 cells (B77 cells; van der Valk et al.,
1987), Rat-1 cells expressing wild-type or truncated NK2 receptor (Alblas
et al., 1995, 1996
), HEK 293 cells, HeLa cells, and mouse embryonic fibroblasts, either expressing or lacking endogenous c-Src (kindly provided by
P. Soriano [Fred Hutchinson Cancer Research Center, Seattle, WA] and
K. Burridge [University of North Carolina, Chapel Hill, NC]), were grown
in DME supplemented with 7.5% fetal calf serum and antibiotics. Cells
were grown to confluency and then exposed to serum-free DME for 16-24 h.
Electrophysiology: Single Electrode Measurements
Electrophysiological recordings were obtained from cells grown in 3-cm
culture dishes using the whole-cell patch-clamp technique as described
(Postma et al., 1996). By analyzing current relaxations evoked by brief
voltage steps, the degree of cell-cell coupling can be monitored continuously by a single patch-clamp electrode (see Results and Fig. 2), as opposed to the dual-electrode techniques used in most coupling studies (for
single-electrode analysis of cell-cell coupling see also Bigiani and Roper,
1995
). Data were collected using an EPC-7 amplifier (List-Medical,
Darmstadt, Germany), interfaced to a personal computer via an A/D converter (TL-1 DMA interface; Axon Instruments, Inc., Foster City, CA).
Voltage-clamp protocols were generated, and data were stored and analyzed using pClamp 6.0 (Axon Instruments, Inc.). Recordings were carried
out using the perforated patch-clamp technique with amphotericin in the
pipette solution (to selectively permeabilize the membrane to monovalent
ions; Rae et al., 1991
; Postma et al., 1996
). Micropipettes were fire-polished and filled with a high K+, low Ca2+ buffer as described (Postma et al.,
1996
). Capacitive transients caused by the patch-pipette were compensated for. Agonists and pharmacological agents were applied either from a
pipette positioned close to the cell(s) under study or by bath perfusion.
Capacitive current transients were fitted using the Chebyshev method.
Based on a a minimal electrical equivalent circuit (Lindau and Neher, 1988
), the current transient recorded from non-coupled cells was fitted
with a single decay time constant (one exponential). However, the transient from coupled cells in a monolayer is considerably more complex; in
first approximation, it can be fitted by two exponentials with time constants
1 and
2 (see Results and Fig. 2; see also Bigiani and Roper, 1995
,
for the case of a coupled cell pair). The first exponential component
largely reflects the charging of the patched cell, and the second component that of the coupled cells. This is further illustrated in the model experiment of Fig. 2. Changes in junctional conductance manifest themselves as changes in time constants and steady-state currents; however, unlike the case of just two coupled cells (Bigiani and Roper, 1995
), junctional conductance in a coupled monolayer cannot readily be calculated
from the capacitive transients and steady-state currents measured. All experiments were done at least in triplicate; results are given as means ± SEM and statistical significance was established using Student's t test.
|
LY Diffusion
Gap junction permeability was determined using both the LY scrape-loading technique (El-Fouly et al., 1987) and the LY microinjection method. For scrape-loading, confluent cells in 3-cm dishes were stimulated with agonist for various periods of time. A scrape line was made on
the monolayer using a surgical blade in medium containing 5 mM LY. LY
was allowed to diffuse for 5 min. Immediately thereafter, the cultures
were washed with PBS, fixed in 3.7% paraformaldehyde, and treated with
antibleach reagent (Vectashield). Quantitative analysis of LY diffusion
was carried out with a confocal microscope coupled to an image analyzer
system (Bio-Rad Laboratories, Hercules, CA), measuring the average
pixel intensity in the direction parallel to the scrape line as a function of
diffusion distance.
In a separate series of experiments, we monitored LY diffusion from a single microinjected Rat-1 cell within a monolayer. Cells were treated with agonist for 5 min and then microinjected with a mixture of LY (100 µM) and ethidium bromide (0.5 mg/ml; for nuclear staining). After microinjection, cells were washed with PBS, and intracellular LY/ethidium bromide fluorescence was monitored immediately thereafter (total LY diffusion time: 2 min).
cDNA Transfection
Rat-1 cells stably transfected with wild-type or truncated NK2 receptor
have been described recently (Alblas et al., 1995, 1996
). For stable transfection of dominant-negative Src into NK2-receptor-expressing Rat-1
cells, 10 µg of pMT2-plasmid containing the complete cDNA of kinase-
inactive Src (SrcK
; mutation Lys295Met; cDNA provided by S. Courtneidge, Sugen, Inc., Redwood City, CA) together with 0.2 µg of pDH
containing the hygromycin resistance gene was transfected using standard calcium phosphate precipitation. After 16 h, cells were split 1:10 and then
exposed to selection medium containing 200 U/ml hygromycin. Hygromycin-resistant clones were isolated and screened for enhanced Src protein
expression in immunoblots using anti-Src monoclonal antibody 327.
Immunoblotting and Cx43 Immunoprecipitations
For immunoblotting, confluent cells were treated with agonist for various periods of time, washed with ice-cold PBS, and lysed in SDS-sample buffer. Proteins were subjected to SDS-PAGE and transferred to nitrocellulose. The filters were blocked with 3% BSA in TBST (10 mM Tris, pH 8.0, 150 mM NaCl, 0.05% Tween 20) and incubated with antibody for 1 h. After washing with TBST, blots were incubated with peroxidase-conjugated rabbit anti-mouse Ig, washed again, and subjected to the ECL procedure. For immunoprecipitation of Cx43, cells were lysed in NP-40 lysis buffer (50 mM Tris-HCl, pH 7.4, 1% NP-40, 0.25 mM Na-deoxycholate, 150 mM NaCl, 2 mM EGTA, 0.1 mM Na3VO4, 10 mM NaF, 0.1 mM PMSF fluoride, 10 mg/ml leupeptin, 10 mg/ml aprotinin). Cell lysates were immunoprecipitated with anti-Cx43 antibody. Precipitated proteins were resolved by SDS-PAGE on 12.5% polyacrylamide gels and either processed for ECL visualization or transferred to nitrocellulose for immunoblotting (see above).
Fluorescence Microscopy
Cells were grown on glass coverslips, fixed in PBS containing 4% paraformaldehyde, and incubated with either anti-Cx43 antibody followed by fluorescent staining using FITC-conjugated rabbit anti-mouse Ig. For F-actin staining, fixed cells were incubated with rhodamine-conjugated phalloidin. Fluorescence was visualized using confocal microscopy.
Src Kinase Assay
Cells were stimulated with agonists for various periods of time, washed
with ice-cold PBS, and solubilized in 0.5 ml of lysis buffer (25 mM Tris pH
7.4, 0.5% NP-40, 150 mM NaCl, 1 mM DTT, 0.1 mM Na3VO4, 10 mM
Na2P2O4, 10 mM NaF, 0.1 mM PMSF fluoride, 10 µg/ml leupeptin, 10 µg/ml
aprotinin) on ice for 10 min. Lysates were precleared twice with normal
mouse serum precoupled to Sepharose-protein A beads (30 min, 4°C). Src
was immunoprecipitated with monoclonal 327 (1 h, 4°C). The precipitates
were washed four times with lysis buffer and washed once with ATP-free
kinase buffer (20 mM Hepes, pH 7.4, 10 mM MgCl2, 1 mM DTT). In vitro
kinase assays were carried out in 30 ml of kinase buffer supplemented
with 0.25 mg/ml enolase, 20 µM ATP and 0.5 mCi/ml [-32P]ATP for 10 min on ice. The reactions were stopped by addition of SDS-sample buffer;
the samples were then boiled and analyzed by SDS-PAGE (10% polyacrylamide). After autoradiography, the amount of 32P present in Src and
enolase was determined by PhosphoImager analysis.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Rapid Changes in Cell-Cell Communication Monitored with a Single Patch-Clamp Electrode
We recently showed that LPA, like serum, rapidly activates a depolarizing Cl current in confluent Rat-1 fibroblasts (Postma et al., 1996
). Surprisingly, activation of this
current is accompanied by a dramatic (~40-fold) fall in
cell input conductance, monitored as the current response
to brief voltage pulses (Fig. 1, A and B). This conductance decrease develops rapidly and is maximal after a few minutes (Fig. 1 B). It thus appears that LPA-induced opening
of Cl
channels is overridden by the simultaneous closure
of other channels. Since confluent fibroblasts are electrically coupled (through Cx43) and thus behave as if the
monolayer were one large cell, the overall input conductance is determined by both transmembrane and cell-to-cell
conductances. Given the finding that LPA increases transmembrane conductance (Cl
-selective), the observed fall
in input conductance must result from a concomitant closure of gap junction channels.
|
Direct support for this notion comes from analysis of the
typical current relaxations in response to a voltage step before and immediately after LPA addition (Fig. 1 C). This
current response consists of an initial capacitive transient
decaying to a steady-state value. It is seen that LPA reduces not only the amplitude of the steady-state current
but also the decay time constant(s) of the capacitive transient (Fig. 1 C). These complex changes in electrical properties of the monolayer can only be explained by acute loss of electrical cell-cell coupling. This is exemplified in a
model experiment, illustrated in Fig. 2 A, which shows the
different current responses recorded from an increasing
number of coupled cells. Time constant and steady-state
currents are seen to increase with increasing number of
contacting adjacent cells, as detailed in the legend to Fig.
2 A. Fig. 2 B shows that the current response of a single
isolated cell is hardly affected by LPA. This demonstrates that the observed electrophysiological changes in LPA-treated monolayers (Fig. 1 C) result from altered cell-cell
coupling, not from altered membrane conductance. Thus,
by analyzing current relaxations in response to brief voltage steps, the degree of cell-cell coupling can be monitored continuously by a single patch-clamp electrode, as
opposed to the dual-electrode techniques used in most
coupling studies. Single-electrode analysis of cell-cell coupling (between two cells) has also been documented by
Bigiani and Roper (1995).
The electrophysiological studies were complemented by LY diffusion experiments, in which we determined the cell-to-cell diffusion distance of intracellularly loaded LY per unit time period. Fig. 3 shows a typical LY scrape-loading experiment revealing that cell-to-cell LY transfer in confluent Rat-1 cells is markedly inhibited by LPA. The results obtained from the LY scrape-loading experiments were supported by microinjection studies, in which we monitored LY diffusion from single cells microinjected with LY (together with ethidium bromide) before and after agonist stimulation (Fig. 4). Under control conditions, LY diffusion from the microinjected cell was very rapid: at 2 min after microinjection, LY fluorescence was detectable in 64 ± 6 surrounding cells (means ± SEM; number of dishes n = 17). After stimulation for 5 min with LPA (1 µM), intercellular LY diffusion was detectable in only 10 ± 3 (n = 11) surrounding cells. Rapid inhibition of cell- cell coupling was not unique for LPA (see below). Strongest inhibition of LY diffusion was observed with the G protein-coupled receptor agonist endothelin, which completely blocked LY diffusion from the microinjected cell in all dishes tested (n = 13; Fig. 4; see also Fig. 5, B and C, for electrophysiological and LY scrape-loading data). In all these experiments, the transmembrane permeability to LY was not affected by agonist stimulation, as both stimulated and control cells showed negligible dye loss over a 15-min period. Thus, the observed inhibition of LY diffusion represents an agonist-induced decrease in gap junction permeability.
|
|
|
Loss of Communication Is Induced by a Variety of G protein-coupled Receptors
We tested a number of agents and agonists for their ability
to inhibit cell-cell communication. Uncoupling was also
induced by AlF4, a direct activator of trimeric G proteins
(Kahn, 1991
), consistent with LPA action being G protein
mediated (Fig. 5 A). Its inhibitory effect develops rather
slowly, consistent with a lag required for AlF4
entry into
the cells.
In addition to LPA, the G protein-coupled receptor agonists endothelin, thrombin as well as a thrombin receptor
agonist peptide (TRP; Jalink and Moolenaar, 1992) all
rapidly inhibit cell-cell coupling in Rat-1 cells, as revealed
by both electrophysiological and LY transfer experiments
and summarized in Fig. 5, B and C (see also Fig. 4). Of
note, these agonists also mimic LPA in evoking a depolarizing Cl
current in Rat-1 cells (Postma et al., 1996
). Pretreatment of the cells with pertussis toxin (PTX) did not
affect agonist action, indicating that Gi proteins are not involved (Fig. 5 C). Activation of endogenous
-adrenergic
receptors by isoproterenol, which raises cAMP in Rat-1 cells
(van Corven et al., 1989), left cell-cell coupling unaltered.
EGF, a potent mitogen for Rat-1 cells (van Corven et al.,
1989), failed to inhibit electrical coupling, although intercellular LY diffusion was somewhat reduced (Fig. 5 C and results not shown). This would suggest that EGF may decrease junctional permeability to larger molecules only, but
further experiments are required to test this hypothesis.
Thus, inhibition of cell-cell communication is a common
response to a subclass of G protein-coupled receptors, notably those that couple to PTX-insensitive G proteins (Gq/11
and G12/13 subfamily). Support for this notion comes from
transfection studies using the human NK-2 receptor for
neurokinin A (NKA; or substance K). When stably expressed in Rat-1 cells, the NK-2 receptor couples to Gq-mediated inositol lipid breakdown and Ca2+ mobilization
(Alblas et al., 1995, 1996
). Ligand stimulation of the transfected NK2 receptor triggers rapid gap junction closure (Fig. 5, B and D) with concomitant activation of the inward Cl
current (Postma et al., 1996
).
Involvement of Cx43
Cx43 is the only gap junction protein thus far identified in
fibroblasts (Goldberg and Lau, 1993). Fig. 6 shows the
characteristic punctate staining of Cx43 at sites of cell-cell
contact. Endothelin treatment does not detectably alter
the localization pattern of Cx43 over a 10-min time period
(Fig. 6) nor the levels of endogenous Cx43 (as determined
by Western blotting; see below).
|
The inhibitory effect of LPA on cell-cell communication was also observed in Cx43-expressing epithelial cells
(HEK 293 cells; results not shown). In contrast, no effect
of LPA on junctional conductance was observed in Cx43-deficient HeLa cells, which are electrically coupled (albeit
weakly; see also Eckert et al., 1993) and do express functional LPA receptors (Jalink et al., 1995
). Fig. 5 E shows
that, while junctional communication in HeLa cells is rapidly blocked by the general anesthetic halothane, LPA
only induces a transient outward current in these cells
without any sign of decreased cell-cell coupling. Taken together, these results are consistent with Cx43 being the
primary target of agonist action.
Recovery of Communication: Regulation by Receptor Desensitization
After the acute loss of communication in response to agonists, cell coupling recovers gradually over time. In the continuous presence of LPA, TRP, or NKA, recovery of communication was usually complete within 1-2 h (Fig. 7); in endothelin-stimulated cells, full recovery took somewhat longer (3-4 h; not shown).
|
How are the kinetics of this recovery regulated? One
possibility is that recovery is due to receptor-mediated signal attenuation ("desensitization"). To examine this point,
we used a mutant NK2 receptor in which the COOH-terminal "desensitization domain" (70 residues) is deleted
(Alblas et al., 1995). When expressed in Rat-1 cells, such
desensitization-defective receptors mediate prolonged inositol lipid breakdown and sustained MAP kinase activation, as opposed to the short-lived responses induced by
wild-type receptors; furthermore, the liganded mutant receptor induces both DNA synthesis and morphological
transformation in response to ligand, whereas wild-type
receptor does not (Alblas et al., 1995
, 1996
).
When Rat-1 cells expressing mutant NK2 receptor were
exposed to ligand, the cells remained uncoupled for a
much longer time period than observed with wild-type receptors (Fig. 7, bottom). Cell-cell coupling had returned to
control values only after ~12 h. These results indicate that
recovery of communication is regulated by receptor desensitization and, furthermore, that there is a correlation
between the duration of receptor-mediated gap junction closure and subsequent morphological transformation (Alblas et al., 1996).
Dissection of Signaling Pathways
Which G protein-effector pathways may underlie inhibition of cell-cell communication? Since agonist-induced
gap junction closure is resistant to PTX (Fig. 5 C), which
blocks LPA- and thrombin-induced Ras-GTP accumulation and MAP kinase activation in Rat-1 cells (van Corven
et al., 1993; Hordijk et al., 1994a; Kranenburg et al., 1997
),
it follows that gap junction closure is independent of Ras-MAP kinase signaling. Consistent with this, treatment of Rat-1 cells with 8Br-cAMP (1 mM) did not affect LPA-
induced gap junction closure (not shown), whereas MAP
kinase activation is completely blocked by 8Br-cAMP
(Hordijk et al., 1994b
).
Strikingly, the G protein-coupled receptors that mediate gap junction closure all couple to inositol lipid breakdown and consequent Ca2+ mobilization in Rat-1 cells.
However, gap junctions did not close in response to ionomycin (25 nM), which raises cytosolic [Ca2+] to micromolar
levels (Postma et al., 1996). Conversely, agonist-induced gap junction closure was not impaired when cells were depleted of intracellular free Ca2+ by addition of ionomycin
in Ca2+-free, La3+-containing medium or by preincubation
with 200 nM thapsigargin (Fig. 8 A, and results not
shown). Furthermore, gap junctions did not close in response to PKC-activating phorbol ester (TPA; 100 nM), as
shown in Fig. 5 C. Prolonged (24 h) pretreatment of the
cells with TPA (1 µM; 24 h) to downregulate PKC did not
prevent cell uncoupling (Fig. 5 C), nor did the PKC inhibitor Ro-31-8220 (5 µM) have any effect. These results contrast with reports that show marked inhibitory effects of
phorbol ester on junctional communication in some cell
types (for review see Goodenough et al., 1996
). Taken together, our results indicate that activation of the Ca2+-PKC pathway is neither necessary nor sufficient for gap
junction closure in Rat-1 cells.
|
Agonist-induced uncoupling also correlates with Cl-mediated membrane depolarization (Postma et al., 1996
).
However, artificial membrane depolarization, induced either by altering the holding potential or by exposing the
cells to high [K+], had no detectable effect on cell coupling
nor on the response to agonists. Thus, gap junction closure
is not secondary to membrane depolarization.
LPA, thrombin, endothelin and NKA also rapidly activate the Ras-related Rho GTPase, leading to the formation of actin stress fibers, which is a PTX-insensitive process (Ridley and Hall, 1992; Alblas et al., 1996
; Postma et al.,
1996
). The bacterial C3 toxin, which modifies and thereby
inactivates Rho (Machesky and Hall, 1996
), did not affect
agonist-induced gap junction closure under conditions
where stress fiber formation was fully inhibited (Fig. 8 B).
This indicates that gap junction closure is independent of
Rho signaling.
Tyrosine Kinase Involvement
We next examined the effects of tyrosine kinase and phosphatase inhibitors. Ortho-vanadate in conjunction with H2O2 (pervanadate) inhibits tyrosine phosphatases in vivo. Fig. 9 A shows that pervanadate treatment mimics agonist stimulation in causing complete inhibition of cell-cell communication in Rat-1 cells, albeit with slower kinetics, consistent with a lag required for vanadate entry into the cells.
|
|
Conversely, agonist-induced gap junction closure is fully
prevented by the tyrosine kinase inhibitors tyrphostin 25 and 47 (Fig. 9 B). In contrast, the Ser/Thr kinase inhibitor
staurosporine (100 nM) has no effect (not shown). The tyrphostins act selectively in that they do not affect activation
of the inward Cl current (Fig. 9 B; Postma et al., 1996
).
These results suggest that G protein-mediated gap junction closure is regulated by a tyrosine kinase pathway.
Involvement of c-Src
Src family tyrosine kinases are attractive candidates for
mediating gap junction closure since expression of active
v-Src (or overexpression of c-Src) in 3T3 cells reduces
junctional communication (Atkinson et al., 1981; Azarnia
et al., 1988
, 1989
; Crow et al., 1990
; Filson et al., 1990
). We
found that in v-Src-expressing Rat-1 cells (van der Valk et
al., 1987), cell-cell communication is almost completely inhibited, while the expression and cell surface distribution
of Cx43 is similar, at least qualitatively, to that in normal
Rat-1 cells (not shown). If endogenous Src regulates G
protein-mediated gap junction closure, then the agonists
used should rapidly activate c-Src. Indeed, LPA, thrombin,
and neuropeptides rapidly activate c-Src in Rat-1 cells and
other fibroblasts (Chen et al., 1994
; Rodriguez-Fernandez
and Rozengurt, 1996
; van Biesen et al., 1996; Kranenburg
et al., 1997
). In addition, a rapid transient increase in c-Src
activity is observed in response to NKA acting on the
transfected NK2 receptor in Rat-1 cells; although the response is relatively small, it is statistically significant (Fig.
10 A).
|
To examine whether c-Src is required for inhibition of
communication, we stably transfected Rat-1 cells with a kinase-inactive form of c-Src (SrcK). This Src mutant, in
which the ATP binding site is inactivated (K295M mutation; Roche et al., 1995
), should act in a dominant-negative manner by binding to proteins that interact with activated
Src family members. Fibroblasts expressing SrcK
are viable but show a somewhat reduced growth rate (not shown; see also Broome and Hunter, 1996
). When compared with
control cells, SrcK
-expressing cells exhibit similar basal
tyrosine phosphorylation patterns, whereas ligand-induced
tyrosine phosphorylation of cellular proteins (Hordijk et
al. 1994a
) is reduced (Fig. 10 B). When analyzed electrophysiologically, SrcK
cells showed normal cell-cell coupling. However, they failed to close their gap junctions in
response to receptor stimulation, as shown in Fig. 10 C. It
is also seen that gap junctions in SrcK
cells could still be
closed (in a reversible manner) by the general anesthetic
halothane. We note that expression of SrcK
does not interfere with activation of the inward Cl
current (Fig. 10
C). Furthermore, we tested fibroblasts derived from Src
/
mice (Bockholt and Burridge, 1995
). In these Src-deficient cells, LPA, thrombin, and endothelin failed to inhibit gap
junctional communication, whereas control cells showed a
normal response, as shown in Fig. 10 D. However, the Src-deficient cells showed no anomalies in LPA-induced inward
currents (not shown) or MAP kinase activation (Kranenburg et al., 1997
). Taken together, these results indicate that
c-Src is required for agonist-induced gap junction closure.
Having established a key role for c-Src in G protein- mediated gap junction closure, we examined the tyrosine phosphorylation state of Cx43 in Rat-1 cells under various conditions. First, we found that vanadate-induced gap junction closure is paralleled by prominent tyrosine phosphorylation of Cx43 (Fig. 11 A). Second, inhibition of junctional communication after v-Src expression is similarly associated with tyrosine phosphorylation of Cx43 (Fig. 11 B). However, our efforts to detect Cx43 tyrosine phosphorylation in response to LPA, TRP, or endothelin were unsuccessful under the conditions used.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
There has been much progress recently in understanding
the biosynthesis and structure of connexin-based gap junction channels, their role in embryonic development and
excitable tissue function, as well as their relationship to
certain human diseases (Bennett et al., 1991; Beyer, 1993
;
Musil, 1994; Paul, 1995
; Goodenough et al., 1996
). One
major unresolved question in the gap junction field is how
junctional communication is regulated by physiological agonists. Although it has long been recognized that junctional conductance and permeability can be modulated by
certain extracellular stimuli, relatively little progress has
been made in identifying receptor-linked signaling pathways that modulate connexin function.
In this study, we report a number of novel findings on
the regulation of Cx43-based cell-cell communication in
fibroblast monolayers. Using a newly developed single-electrode technique in conjunction with dye transfer and
biochemical experiments, we have demonstrated that Cx43-
based communication is acutely inhibited after stimulation
of a subclass of G protein-coupled receptors and characterized the underlying signal transduction events. Given its
resistance to PTX, receptor action must be mediated by
the Gq/11 or G12/13 subfamily of heterotrimeric G proteins. Regulation of cell-cell communication via G protein signaling has not been systematically examined to date. One
relevant example concerns anandamide, an arachidonate
derivative, which inhibits gap junctions in astrocytes in a
PTX-sensitive manner, but otherwise its mode of action is
unknown (Venance et al., 1995).
We find that G protein-induced gap junction closure
cannot be explained by known second messengers nor by
the membrane depolarization that consistently accompanies gap junction closure. Our results obtained with the
Rho-inactivating C3 toxin also rule out a role for receptor-mediated Rho signaling in gap junction closure. Previous studies have assigned a central role to PKC and MAP kinase in the regulation of Cx43-based communication (Kanemitsu and Lau, 1993; Hii et al., 1994
; for review see Goodenough et al., 1996
), while MAP kinase can phosphorylate
Cx43 in vitro (Warn-Cramer et al., 1996
). However, the
lack of effect of phorbol ester, PKC inhibitors, PTX, and
8Br-cAMP strongly argues against the involvement of these
Ser/Thr kinases in agonist-induced gap junction closure in
Rat-1 fibroblasts. That MAP kinase does not mediate gap
junction closure in Rat-1 cells is reinforced by the finding
that EGF, a strong activator of the Ras-MAP kinase pathway (van Corven et al., 1993; Hordijk et al., 1994a
; Kranenburg et al., 1997
) has only a minor effect on cell-cell
coupling (Fig. 5, B and C).
Instead, it appears that a c-Src tyrosine kinase pathway
links G protein-coupled receptors to gap junction closure.
This conclusion is based on the following findings. First, G
protein-mediated gap junction closure is prevented by tyrosine kinase inhibitors (tyrphostin 25 and 47) but not by
the Ser/Thr kinase inhibitor stauroporine, while it can be
mimicked by pervanadate, a tyrosine phosphatase inhibitor; also in hamster fibroblasts, pervanadate (but not other
phosphatase inhibitors) decreases cell-cell communication (Husoy et al. 1993). Second, G protein-mediated gap junction closure is accompanied by rapid activation of c-Src
(Chen et al., 1994
; van Biesen et al., 1996; Dikic et al.,
1996
; Rodriguez-Fernandez and Rozengurt, 1996
; Kranenburg et al., 1997
). Third, expression of constitutively active
Src inhibits communication. Fourth, dominant-negative c-Src
protects gap junctions from being closed by G protein-
coupled receptors. Finally, agonist-induced gap junction
closure is not observed in Src-deficient fibroblasts. The latter observation is of note since one might have anticipated
that other Src family members, notably Fyn or Yes, would
compensate for the lack of c-Src.
How does activation of the G protein-Src kinase pathway lead to inhibition of Cx43 function? One attractive
possibility is that inhibition is due to Src-mediated tyrosine
phosphorylation of Cx43. Indeed, enhanced tyrosine phosphorylation of Cx43, correlating with loss of communication, is readily detectable in both v-Src-expressing and
vanadate-treated cells (Fig. 11, A and B). Furthermore, it has been shown that mutation of a specific tyrosine residue in Cx43 (Tyr265) abolishes both inhibition of communication and tyrosine phosphorylation induced by v-Src in
a Xenopus oocyte expression system (Swenson et al.,
1990). The finding that Src family kinases are enriched in
cell-cell contacts (Tsukita et al., 1991
) would be consistent
with a direct action of Src kinases on Cx43. However, we
have not yet been able to detect enhanced tyrosine phosphorylation of Cx43 in response to receptor stimulation.
While this failure may well be due to technical limitations
(e.g., low stoichiometry of phosphorylation or the transient nature of such phosphorylation event), it remains
possible that c-Src acts in a more indirect manner, not involving tyrosine phosphorylation of Cx43. The possibility that enhanced serine phosphorylation of Cx43 is involved
in gap junction closure deserves some discussion. Serine
phosphorylation of Cx43 is thought to regulate intracellular trafficking of Cx43 and correlates with gap junction assembly and maintenance (Musil and Goodenough, 1991
;
Goodenough et al., 1996
). In addition, gap junction closure is often accompanied by enhanced serine phosphorylation of Cx43, which gives rise to a reduced electrophoretic
mobility of the protein (for example see Kanemitsu and
Lau, 1993
; Hii et al., 1994
). Although we do detect agonist-induced mobility shifts of Cx43 in Western blots, our analysis reveals that there is no simple correlation between
these mobility shifts (which occur relatively slowly) and gap
junction closure as judged by kinetic and pharmacological
criteria (Giepmans, B.N.G., unpublished observations).
The present study also addresses the mechanism responsible for the gradual recovery of communication occurring
in the continuous presence of agonist. Such recovery is
commonly observed in response to receptor stimulation.
By taking advantage of a truncated, desensitization-defective NK2 receptor, we find that recovery of Cx43-based
communication is controlled in large part at the level of
receptor desensitization, which in turn is regulated by
ligand-induced Ser/Thr phosphorylation of the receptor's
COOH-terminal tail (Alblas et al., 1995, 1996
). Mutant
NK2 receptor not only mediates prolonged uncoupling,
but it also mediates ligand-induced morphological transformation, as indicated by the deregulated, criss-cross
growth pattern that is observed after NKA addition to mutant NK2 receptor-expressing Rat-1 cells (Alblas et al.,
1996
). Thus, there is a striking correlation between the duration of junctional uncoupling and phenotypic transformation after (mutant) receptor stimulation, although cause-
effect relationships remain to be established.
It is tempting to speculate that temporary inhibition of
junctional communication is critical during normal wound
healing: after traumatic injury cells at the wound margin
are acutely exposed to agonists, including platelet-derived
LPA, thrombin, and endothelin, which shut off cell-cell
communication to prevent intercellular "leakage" of signaling molecules. In other words, by rapidly closing their
gap junctions cells at the wound edge may not arouse their quiescent neighbors. Further experiments are needed to
test this model. Receptor-mediated isolation of individual
cells from surrounding cells may also be relevant to the
concept of compartmental boundaries during embryonic
development (Kalimi and Lo, 1989; Godwin et al., 1993
).
Several outstanding questions remain to be addressed. In particular, what is the identity of the G protein subunit(s)
that mediate(s) gap junction closure? What is the nature
of the responsible G protein-linked effector(s)? And, does
c-Src act in a direct or indirect manner on Cx43? These issues are currently under investigation.
![]() |
Footnotes |
---|
Received for publication 17 June 1997 and in revised form 30 December 1997.
Jacqueline Alblas' current address is Department of Pulmonary Diseases, University Hospital Utrecht, 3584 CX Utrecht, The Netherlands.We thank L. Oomen for expert assistance with confocal microscopy, I. van Etten for cDNA transfections, S. Courtneidge for providing Src cDNAs, and P. Soriano and K. Burridge for Src-deficient fibroblasts.
This work was supported by the Dutch Cancer Society and the Netherlands Organization for Scientific Research.
![]() |
Abbreviations used in this paper |
---|
Cx43, connexin43; Et, endothelin; LPA, lysophosphatidic acid; LY, Lucifer yellow; MAP, mitogen-activated protein; NKA, neurokinin A; PKC, protein kinase C; PTX, pertussis toxin; TRP, thrombin receptor-activating peptide.
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
Alblas, J.,
I. van Etten,
A. Khanum, and
W.H. Moolenaar.
1995.
C-terminal truncation of the neurokinin-2 receptor causes enhanced and sustained agonist-induced signaling.
J. Biol. Chem.
270:
8944-8951
|
2. | Alblas, J., I. van Etten, and W.H. Moolenaar. 1996. Truncated, desensitization-defective neurokinin receptors mediate sustained MAP kinase activation, cell growth and transformation by a Ras-independent mechanism. EMBO (Eur. Mol. Biol. Organ.) J. 15: 3351-3360 [Abstract]. |
3. | Atkinson, M.M., A.S. Menko, R.G. Johnson, J.R. Sheppard, and J.D. Sheridan. 1981. Rapid and reversible reduction of junctional permeability in cells infected with a temperature-sensitive mutant of avian sarcoma virus. J. Cell Biol. 91: 573-578 [Abstract]. |
4. | Azarnia, R., S. Reddy, T.E. Kmiecik, D. Shalloway, and W.R. Loewenstein. 1988. The cellular src gene product regulates junctional cell-to-cell communication. Science 239: 398-401 |
5. | Azarnia, R., M. Mitcho, D. Shalloway, and W.R. Loewenstein. 1989. Junctional intercellular communication is cooperatively inhibited by oncogenes in transformation. Oncogene. 4: 1161-1168 |
6. | Bennett, M.V.L., L.C. Barrio, T.A. Bargiello, D.C. Spray, E. Hertzberg, and J.C. Saez. 1991. Gap junctions: new tools, new answers, new questions. Neuron 6: 305-320 |
7. | Beyer, E.C. 1993. Gap junctions. Int. Rev. Cytol. 137C:1-37. |
8. | Bigiani, A., and S.D. Roper. 1995. Estimation of the junctional resistance between electrically coupled receptor cells in Necturus taste buds. J. Gen. Physiol. 106: 705-725 [Abstract]. |
9. | Bockholt, S.M., and K. Burridge. 1995. An examination of focal adhesion formation and tyrosine phosphorylation in fibroblasts isolated from src-, fyn-, and yes- mice. Cell Adhes. Commun. 3: 91-100 |
10. |
Broome, M.A., and
T. Hunter.
1996.
Requirement for c-Src catalytic activity
and the SH3 domain in platelet-derived growth factor BB and epidermal
growth factor mitogenic signaling.
J. Biol. Chem.
271:
16798-16806
|
11. | Burghardt, R.C., R. Barhoumi, T.C. Sewall, and J.A. Bowen. 1995. Cyclic AMP induces rapid increases in gap junction permeability and changes in the cellular distribution of connexin43. J. Membr. Biol. 148: 243-253 |
12. |
Chen, Y.H.,
J. Pouyssegur,
S.A. Courtneidge, and
E. Van Obberghen-Schilling.
1994.
Activation of Src family kinases by the G protein-coupled thrombin receptor in growth-responsive fibroblasts.
J. Biol. Chem.
269:
27372-27377
|
13. | Crow, D.S., E.C. Beyer, D.L. Paul, S.S. Kobe, and A.F. Lau. 1990. Phosphorylation of connexin43 gap junction protein in uninfected and Rous sarcoma virus-transformed mammalian fibroblasts. Mol. Cell. Biol. 10: 1754-1763 |
14. | Dikic, I., G. Tokiwa, S. Lev, S.A. Courtneidge, and J. Schlessinger. 1996. A role for Pyk2 and src in linking G-protein-coupled receptors with MAP kinase activation. Nature. 383: 547-550 |
15. | Eckert, R., A. Dunina-Barkovskaya, and D.F. Huelser. 1993. Biophysical characterization of gap-junction channels in HeLa cells. Pfluegers Arch. 424: 335-342 |
16. | El-Fouly, M.H., J.E. Trosko, and C.C. Chang. 1987. Scrape-loading and dye transfer. A rapid and simple technique to study gap junctional intercellular communication. Exp. Cell Res. 168: 422-430 |
17. | Filson, A.J., R. Azarnia, E.C. Beyer, W.R. Loewenstein, and J.S. Brugge. 1990. Tyrosine phosphorylation of gap junction protein correlates with inhibition of cell-to-cell communication. Cell Growth Diff. 1: 661-668 [Abstract]. |
18. | Godwin, A.J., L.M. Green, M.P. Walsh, J.R. McDonald, D.A. Walsh, and W.H. Fletcher. 1993. In situ regulation of cell-cell communication by the cAMP-dependent protein kinase and protein kinase C. Mol. Cell. Biochem. 127/128: 293-307. |
19. | Goldberg, G.S., and A.F. Lau. 1993. Dynamics of connexin43 phosphorylation in pp60v-src-transformed cells. Biochem. J. 295: 735-742 |
20. | Goodenough, D.A., J.A. Goliger, and D.L. Paul. 1996. Connexins, connexons, and intercellular communication. Annu. Rev. Biochem. 65: 475-502 |
21. | Hii, C.S.T., S.-Y. Oh, S.A. Schmidt, K.J. Clark, and A.W. Murray. 1994. Lysophosphatidic acid inhibits gap-junctional communication and stimulates phosphorylation of connexin-43 in WB cells: possible involvement of the mitogen-activated protein kinase cascade. Biochem. J. 303: 475-479 |
22. |
Hordijk, P.L.,
I. Verlaan,
E.J. van Corven, and
W.H. Moolenaar.
1994a.
Protein tyrosine phosphorylation induced by lysophosphatidic acid in Rat-1 fibroblasts.
J. Biol. Chem.
269:
645-651
|
23. |
Hordijk, P.L.,
I. Verlaan,
K. Jalink,
E.J. van Corven, and
W.H. Moolenaar.
1994b.
cAMP abrogates the p21ras-MAP kinase pathway in fibroblasts.
J. Biol. Chem.
269:
3534-3538
|
24. | Hotz-Wagenblatt, A., and D. Shalloway. 1993. Gap junctional communication and neoplastic transformation. Crit. Rev. Oncog 4: 541-558 |
25. | Husoy, T., S.O. Mikalsen, and T. Sanner. 1993. Phosphatase inhibitors, gap junctional intercellular communication and 125I-EGF binding in hamster fibroblasts. Carcinogenesis. 14: 2257-2265 [Abstract]. |
26. | Jalink, K., and W.H. Moolenaar. 1992. Thrombin receptor activation causes rapid neural cell rounding and neurite retraction independent of classic second messengers. J. Cell Biol. 118: 411-419 [Abstract]. |
27. | Jalink, K., T. Hengeveld, S. Mulder, F.R. Postma, M.-F. Simon, H. Chap, G.A. van der Marel, J.H. van Boom, W.J. van Blitterswijk, and W.H. Moolenaar. 1995. Lysophosphatidic acid-induced Ca2+ mobilization in human A431 cells: structure-activity analysis. Biochem. J. 307: 609-616 |
28. |
Kahn, R.A..
1991.
Fluoride is not an activator of the smaller (20-25 kD) GTP-binding proteins.
J. Biol. Chem.
266:
15595-15597
|
29. | Kalimi, G.H., and C.W. Lo. 1989. Communication compartments in the gastrulating embryo. J. Cell Biol. 107: 241-255 [Abstract]. |
30. | Kanemitsu, M.Y., and A.F. Lau. 1993. Epidermal growth factor stimulates the disruption of gap junctional communication and connexin43 phosphorylation independent of 12-O-tetradecanoyl-phorbol 13-acetate-sensitive protein kinase C: the possible involvement of mitogen-activated protein kinase. Mol. Biol. Cell 4: 827-848 . |
31. |
Kranenburg, O.,
I. Verlaan,
P.L. Hordijk, and
W.H. Moolenaar.
1997.
Gi-mediated activation of the Ras-MAP kinase pathway involves a 100 kD tyrosine-phosphorylated Grb2 SH3-binding protein, but not Src nor Shc.
EMBO (Eur. Mol. Biol. Organ.) J.
16:
3097-3105
|
32. | Kumar, N.M., and N.B. Gilula. 1996. The gap junction communication channel. Cell 84: 381-388 |
33. | Lasater, E.M.. 1987. Retinal horizontal cell gap junctional conductance is modulated by dopamine through a cAMP-dependent protein kinase. Proc. Natl. Acad. Sci. USA. 84: 7319-7323 [Abstract]. |
34. | Lindau, M., and E. Neher. 1988. Patch-clamp techniques for time-resolved capacitance measurements in single cells. Pfluegers Arch. 411: 137-146 |
35. | Machesky, L.M., and A. Hall. 1996. Rho: a connection between membrane signaling and the cytoskeleton. Trends Cell Biol. 6: 304-310 . |
36. | Maldonado, P.E., B. Rose, and W.R. Loewenstein. 1988. Growth factors modulate cell-to-cell communication. J. Membr. Biol. 106: 203-210 |
37. | Mesnil, M., V. Kurtovskikh, C. Piccoli, C. Elfgang, O. Traub, K. Willecke, and H. Yamasaki. 1995. Negative growth control of HeLa cells by connexin genes: connexin species specificity. Cancer Res. 55: 629-639 |
38. | Moolenaar, W.H., O. Kranenburg, F.R. Postma, and G.C.M. Zondag. 1997. Lysophosphatidic acid: G protein signaling and cellular responses. Curr. Opin. Cell Biol. 9: 168-173 |
39. | Musil, L.S., and D.A. Goodenough. 1991. Biochemical analysis of connexin43 intracellular transport, phosphorylation and assembly into gap junction plaques. J. Cell Biol. 115: 1357-1374 [Abstract]. |
40. | Paul, D.L.. 1995. New functions for gap junctions. Curr. Opin. Cell Biol. 7: 665-672 |
41. | Postma, F.R., K.J. Jalink, T. Hengeveld, A.G.M. Bot, J. Alblas, H.R. de Jonge, and W.H. Moolenaar. 1996. Serum-induced membrane depolarization in quiescent fibroblasts: activation of a chloride conductance through the G protein-coupled LPA receptor. EMBO (Eur. Mol. Biol. Organ.) J. 15: 63-72 [Abstract]. |
42. | Rae, J., K. Cooper, P. Gates, and M. Watsky. 1991. Low access resistance perforated patch recordings using amphotericin B. J. Neurosci. Methods. 37: 15-26 |
43. | Ridley, A.J., and A. Hall. 1992. The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors. Cell 70: 389-399 |
44. | Roche, S., M. Koegl, M.V. Barone, M.F. Roussel, and S.A. Courtneidge. 1995. DNA synthesis induced by some but not all growth factors requires Src family protein tyrosine kinases. Mol. Cell. Biol. 15: 1102-1109 [Abstract]. |
45. |
Rodriguez-Fernandez, J.L., and
R. Rozengurt.
1996.
Bombesin, bradykinin, vasopressin, and phorbol esters rapidly and transiently activate Src family tyrosine kinases in Swiss 3T3 cells.
J. Biol. Chem.
271:
27895-27901
|
46. | Spray, D.C. 1994. Physiological and pharmacological regulation of gap junction channels. In Molecular Mechanisms of Epithelial Cell Junctions: From Development to Disease. S. Siti, editor. R.G. Landes Company, Austin, TX. 195-215. |
47. | Swenson, K.I., H. Piwnica-Worms, H. McNamee, and D.J. Paul. 1990. Tyrosine phosphorylation of the gap junction protein connexin43 is required for the pp60v-src-induced inhibition of communication. Cell Regul. 1: 989-1002 |
48. | Tsukita, S., K. Oishi, T. Akiyama, Y. Yamanashi, T. Yamamoto, and S. Tsukita. 1991. Specific proto-oncogenic tyrosine kinases of src family are enriched in cell-to-cell adherens junctions where the level of tyrosine phosphorylation is elevated. J. Cell Biol. 113: 867-879 [Abstract]. |
49. | Van Biesen, T., L.M. Luttrell, B.E. Hawes, and R.J. Lefkowitz. 1996. Mitogenic signaling via G protein-coupled receptors. Endocr. Rev. 17: 698-714 |
50. | Van Corven, E., A. Groenink, K. Jalink, T. Eichholtz, and W.H. Moolenaar. 1989. Lysophosphatidate-induced cell proliferation: identification and dissection of signaling pathways mediated by G proteins. Cell. 59: 45-54 |
51. | Van Corven, E.J., P.L. Hordijk, R.H. Medema, J. L. Bos, and W.H. Moolenaar. 1993. Pertussis toxin-sensitive activation of p21ras by G protein-coupled receptor agonists in fibroblasts. Proc. Natl. Acad. Sci. USA 90: 1257-1261 [Abstract]. |
52. |
Van der Valk, J.,
I. Verlaan,
S.W. de Laat, and
W.H. Moolenaar.
1987.
Expression of pp60v-src alters the ionic permeability of the plasma membrane in rat
cells.
J. Biol. Chem.
262:
2431-2434
|
53. | Venance, L., D. Piomelli, J. Glowinski, and C. Giaume. 1995. Inhibition by anandanmide of gap junctions and intercellular calcium signaling in strital astrocytes. Nature 376: 590-594 |
54. |
Warn-Cramer, B.J.,
P.D. Lampe,
W.E. Kurata,
M.Y. Kanemitsu,
L.W.L. Loo,
W. Eckhart, and
A.F. Lau.
1996.
Characterization of the mitogen-activated
protein kinase phosphorylation sites on the connexin-43 gap junction protein.
J. Biol. Chem.
271:
3779-3786
|