(Received for publication, September 17, 1996, and in revised form, January 29, 1997)
From the Departments of Molecular Physiology and
Biological Physics, § Pathology, and ¶ Internal
Medicine, University of Virginia Health Sciences Center,
Charlottesville, Virginia 22906-0011
We determined the relationship between the
localization of rhoA and Ca2+ sensitization of
force in smooth muscle. In -toxin-permeabilized rabbit portal vein
at pCa 6.5, the particulate hydrophobic fraction of
rhoA (10 ± 1.6% of the total) was significantly
increased by phenylephrine to 18 ± 5.5% at 5 min, by
AlF4
to 26 ± 8.4% at 20 min, and dose-dependently up to 62 ± 9.5% by
guanosine 5
-O-(3-thiotriphosphate) (GTP
S; 0.3-50
µM). Translocation of rhoA was selective
(Rac1 and Cdc42 were not translocated) and was quantitatively
correlated (up to ~50%; r = 0.91, p < 0.05) with Ca2+ sensitization; high
GTP
S concentrations (
10 µM) further increased translocation without increasing force. The initial recruitment of
rhoA to the membrane paralleled the time course of
contraction, but sensitization could be reversed without a decrease in
particulate rhoA. High [Ca2+]
(pCa 4.5) also increased particulate rhoA to
31 ± 5.8%. Membrane-associated rhoA in unstimulated
portal vein was a good substrate for in vitro ADP-ribosylation, whereas the large amount translocated by GTP
S was
not. We conclude that 1) translocation of rhoA plays a
causal role in Ca2+ sensitization, and 2) membrane-bound
rhoA can exist in two or more states.
Phosphorylation of the regulatory light chain of myosin
(MLC20)1 by a
calcium/calmodulin-dependent protein kinase is the primary determinant of force developed by smooth muscle. However, this phosphorylation can also be increased ("Ca2+
sensitization") at constant [Ca2+] by a
G-protein-coupled mechanism (1-4) that inhibits the trimeric phosphatase (SMPP-1M) (5-7) that dephosphorylates MLC20.
The Ca2+-sensitizing effect of recombinant p21rhoA
and the inhibition of agonist-induced Ca2+ sensitization by
selective ADP-ribosylation of p21rhoA (either recombinant or
endogenous) have implicated this monomeric G-protein in
Ca2+ sensitization (8-11). Because bacterially expressed
p21rhoA that lacks the prenylated C terminus required for
membrane association (12) did not show significant
Ca2+-sensitizing activity and even active
(geranylgeranylated) p21rhoA failed to
Ca2+-sensitize preparations heavily permeabilized with
Triton X-100 (11), we suggested that association of p21rhoA
with the plasma membrane may be required for its
Ca2+-sensitizing effect (11). Recruitment of cytosolic
proteins to the membrane is an important component of several other
signaling systems, such as the Raf-Ras pathway (13) and protein kinase C cascades (conventional and novel) (14). The purpose of this study was
to determine whether p21rhoA signaling of Ca2+
sensitization also involves its translocation to the cell membrane in vivo. We now show that GTPS-induced translocation of
p21rhoA is quantitatively and kinetically associated with
Ca2+ sensitization of smooth muscle and provide evidence of
more than one conformational state of membrane-associated
p21rhoA.
Small strips (200 µm wide and 3 mm long) of rabbit portal vein and ileum longitudinal smooth muscle were dissected, and isometric tension was measured as published (15-17).
Separation of Particulate and Cytosolic FractionsA minimum
of 10 small (200 µm wide and 3 mm long) strips of rabbit portal vein
or ileum longitudinal smooth muscle were used to provide sufficient
protein for reliable separation of cytosolic and particulate fractions.
Stimulated and control strips were homogenized in ice-cold
homogenization buffer (10 mM Tris-HCl, pH 7.5, 5 mM MgCl2, 2 mM EDTA, 250 mM sucrose, 1 mM dithiothreitol, 1 mM 4-(2-aminoethyl)bezenesulfonyl fluoride, 20 µg/ml
leupeptin, and 20 µg/ml aprotinin) and centrifuged at 100,000 × g for 30 min at 4 °C (OptimaTM TLX ultracentrifuge, TLA
120.1 rotor, Beckman Instruments), and the supernatant was collected as
the cytosolic fraction. Pellets were resuspended, and membrane proteins
were extracted by incubation for 30 min in homogenization buffer
containing 1% Triton X-100 and 1% sodium cholate or only 2% Triton
X-114. The latter buffer was used to avoid the increase in the cloudy point of Triton X-114 by a second detergent. The extract was
centrifuged at 800 × g for 10 min. The supernatant was
collected and is referred to as the particulate fraction, and the
pellet was collected and is referred to as the detergent-insoluble
particulate fraction. Cytosolic, particulate, and detergent-insoluble
particulate fraction proteins were separated by SDS-polyacrylamide gel
electrophoresis. Only the cytosolic and particulate p21rhoA
proteins are shown in most of the figures, as no immunoblot-detectable p21rhoA was found in the detergent-insoluble particulate
fraction. The absence of p21rhoA in the detergent-insoluble
particulate fraction verified the completion of the extraction of
membrane p21rhoA proteins and completion of homogenization.
Prompt termination of the reaction in homogenization buffer was
verified by the absence of translocation of p21rhoA when
control strips were homogenized in GTPS (50 µM)-containing homogenization buffer.
Precondensed Triton X-114 stock solution was added to tissue homogenates or cytosolic fractions to a final concentration of ~2%, and proteins were extracted by incubation for 30 min on ice with occasional mixing (18). The mixture was centrifuged at 10,000 × g for 10 min at 4 °C; the pellet was solubilized in sample buffer; and proteins were separated by SDS-polyacrylamide gel electrophoresis to determine cellular proteins insoluble in nonionic detergent. The supernatant was collected in a fresh tube and warmed to 37 °C in a water bath until the solution became cloudy (for ~5 min). Phase separation was achieved by centrifuging the solution in a tabletop centrifuge for 10 min at 800 × g at room temperature. The upper aqueous phase contains soluble proteins, and the lower, detergent-enriched phase contains proteins bearing hydrophobic domains.
Western BlotsAfter transfer to polyvinylidene difluoride membrane, the membranes were blocked with 5% nonfat dry milk in phosphate-buffered saline containing 0.05% Tween 20 for 1 h and then incubated with primary antibody for 3 h and secondary antibody for 1 h at room temperature. Blots were detected with enhanced chemiluminescence (ECL, Amersham Corp.) and quantitated by densitometry using a Bio-Rad GS-670 imaging densitometer. Optimal primary antibody concentration was determined by antibody titration (1:100, 1:500, 1:1000, and 1:5000) using a Mini-protein II multiscreen apparatus (Bio-Rad). Preliminary experiments established that the amount of protein loaded was within the range of linearity of the assays. The percent of particulate p21rhoA was calculated according to particulate p21rhoA/(particulate + cytosolic p21rhoA).
Monoclonal anti-p21rhoA antibody (Santa Cruz Biotechnology,
Inc., Santa Cruz, CA) generated against amino acids 120-150 of human p21rhoA was used at a 1:2500 dilution. Polyclonal
anti-Gq/11, anti-Rac1, and anti-Cdc42 antibodies (Santa
Cruz Biotechnology, Inc.) generated against amino acids 341-359 common
to G
q and G
11, amino acids 178-191 of
Rac1, and amino acids 166-182 of Cdc42 were used at 1:5000, 1:500 and
1:1000 dilutions, respectively.
The detergent concentration and the volumes
of cytosolic and particulate fractions were adjusted to identical
values, and the following reagents were added: 200 µM
GTP, 10 mM dithiothreitol, 2 mM thymidine, and
1 µg/ml Clostridium botulinum exoenzyme C3. After
initiation of ADP-ribosylation by addition of [32P]NAD
(final concentration of 50 µCi/ml), the mixture (total volume of 100 µl) was incubated for 30 min at 30 °C. The reaction was stopped by
trichloroacetic acid (24%, 250 µl) and deoxycholate (2%, 6 µl),
and the final volume was adjusted to 1 ml with water. After
centrifugation (5000 × g, 10 min), the supernatant was
carefully removed, and the pellet was resuspended in 20 µl of 2 × sample buffer. 10 µl of 1 M Tris base was added to
neutralize the pH. Samples were heated at 85 °C for 5 min, and the
proteins were separated by SDS-polyacrylamide gel electrophoresis.
ADP-ribosylation of p21rhoA in -escin-permeabilized strips
was carried out as described previously (11).
Details of the solutions used for study of permeabilized strips were
described previously (15-17). The pretreatment with A23187 and the
presence of 10 mM EGTA assured (15-17) that the changes in
force and MLC20 phosphorylation observed under these
conditions were not due to changes in [Ca2+]. -Toxin
was purchased from List Biological Laboratories Inc. (Campbell, CA).
GTP
S was from Boehringer (Mannheim, Germany). ADP-ribosyltransferase
C3, tautomycin, and A23187 were from Calbiochem. [32P]NAD
(30 Ci/mmol) was from DuPont NEN. Statistical comparisons were made
using Student's t test; all values are given as mean ± S.E.
Phenylephrine (PE; 100 µM) plus GTP
(10 µM) increased force from 13 ± 1.1%
(n = 18) to 41 ± 5.4% (n = 6, p < 0.001) of the maximal Ca2+-induced
contraction in -toxin-permeabilized rabbit portal vein strips at
constant free Ca2+ (pCa 6.5). Such contractions
are the result of increased MLC20 phosphorylation (2, 4).
Force development was accompanied by translocation of p21rhoA
from the cytosol to the particulate fraction. The particulate fraction
contained 10 ± 1.6% (n = 23) of the total
p21rhoA in control pCa 6.5 solution, increasing to
18 ± 5.5% (n = 9, p < 0.05)
upon stimulation with PE plus GTP (Fig.
1A).
GTPS translocated p21rhoA from the cytosol to the
particulate fraction in a concentration-dependent manner
(Fig. 1B; r = 0.9995, p < 0.001) from 10 ± 1.6% (n = 23) to 21 ± 4.2% (n = 8; 0.3 µM), to 31 ± 3.7% (n = 6; 1 µM), to 50 ± 6.9%
(n = 2; 10 µM), and 62 ± 9.5%
(n = 4; 50 µM). The translocation of
p21rhoA induced by GTP
S was accompanied by Ca2+
sensitization of force from 13% ± 1.1% (n = 18;
pCa 6.5) to 33 ± 4.7% (n = 6; 0.3 µM), to 49 ± 2.8% (n = 10; 1 µM), to 62 ± 2.1% (n = 8; 10 µM), and to 64 ± 1.4% (n = 8; 50 µM), respectively.
If PE-induced activation and translocation of p21rhoA mediates
Ca2+ sensitization in portal vein smooth muscle, then
ADP-ribosylation of p21rhoA would be expected to inhibit this
effect (see the Introduction). ADP-ribosylation of p21rhoA with
C3 (see "Materials and Methods") (19, 20) significantly inhibited
the Ca2+ sensitization of force in -escin-permeabilized
rabbit portal vein by PE plus GTP from 38 ± 3.1%
(n = 3) to 7 ± 0.7% (n = 3, p < 0.01) and by GTP
S (50 µM) from
52 ± 2.8% (n = 3) to 33 ± 2.0%
(n = 3, p < 0.05).
Several agonists acting on receptors coupled to heterotrimeric
G-protein can both activate the phosphatidylinositol cascade and cause
Ca2+ sensitization, as does
AlF4, an agent previously thought to
activate heterotrimeric, but not monomeric, G-proteins (21). However,
recently, we (11) and others (9) observed that ADP-ribosylation of
endogenous p21rhoA also inhibited Ca2+
sensitization of force by AlF4
.
Therefore, we determined whether AlF4
also translocated p21rhoA in portal vein.
AlF4
(10 mM NaF + 30 µM AlCl3, 20 min) increased force from
13 ± 1.1% (n = 18) to 53 ± 3.1%
(n = 4) of the maximal Ca2+-induced
contraction and particulate p21rhoA from the control value of
10 ± 1.6% (n = 23) to 26 ± 8.4%
(n = 4, p < 0.01) (Fig.
1A).
As shown in Fig. 1A, the translocation of p21rhoA,
up to 50%, was quantitatively correlated (r = 0.91, p < 0.05) with Ca2+ sensitization of force
induced by agonist (PE plus GTP),
AlF4, and various concentrations of
GTP
S. Higher concentrations (
10 µM) of GTP
S
caused further translocation of p21rhoA without further
increase in force, indicating a "ceiling effect."
To ascertain whether the observed translocation is -adrenergic
receptor-specific, we also determined the effect of a muscarinic agonist on the localization of p21rhoA in permeabilized ileum
smooth muscles. Surprisingly, a high percentage (61 ± 6.8%,
n = 14) of p21rhoA was located in the
particulate fraction of unstimulated ileum. This high basal level of
particulate p21rhoA was not due to Ca2+
(submaximal, pCa 6.5) because 59 ± 1.4%
(n = 2) of p21rhoA was particulate even at
cytoplasmic [Ca2+] < pCa 8 (no free
Ca2+ added, 10 mM EGTA present). In contrast,
inclusion of the muscarinic antagonist atropine (10 µM)
during and following dissection decreased particulate p21rhoA
to 28 ± 11.6% (n = 6, p < 0.05). This significant decrease in particulate p21rhoA by a
highly specific muscarinic antagonist indicates that acetylcholine released from nerve endings in the richly innervated ileum causes translocation of p21rhoA to the particulate fraction and that
such translocation is not limited to the action of
-adrenergic
agonists.
To determine the specificity of translocation
of p21rhoA by GTPS, we also determined the distribution of
two other Rho family proteins, Rac1 and Cdc42, under conditions
identical to those used for determining the partitioning of
p21rhoA. GTP
S (50 µM, 20 min) (Fig.
2) had no significant effect on the amount of either
Rac1 or Cdc42 present in the particulate fraction: 46 ± 2.9%
(n = 5) of Rac1 and 14 ± 4.1% (n = 5) of Cdc42 in pCa 6.5 solution and 47 ± 4.2%
(n = 6, p > 0.005) of Rac1 and 19 ± 4.8% (n = 6, p > 0.05) of Cdc42
after stimulation with GTP
S. Unlike p21rhoA and Cdc42, which
were not detected in the detergent-insoluble particulate fraction,
23 ± 7.0% (n = 5) of Rac1 was in the
detergent-insoluble particulate fraction, and this fraction was also
not changed by GTP
S (25 ± 5.7%, n = 6, p > 0.05).
Relaxation of Phenylephrine-induced Ca2+ Sensitization Is Unaccompanied by the Return of p21rhoA from the Particulate to the Cytosolic Fraction
To determine whether the return of p21rhoA from the particulate to the cytosolic fraction is required for the reversal of Ca2+ sensitization, the distribution of p21rhoA after "washout" of the agonist was determined. After increasing the steady-state pCa 6.5-induced contraction by PE plus GTP from 7 ± 2.2% (n = 3) to 32 ± 1.2% (n = 3, p < 0.001), the muscles were transferred into relaxing solution (no added Ca2+, PE, or GTP and containing 1 mM EGTA) and exchanged three times for a total of 25 min. At this time, the pCa 6.5-induced contraction was not significantly different from before exposure to PE plus GTP (9 ± 0.6%, n = 3), indicating that the muscles were no longer Ca2+-sensitized, but 21 ± 4.6% (n = 9) of p21rhoA still remained in the particulate fraction; this was not significantly different from that found in the presence of PE (18 ± 5.5%, n = 9, 5 min, p > 0.05). Even when strips were washed in 10 mM EGTA-containing solution for 60 min, the translocated p21rhoA remained in the particulate fraction (data not shown).
Time Courses of GTPThe time courses of
GTPS-induced potentiation of force and translocation of
p21rhoA were determined to evaluate whether they were
kinetically consistent with the potential role of p21rhoA as a
mediator of agonist-induced Ca2+ sensitization. As shown in
Fig. 3 (A and B), within 1 min
following addition of GTP
S to permeabilized portal vein smooth
muscle at pCa 6.5, force reached 21 ± 4.2%
(n = 10) of the maximal GTP
S-induced contraction;
this was accompanied by translocation of p21rhoA to the
particulate fraction, increasing from the control value of 10 ± 1.6% (n = 23) to 32 ± 9.7% (n = 6, p < 0.0001). Thus, within the time resolution of
this study, the kinetics of translocation of p21rhoA were
consistent with its role in GTP
S-induced Ca2+
sensitization of force. However, the later time course of
GTP
S-induced p21rhoA translocation was slower than that of
force development: contraction peaked at 5 min, at which time
~51 ± 4% (n = 6) of p21rhoA was in the
particulate fraction, whereas p21rhoA continued to translocate,
reaching its peak of 62 ± 9.5% (n = 4) at 20 min, consistent with the ceiling effect in the translocation-force relationship (Fig. 1A).
We also determined the time course of translocation of
Gq/11, the heterotrimeric G-protein implicated in
the activation of phospholipase C, a major contributor to
pharmacomechanical coupling in smooth muscle (reviewed in Ref. 4).
Under control conditions (pCa 6.5), 86 ± 1.7%
(n = 25) of the total G
q/11 was in the
particulate fraction, and this was reduced by GTP
S to 60 ± 12% (n = 6, p < 0.01) at 1 min and
70 ± 8.8% (n = 6, p < 0.05) at
5 min (Fig. 3, A and B). In contrast to
p21rhoA, the translocation of G
q/11 was
transient: by 60 min, the previously translocated protein had returned
to the particulate fraction (Fig. 3, A and
B).
The
hydrophobic domain of cytosolic, geranylgeranylated p21rhoA is
masked by bound Rho-GDI, and activated p21rhoA is thought to
bind to the cell membrane through the unmasked hydrophobic
geranylgeranyl group exposed by the release of Rho-GDI (22). Because
the particulate fraction obtained through centrifugation may contain
both hydrophobic (membrane) and nonhydrophobic (e.g. cytoskeletal) components, we determined by phase separation with Triton
X-114 whether the p21rhoA translocated to the particulate
fraction by GTPS in vivo was hydrophobic, as indicated by
partitioning into Triton X-114. Indeed, GTP
S (50 µM,
30 min) increased the fraction of p21rhoA partitioned into the
detergent phase when whole homogenates were treated with Triton X-114.
p21rhoA in the Triton X-114 phase increased from the control
value of 22 ± 6.6% (n = 11) to 81 ± 1.8%
(n = 6, p < 0.0001), indicating that
most of the particulate p21rhoA was associated with hydrophobic
(presumably membrane) components.
To further evaluate whether cytosolic p21rhoA (complexed with
Rho-GDI) is hydrophilic, whereas particulate p21rhoA is
hydrophobic, the whole homogenate was first separated into cytosolic
and particulate fractions, which were subsequently phase-separated with
Triton X-114 (see "Materials and Methods"). As shown in Fig. 4, only 5 ± 1.8% (n = 9) of the
cytosolic p21rhoA partitioned into the Triton X-114 phase. In
contrast, most of the particulate p21rhoA partitioned into the
Triton X-114 phase, with only 7 ± 2.5% (n = 8)
partitioning into the aqueous phase. Again, there was a dramatic increase from 11 ± 8.6% (n = 3) to 63 ± 13.5% (n = 3, p < 0.05) in the amount
of p21rhoA in the Triton X-114-treated particulate fraction of
GTPS (50 µM, 20 min)-stimulated muscles. The small
quantities of cytosolic p21rhoA partitioning into the Triton
X-114 phase and particulate p21rhoA partitioning into the
aqueous phase may result from "carryover" during the experimental
procedure.
Translocated p21rhoA Is Not a Good Substrate for C3-catalyzed ADP-ribosylation
The cytosolic and particulate
fractions of control and GTPS-stimulated tissues were incubated with
C3 and [32P]NAD (see "Materials and Methods") to
determine if the translocated p21rhoA is a good substrate for
C3-catalyzed ADP-ribosylation. In unstimulated strips, the largely
cytosolic p21rhoA (Figs. 1, 2, and 5) was only
minimally ADP-ribosylated; a very faint band was detected in the
autoradiograph (Fig. 5, upper left panel), whereas the very
small amount of particulate p21rhoA was highly ADP-ribosylated
(lower left panel). This is consistent with previous results
showing that cytosolic p21rhoA is complexed with Rho-GDI and
that the complex is a poor substrate for C3-catalyzed ADP-ribosylation
(23, 24), but becomes a better substrate after its dissociation from
Rho-GDI. In contrast, the large amount of particulate p21rhoA
in GTP
S-stimulated tissues (Figs. 1, 2, 3 and 5) was only minimally ADP-ribosylated (Fig. 5, right panels). This lower level of
ADP-ribosylation of particulate p21rhoA in GTP
S-stimulated
strips was not due to the loss of a membrane component to the cytosol
because ADP-ribosylation was significantly less even in total tissue
homogenates containing both cytosolic and particulate fractions of
GTP
S-treated tissue compared with controls (data not shown), and
cytosolic p21rhoA in GTP
S-stimulated tissue was still not a
good substrate for ADP-ribosylation (Fig. 5, right panels).
This was also not the result of reassociation with Rho-GDI because the
latter was not detectable in any of the particulate fractions. These
results suggest that membrane-associated p21rhoA exists in at
least two states: a resting state that is a good substrate and an
activated (and/or inactivated; see "Discussion") state that is a
poor substrate for C3-catalyzed ADP-ribosylation.
Effect of Ca2+ and of the Phosphatase Inhibitor Tautomycin on the Localization of p21rhoA
To
determine whether Ca2+ alone can induce translocation of
p21rhoA, -toxin-permeabilized rabbit portal vein strips were
incubated in Ca2+-free solution (no Ca2+ added
and containing 10 mM EGTA) for 15 min, homogenized, and fractionated. The particulate fraction under this Ca2+-free
condition contained 9 ± 3.0% (n = 6) of the
total p21rhoA, which is not significantly different from strips
incubated in pCa 6.5 solution: 10 ± 1.6%
(n = 23, p > 0.05). However,
increasing Ca2+ to very high levels (pCa 4.5, 15 min) increased the p21rhoA content of the particulate fraction
to 31 ± 5.8% (n = 10, p < 0.01). This translocation of p21rhoA induced by pCa
4.5 was not due to the release of norepinephrine from nerve endings
because inclusion of the
-adrenergic blocker prazosin (10 µM) in the solutions during permeabilization and thereafter had no effect on the pCa 4.5-induced
translocation of p21rhoA to the particulate fraction (39 ± 6.9%, n = 8, p > 0.05). To exclude
the possibility that the translocation of p21rhoA by
pCa 4.5 was due to trapping of cytosolic p21rhoA in
the cytoskeletal components of the contracted tissue, cytosolic and
particulate fractions of pCa 4.5-stimulated tissues were
phase-separated by Triton X-114. Similar to the GTP
S-stimulated
tissues, 89 ± 8.6% (n = 3) of p21rhoA in
the particulate fraction (29.0 ± 13.9% (n = 3)
of the total p21rhoA) was partitioned into the Triton X-114
phase, suggesting that high [Ca2+] alone can translocate
p21rhoA to the membrane.
To determine whether p21rhoA translocated by high [Ca2+] returns to the cytosol after removal of Ca2+, portal vein strips stimulated with pCa 4.5 for 15 min were washed five times in Ca2+-free solution (no Ca2+ added and containing 10 mM EGTA) for a total of 60 min, and the distribution of p21rhoA was determined. Even after this extensive wash, the same amount (38 ± 2%, n = 3) of translocated p21rhoA remained in the particulate fraction.
The Ca2+ dependence of GTPS-induced translocation of
p21rhoA was also determined by adding GTP
S to
-toxin-permeabilized smooth muscle in the presence and absence of
Ca2+. GTP
S (50 µM, 60 min) caused similar
p21rhoA translocation in Ca2+-free, 10 mM EGTA-containing solution as in pCa 6.5 solution: p21rhoA in the particulate fraction was 65 ± 6.9% (n = 6) versus 62 ± 9.5%
(n = 4, p > 0.05), respectively.
Tautomycin, a potent inhibitor of protein phosphatases 1 and 2A, (25) causes substantial MLC20 phosphorylation and smooth muscle contraction even in the absence of Ca2+ (26) by inhibiting the catalytic subunit of SMPP-1M. We also wished to localize p21rhoA in tautomycin-stimulated tissues to determine whether phosphorylation of a protein that can be dephosphorylated by phosphatase 1 or 2A is involved in regulating translocation of p21rhoA. No significant translocation of p21rhoA was detected in tautomycin-stimulated tissue at pCa 6.5 (data not shown), although it caused 86 ± 6.3% (n = 3) of the maximal Ca2+-induced contraction.
The relationships between the extent and time course of
translocation of p21rhoA to the particulate fraction (Figs. 1
and 3) and enhancement of force at constant [Ca2+] are
consistent with a causal role of p21rhoA recruitment to the
membrane in Ca2+ sensitization. This conclusion is also
supported by the hydrophobicity of particulate p21rhoA
(partitioning into Triton X-114; this study), the abolition of the
Ca2+-sensitizing effect of recombinant p21rhoA by
extensive permeabilization of smooth muscle with detergent, and the
inactivity of nonprenylated p21rhoA (11). Similarly,
GTPS-induced activation of NADPH oxidase in neutrophils involves the
dissociation of p21rho from Rho-GDI and its
translocation to the membrane by a mechanism that requires a heat- and
trypsin-labile membrane component (27). The translocation was limited
to p21rhoA in our study: the localization of two other Rho
family proteins, Rac1 and Cdc42, was not affected by GTP
S under
conditions causing extensive translocation of p21rhoA.
The -adrenergic agonist phenylephrine, GTP
S, and
AlF4
induced translocation of
p21rhoA, whereas the muscarinic antagonist atropine inhibited
the translocating effect of endogenous acetylcholine. The significant
reduction by atropine of the large amount of particulate
p21rhoA in "unstimulated" ileum smooth muscle and the
translocation of p21rhoA by high [Ca2+] indicate
that preparatory conditions alone can affect its localization. Thus,
caution is required in interpreting results of fractionation obtained
in the presence of high [Ca2+] and/or locally stored and
released transmitters.
Because agonists acting on heptameric serpentine receptors coupled to
trimeric G-proteins induced Ca2+ sensitization and because
AlF4 was not reported to interact with
Ras family proteins (21), we had previously thought that the
Ca2+ sensitization by AlF4
was mediated by a trimeric G-protein (4). Subsequently, we (11) and
others (9) found that ADP-ribosylation of p21rhoA inhibited
AlF4
-induced Ca2+
sensitization, and we now show (Fig. 1) that
AlF4
, like agonists and GTP
S, also
translocates p21rhoA to the membrane. Furthermore, it has now
been shown that although AlF4
cannot
interact with another monomeric G-protein (elongation factor G) in
solution, such an interaction can occur when elongation factor G is
associated with ribosomes (28). On the other hand, other reports show
p21rhoA to be a downstream effector of G
12- and
G
13-induced stress fiber formation (29) and of
G
q-initiated hypertrophic signaling to cardiomyocytes
(30). Neither these G
subunits nor agonist-induced
Ca2+ sensitization (11) is sensitive to pertussis toxin,
suggesting that they could act as upstream initiators (activated by
AlF4
) of a p21rhoA-mediated
cascade. At present, we consider it equally likely that a complex of
p21rhoA with a cytosolic protein can directly interact with
AlF4
.
A larger fraction of cytoplasmic p21rhoA (>60% of the total)
could be translocated by GTPS (50 µM) to the membrane
than required for maximal Ca2+ sensitization of force. This
ceiling effect presumably reflects the fact that the extent (~50%)
(2) of inhibition of SMPP-1M attainable by G-protein-coupled mechanisms
is achieved by activating only ~50% of endogenous p21rhoA
(Fig. 1A). The p21rhoA translocated to smooth muscle
membranes did not return to the cytosol during 60 min of incubation
without the agonist, although Ca2+ sensitization of force
by this time was already reversed. This is consistent with the finding
that activated p21rhoA associated with the particulate fraction
is transformed, with time, into an inactivated membrane-bound form,
and/or its downstream effector(s) is down-regulated (31). According to
a recent report, rho translocation in cultured fibroblasts
is rapidly reversible (32). The difference between these and our
findings may be tissue-dependent or indicate an inhibitory
effect of permeabilization (this study) on the reversibility of
translocation.
The large cytosolic fraction of p21rhoA present in unstimulated
smooth muscle was not ADP-ribosylated, whereas ADP-ribosylation (Fig.
5) of the small amount of membrane-associated p21rhoA was
extensive and sufficient to inhibit the Ca2+-sensitizing
effect of the -adrenergic agonist PE and of GTP
S. In other types
of smooth muscle, ADP-ribosylation of endogenous p21rhoA only
slowed GTP
S-induced Ca2+ sensitization without reducing
its amplitude (rabbit mesenteric artery). It also inhibited
Ca2+ sensitization by carbachol (ileum smooth muscle) (9,
11) and the increase in MLC20 phosphorylation induced by
GTP
S in cultured smooth muscle cells (33).
The lack of correlation between p21rhoA detected by Western blotting and ADP-ribosylation, respectively, in several tissues (Ref. 24 and this study) raises the possibility that the variable and often incomplete (agonist- and tissue-dependent) inhibition of Ca2+ sensitization by in situ ADP-ribosylation may reflect slow recruitment of p21rhoA from a cytosolic pool protected from ADP-ribosylation by Rho-GDI. Alternatively, other p21rhoA-independent pathways of Ca2+ sensitization may account for incomplete inhibition of Ca2+ sensitization following ADP-ribosylation of endogenous p21rhoA.
The variable availability of particulate p21rhoA for
ADP-ribosylation by C3 (Fig. 5) suggests that membrane-associated
p21rhoA can exist in more than one (conformational) state. One
state is membrane-associated under resting conditions, available for ADP-ribosylation and presumably ready, "with the trigger cocked," to be activated by an agonist, GTPS, or
AlF4
. This would account for the more
rapid rate of Ca2+ sensitization achieved by agonists or
GTP
S than by exogenous p21rhoA (11). Another state (or
states) is much less available for ADP-ribosylation, as indicated by
the large quantity of p21rhoA that was translocated by GTP
S
to the membrane, but not ADP-ribosylated (Fig. 5). This may represent a
combination of transitional states being activated through membrane
association, as well as a third membrane-bound but inactivated state of
p21rhoA (31). However, we cannot completely rule out the
possibility that a very small fraction of activated p21rhoA
that was not translocated is involved in Ca2+
sensitization.
Tautomycin, a potent inhibitor of protein phosphatases 1 and 2, did not translocate p21rhoA, although it had a large "Ca2+-sensitizing" effect on contraction (see also Ref. 26). Therefore, it is unlikely that phosphorylation of a site susceptible to dephosphorylation by these phosphatases plays a role in the translocation of p21rhoA. The possibility of a role of tyrosine phosphorylation in this process is currently under study.
Ca2+-sensitizing mechanisms, whether mediated by protein kinase C (34, 35, 41) or other effectors, appear to converge to inhibit dephosphorylation of a cytosolic SMPP-1M substrate, MLC20 (2). Smooth muscle myosin light chain phosphatase is not known to be associated with the plasma membrane. Therefore, p21rhoA-coupled Ca2+ sensitization is thought to involve the participation of additional downstream messengers/mechanisms, such as inhibitory phosphorylation of the regulatory subunit of SMPP-1M by a p21rhoA-regulated and/or SMPP-1M-associated kinase (36-40) or an atypical protein kinase C (or a related kinase) (41), and/or activation of a phosphatase inhibitor (1). SMPP-1M can also be inhibited directly by arachidonic acid (42). It is likely that multiple mechanisms, including some mediated by p21rhoA, are involved in Ca2+ sensitization.
We thank Barbara Nordin for preparation of the manuscript and Jama Coartney for preparation of the figures.