Translocation of rhoA Associated with Ca2+ Sensitization of Smooth Muscle
1997; Elsevier BV; Volume: 272; Issue: 16 Linguagem: Inglês
10.1074/jbc.272.16.10704
ISSN1083-351X
AutoresMing Gong, Hideyoshi Fujihara, Avril V. Somlyo, Andrew P. Somlyo,
Tópico(s)Neuroscience and Neuropharmacology Research
ResumoWe determined the relationship between the localization of rhoA and Ca2+ sensitization of force in smooth muscle. In α-toxin-permeabilized rabbit portal vein at pCa 6.5, the particulate hydrophobic fraction ofrhoA (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) (GTPγS; 0.3–50 μm). Translocation ofrhoA 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 GTPγS concentrations (≥10 μm) further increased translocation without increasing force. The initial recruitment ofrhoA 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 vitroADP-ribosylation, whereas the large amount translocated by GTPγS was not. We conclude that 1) translocation of rhoA plays a causal role in Ca2+ sensitization, and 2) membrane-boundrhoA can exist in two or more states. We determined the relationship between the localization of rhoA and Ca2+ sensitization of force in smooth muscle. In α-toxin-permeabilized rabbit portal vein at pCa 6.5, the particulate hydrophobic fraction ofrhoA (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) (GTPγS; 0.3–50 μm). Translocation ofrhoA 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 GTPγS concentrations (≥10 μm) further increased translocation without increasing force. The initial recruitment ofrhoA 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 vitroADP-ribosylation, whereas the large amount translocated by GTPγS was not. We conclude that 1) translocation of rhoA plays a causal role in Ca2+ sensitization, and 2) membrane-boundrhoA can exist in two or more states. Phosphorylation of the regulatory light chain of myosin (MLC20) 1The abbreviations used are: MLC, myosin light chain; GTPγS, guanosine 5′-O-(3-thiotriphosphate); PE, phenylephrine; GDI, guanine nucleotide dissociation inhibitor. 1The abbreviations used are: MLC, myosin light chain; GTPγS, guanosine 5′-O-(3-thiotriphosphate); PE, phenylephrine; GDI, guanine nucleotide dissociation inhibitor. 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 (1Somlyo A.P. Kitazawa T. Himpens B. Matthijs G. Horiuti K. Kobayashi S. Goldman Y.E. Somlyo A.V. Adv. Protein Phosphatases. 1989; 5: 181-195Google Scholar, 2Kitazawa T. Masuo M. Somlyo A.P. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9307-9310Crossref PubMed Scopus (281) Google Scholar, 3Hartshorne D.R. Johnson L.R. Physiology of the Gastrointestinal Tract. Raven Press, Ltd., New York1987: 423-482Google Scholar, 4Somlyo A.P. Somlyo A.V. Nature. 1994; 372: 231-236Crossref PubMed Scopus (1712) Google Scholar) that inhibits the trimeric phosphatase (SMPP-1M) (5Alessi D. MacDougall L.K. Sola M.M. Ikebe M. Cohen P. Eur. J. Biochem. 1992; 210: 1023-1035Crossref PubMed Scopus (324) Google Scholar, 6Shimizu H. Ito M. Miyahara M. Ichikawa K. Okubo S. Konishi T. Naka M. Tanaka T. Hirano K. Hartshorne D.J. Nakano T. J. Biol. Chem. 1994; 269: 30407-30411Abstract Full Text PDF PubMed Google Scholar, 7Shirazi A. Iizuka K. Fadden P. Mosse C. Somlyo A.P. Somlyo A.V. Haystead T.A.J. J. Biol. Chem. 1994; 269: 31598-31606Abstract Full Text PDF PubMed Google Scholar) that dephosphorylates MLC20. The Ca2+-sensitizing effect of recombinant p21rhoAand 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 (8Hirata K. Kikuchi A. Sasaki T. Kuroda S. Kaibuchi K. Matsuura Y. Seki H. Saida K. Takai Y. J. Biol. Chem. 1992; 267: 8719-8722Abstract Full Text PDF PubMed Google Scholar, 9Fujita A. Takeuchi T. Nakajima H. Nishio H. Hata F. J. Pharmacol. Exp. Ther. 1995; 274: 555-561PubMed Google Scholar, 10Itagaki M. Komori S. Unno T. Syuto B. Ohashi H. Jpn. J. Pharmacol. 1995; 67: 1-7Crossref PubMed Scopus (31) Google Scholar, 11Gong M.C. Iizuka K. Nixon G. Browne J.P. Hall A. Eccleston J.F. Sugai M. Kobayashi S. Somlyo A.V. Somlyo A.P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1340-1345Crossref PubMed Scopus (264) Google Scholar). Because bacterially expressed p21rhoA that lacks the prenylated C terminus required for membrane association (12Hori Y. Kobe J. Med. Sci. 1992; 38: 79-92PubMed Google Scholar) did not show significant Ca2+-sensitizing activity and even active (geranylgeranylated) p21rhoA failed to Ca2+-sensitize preparations heavily permeabilized with Triton X-100 (11Gong M.C. Iizuka K. Nixon G. Browne J.P. Hall A. Eccleston J.F. Sugai M. Kobayashi S. Somlyo A.V. Somlyo A.P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1340-1345Crossref PubMed Scopus (264) Google Scholar), we suggested that association of p21rhoAwith the plasma membrane may be required for its Ca2+-sensitizing effect (11Gong M.C. Iizuka K. Nixon G. Browne J.P. Hall A. Eccleston J.F. Sugai M. Kobayashi S. Somlyo A.V. Somlyo A.P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1340-1345Crossref PubMed Scopus (264) Google Scholar). Recruitment of cytosolic proteins to the membrane is an important component of several other signaling systems, such as the Raf-Ras pathway (13Marshall C.J. Nature. 1996; 383: 127-128Crossref PubMed Scopus (99) Google Scholar) and protein kinase C cascades (conventional and novel) (14Hug H. Sarre T.F. Biochem. J. 1993; 291: 329-343Crossref PubMed Scopus (1213) Google Scholar). The purpose of this study was to determine whether p21rhoA signaling of Ca2+sensitization also involves its translocation to the cell membranein vivo. We now show that GTPγS-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 (15Kitazawa T. Kobayashi S. Horiuti K. Somlyo A.V. Somlyo A.P. J. Biol. Chem. 1989; 264: 5339-5342Abstract Full Text PDF PubMed Google Scholar, 16Kobayashi S. Kitazawa T. Somlyo A.V. Somlyo A.P. J. Biol. Chem. 1989; 264: 17997-18004Abstract Full Text PDF PubMed Google Scholar, 17Kobayashi S. Gong M.C. Somlyo A.V. Somlyo A.P. Am. J. Physiol. 1991; 260: C364-C370Crossref PubMed Google Scholar). A 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 (Optima™ 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 p21rhoAproteins 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 GTPγS (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 (18Bordier C. J. Biol. Chem. 1981; 256: 1604-1607Abstract Full Text PDF PubMed Google Scholar). 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. After 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-Gαq/11, anti-Rac1, and anti-Cdc42 antibodies (Santa Cruz Biotechnology, Inc.) generated against amino acids 341–359 common to Gαq and Gα11, 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 μmGTP, 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 β-escin-permeabilized strips was carried out as described previously (11Gong M.C. Iizuka K. Nixon G. Browne J.P. Hall A. Eccleston J.F. Sugai M. Kobayashi S. Somlyo A.V. Somlyo A.P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1340-1345Crossref PubMed Scopus (264) Google Scholar). Details of the solutions used for study of permeabilized strips were described previously (15Kitazawa T. Kobayashi S. Horiuti K. Somlyo A.V. Somlyo A.P. J. Biol. Chem. 1989; 264: 5339-5342Abstract Full Text PDF PubMed Google Scholar, 16Kobayashi S. Kitazawa T. Somlyo A.V. Somlyo A.P. J. Biol. Chem. 1989; 264: 17997-18004Abstract Full Text PDF PubMed Google Scholar, 17Kobayashi S. Gong M.C. Somlyo A.V. Somlyo A.P. Am. J. Physiol. 1991; 260: C364-C370Crossref PubMed Google Scholar). The pretreatment with A23187 and the presence of 10 mm EGTA assured (15Kitazawa T. Kobayashi S. Horiuti K. Somlyo A.V. Somlyo A.P. J. Biol. Chem. 1989; 264: 5339-5342Abstract Full Text PDF PubMed Google Scholar, 16Kobayashi S. Kitazawa T. Somlyo A.V. Somlyo A.P. J. Biol. Chem. 1989; 264: 17997-18004Abstract Full Text PDF PubMed Google Scholar, 17Kobayashi S. Gong M.C. Somlyo A.V. Somlyo A.P. Am. J. Physiol. 1991; 260: C364-C370Crossref PubMed Google Scholar) that the changes in force and MLC20 phosphorylation observed under these conditions were not due to changes in [Ca2+]. α-Toxin was purchased from List Biological Laboratories Inc. (Campbell, CA). GTPγS 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 α-toxin-permeabilized rabbit portal vein strips at constant free Ca2+ (pCa 6.5). Such contractions are the result of increased MLC20 phosphorylation (2Kitazawa T. Masuo M. Somlyo A.P. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9307-9310Crossref PubMed Scopus (281) Google Scholar, 4Somlyo A.P. Somlyo A.V. Nature. 1994; 372: 231-236Crossref PubMed Scopus (1712) Google Scholar). Force development was accompanied by translocation of p21rhoAfrom 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.1 A). GTPγS translocated p21rhoA from the cytosol to the particulate fraction in a concentration-dependent manner (Fig. 1 B; 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 GTPγS 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") (19Narumiya S. Morii N. Cell. Signalling. 1993; 5: 9-19Crossref PubMed Scopus (50) Google Scholar, 20Aktories K. Hall A. Trends Pharmacol. Sci. 1989; 10: 415-418Abstract Full Text PDF PubMed Scopus (118) Google Scholar) significantly inhibited the Ca2+ sensitization of force in β-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 GTPγS (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 (21Kahn R.A. J. Biol. Chem. 1991; 266: 15595-15597Abstract Full Text PDF PubMed Google Scholar). However, recently, we (11Gong M.C. Iizuka K. Nixon G. Browne J.P. Hall A. Eccleston J.F. Sugai M. Kobayashi S. Somlyo A.V. Somlyo A.P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1340-1345Crossref PubMed Scopus (264) Google Scholar) and others (9Fujita A. Takeuchi T. Nakajima H. Nishio H. Hata F. J. Pharmacol. Exp. Ther. 1995; 274: 555-561PubMed Google Scholar) 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.1 A). As shown in Fig. 1 A, 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 GTPγS. Higher concentrations (≥10 μm) of GTPγS caused further translocation of p21rhoA without further increase in force, indicating a "ceiling effect." To ascertain whether the observed translocation is α-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 p21rhoAto 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 α-adrenergic agonists. To determine the specificity of translocation of p21rhoA by GTPγS, 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. GTPγS (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 GTPγS. 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 GTPγS (25 ± 5.7%, n = 6,p > 0.05). 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-statepCa 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). The time courses of GTPγS-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 GTPγS to permeabilized portal vein smooth muscle at pCa 6.5, force reached 21 ± 4.2% (n = 10) of the maximal GTPγS-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 GTPγS-induced Ca2+sensitization of force. However, the later time course of GTPγS-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. 1 A). We also determined the time course of translocation of Gαq/11, the heterotrimeric G-protein implicated in the activation of phospholipase C, a major contributor to pharmacomechanical coupling in smooth muscle (reviewed in Ref. 4Somlyo A.P. Somlyo A.V. Nature. 1994; 372: 231-236Crossref PubMed Scopus (1712) Google Scholar). Under control conditions (pCa 6.5), 86 ± 1.7% (n = 25) of the total Gαq/11 was in the particulate fraction, and this was reduced by GTPγS 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 Gαq/11 was transient: by 60 min, the previously translocated protein had returned to the particulate fraction (Fig. 3, A andB). 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 (22Mohr C. Just I. Hall A. Aktories K. FEBS Lett. 1990; 275: 168-172Crossref PubMed Scopus (25) Google Scholar). 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 GTPγS in vivo was hydrophobic, as indicated by partitioning into Triton X-114. Indeed, GTPγS (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 GTPγS (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. The cytosolic and particulate fractions of control and GTPγS-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 (23Bourmeyster N. Stasia M.-J. Garin J. Gagnon J. Boquet P. Vignais P.V. Biochemistry. 1992; 31: 12863-12869Crossref PubMed Scopus (58) Google Scholar, 24Fritz G. Lang P. Just I. Biochim. Biophys. Acta. 1994; 1222: 331-338Crossref PubMed Scopus (20) Google Scholar), but becomes a better substrate after its dissociation from Rho-GDI. In contrast, the large amount of particulate p21rhoAin GTPγS-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 GTPγS-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 GTPγS-treated tissue compared with controls (data not shown), and cytosolic p21rhoA in GTPγS-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. To determine whether Ca2+ alone can induce translocation of p21rhoA, α-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 α-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 bypCa 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 GTPγS-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 GTPγS-induced translocation of p21rhoA was also determined by adding GTPγS to α-toxin-permeabilized smooth muscle in the presence and absence of Ca2+. GTPγS (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, (25MacKintosh C. Klumpp S. FEBS Lett. 1990; 277: 137-140Crossref PubMed Scopus (185) Google Scholar) causes substantial MLC20 phosphorylation and smooth muscle contraction even in the absence of Ca2+ (26Gong M.C. Cohen P. Kitazawa T. Ikebe M. Masuo M. Somlyo A.P. Somlyo A.V. J. Biol. Chem. 1992; 267: 14662-14668Abstract Full Text PDF PubMed Google Scholar) 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. 1and 3) and enhancement of force at constant [Ca2+] are consistent with a causal role of p21rhoA recruitment to the membrane in Ca2+
Referência(s)