©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Primary Structure of a Subunit of G Protein, , and Its Phosphorylation by Protein Kinase C (*)

(Received for publication, August 14, 1995; and in revised form, September 22, 1995)

Rika Morishita Hiroshi Nakayama (1) Toshiaki Isobe (1) Takahiko Matsuda (2) Yuichi Hashimoto (2) Toshiyuki Okano (2) Yoshitaka Fukada (2) Keiko Mizuno (3) Shigeo Ohno (3) Osamu Kozawa Kanefusa Kato Tomiko Asano (§)

From the  (1)Department of Biochemistry, Institute for Developmental Research, Aichi Human Service Center, Kasugai, Aichi 480-03, the Department of Chemistry, Faculty of Science, Tokyo Metropolitan University, Hachioji 192-03, the (2)Department of Life Sciences, College of Arts and Sciences, University of Tokyo, Komaba, Tokyo 153, and the (3)Department of Molecular Biology, Yokohama City University, School of Medicine, Kanazawa-ku, Yokohama 236, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

We have determined the primary structure of a novel subunit (, previously designated ) of G protein purified from bovine spleen. The mature protein composed of 68 amino acids had acetylated serine at the N terminus and geranylgeranylated/carboxylmethylated cysteine at the C terminus. This was consistent with the C-terminal prenylation signal in the amino acid sequence, which was predicted from cDNA isolated from a bovine spleen cDNA library. Western blots with the specific antibody against showed that is present in all tissues examined. Among various subunits ((1), (2), (3), (7), and ), has a unique property to be phosphorylated by protein kinase C. The phosphorylated amino acid residue was Ser^1 (or Ser^2). The phosphorylated beta associated with G(o)alpha more tightly than the unphosphorylated form. Exposure of Swiss 3T3 and aortic smooth muscle cells to phorbol 12-myristate 13-acetate and NaF induced phosphorylation of . Stimulation of aortic smooth muscle cells with natural vasoactive agents such as angiotensin II and vasopressin also induced phosphorylation of . The extent of phosphorylation of betain vitro was suppressed by a complex formation with G(o)alpha, which was relieved by the addition of guanosine 5`-O-(3-thiotriphosphate) or aluminum fluoride. These results strongly suggest that is phosphorylated by protein kinase C during activation of receptor(s) and G protein(s) in living cells.


INTRODUCTION

Heterotrimeric G proteins (^1)play a major role in the transduction of signals from cell surface receptors to cellular effectors(1) . G proteins are composed of alpha, beta, and subunits, and the beta and subunits form a tight complex under non-denaturing conditions. Upon activation, the GTP-bound alpha subunit interacts directly with various effectors. The beta complex also regulates several effectors, which include K channels, adenylyl cyclase, phospholipase A(2), phospholipase C-beta, receptor kinases, and phosphatidylinositol 3-kinase(1, 2) .

Analysis of purified proteins and cloned cDNAs has revealed the existence of multiple forms of beta and subunits in addition to many isoforms of the alpha subunit(1, 2) . The amino acid sequences of the five isoforms of beta subunit (beta(1)-beta(5)) are 53-90% identical to one another(1, 2) , while nine kinds of mammalian subunit, (1), (2), (3), (4), (5), (7), two (8)s(3, 4, 5, 6, 7, 8, 9, 10, 11) , and (previously designated , (12) ), have more diverged sequences. Functional differences among various forms of beta complexes have been attributed to the rather than to the beta subunit(12, 13, 14, 15) . Especially, the biological properties of beta(1), a beta complex of transducin, is noticeably different from those of the other beta complexes(12, 13, 14, 15) . Although the properties of the latter beta complexes resemble one another, their tissue distribution varies. The (1) and one of the (8)s are specifically expressed in retinal rods and cones, respectively(3, 4, 11) . Another (8) is expressed only in olfactory and vomeronasal neuroepithelia(10) , while the (3) is localized only in the brain(7, 9, 16, 17) . By contrast, (2), (5), and (7) are distributed in a variety of tissues(7, 9, 16, 17) .

It is well known that many hormones, neurotransmitters, and growth factors activate certain isozymes of phospholipase C that hydrolyze phosphatidylinositol 4,5-bisphosphate to produce inositol 1,4,5-trisphosphate and diacylglycerol. They regulate the release of Ca ions from intracellular stores and the activity of protein kinase C (PKC), respectively(18, 19) . Ca ions and PKC play crucial roles in the signal transduction mechanisms(19) . In many cell lines, the agonist-induced phospholipase C reactions are inhibited by phorbol esters such as PMA, a potent activator of PKC(19) . Such inhibition by PKC activators appears to provide a feedback control on agonist-stimulated phospholipase C. The activation of PKC can also affect the function of the receptor-adenylyl cyclase reactions in different cell types(19) . Although the mechanisms of this inhibition by PKC has not been clarified, several groups have investigated the effect of PKC on G proteins(20, 21, 22, 23, 24) . Treatment of human platelets with PMA or thrombin induced the selective phosphorylation of G(z)alpha and attenuated the ability of agonists both to suppress formation of cAMP and to stimulate hydrolysis of phosphoinositides(21) . Similarly, PMA and agonists such as glucagon, vasopressin, and angiotensin II caused to phosphorylate Galpha and to attenuate to inhibit adenylyl cyclase in hepatocytes(22) . However, there is no direct evidence to show that phosphorylation of alpha subunits of G or G(z) indeed decreases their physiological activities. In addition to alpha subunit, phosphorylation of unknown isoforms of beta and subunits of G protein was observed in vitro(23, 24) , but it has not been shown that PKC phosphorylates these subunits in intact cells nor that the phosphorylation changes their biological activities.

In this study, we found that a novel subunit of G protein, , was a good substrate for PKC among various forms of examined in vitro. The phosphorylation of by PKC was confirmed in intact cells, supporting its physiological relevance.


EXPERIMENTAL PROCEDURES

Materials

Five forms of the beta complex (designated beta(1), beta(2), beta(3), beta(7), and beta) were purified from bovine retina (25) and brain and spleen(12) . Each beta complex mainly contained beta(1)(12) . The and (5) subunits were isolated from purified beta complexes of bovine spleen by a reversed-phase HPLC(16, 26) . G(o)alpha was purified from bovine brain by the method of Asano et al.(27) . Purified G(o)alpha was conjugated to EAH-Sepharose 4B (Pharmacia Biotech Inc.) with 1-ethyl-3-(dimethylaminopropyl)-carbodiimide, and the amount of immobilized G(o)alpha was estimated to be approximately 1 mg/ml gel.

PKC was purified from rat or rabbit brains by the method of Kitano et al.(28) with a modification. PKCbeta was separated from PKC fraction of the rabbit brain by a hydroxyapatite column(29) . Other isozymes of PKC (PKCalpha, , , and ) were highly purified from recombinant baculovirus-infected Sf21 cells(30) . 1 unit of PKC is defined as the amount of enzyme that catalyzes the transfer of 1 nmol of phosphate from ATP to histone type III-S per min at 30 °C.

A peptide N-acetyl-SSKTASTNNC corresponding to residues Ser^1-Asn^9 of appended with cysteine for coupling purpose was synthesized. Antisera against were raised in rabbits by the injection of the synthetic peptide conjugated to keyhole limpet hemocyanin. The antibody was purified from antisera by the use of a column of Sepharose to which peptide had been covalently coupled. Antibodies against the other subunits have been previously generated with the individual peptides(17) . Antibodies against (2), (3), and (5) were specific for the respective subunit, while the antibody against (7), raised with the C-terminal peptide of Gly-Cys, reacted not only with (7) but also with (2), (3), and (17) .

Enzymatic Cleavage of and Purification of Peptide Fragments

Approximately 20 µg of isolated were digested by incubation at 37 °C for 16 h with 2 µg of Staphylococcus aureus V8 protease in 20 mM Tris-HCl (pH 7.5), 0.1 mM EDTA, and 0.2% SDS. After incubation, the reaction mixture was loaded onto a reversed-phase HPLC column to separate peptide fragments(16) . Arginyl endopeptidase (Arg-C, Boehringer) digestion of (ca. 2 µg) was performed at 37 °C for 16 h in 0.1 M Tris-HCl (pH 8.1) at an enzyme/substrate ratio of 1/20. A portion of the digest was analyzed directly by a capillary column liquid chromatography-mass spectrometry (LC/MS) system for peptide mapping and amino acid sequencing.

Approximately 40 µg of beta complex were incubated at 30 °C for 30 min with 40 µg of µ-calpain (from porcine erythrocytes, >120 units/mg protein; Nacalai Tesque) in 50 mM Hepes-NaOH (pH 7.4), 1 mM dithiothreitol, 1 mM CaCl(2), and 0.06% sodium cholate. The reaction was terminated by the addition of Lubrol PX, EGTA, and E64 (an inhibitor of calpain) at final concentrations of 0.05%, 7 mM and 45 µg/ml, respectively. The reaction mixture was then dialyzed against 10 mM Tris-HCl (pH 7.5), 0.1 mM dithiothreitol, and 0.05% Lubrol PX and loaded onto a reversed-phase Cosmosil 5C8 column (4.6 times 150 mm; Nacalai Tesque). Proteolytic fragments were eluted with a linear gradient of 10-90% acetonitrile in 0.1% trifluoroacetic acid.

Phosphorylated and unphosphorylated beta (0.25 µg of protein) were incubated at 30 °C for 30 min with various amounts of µ-calpain in the same medium mentioned above. The reaction was stopped by the addition of an equal volume of 2 times sample buffer for Tricine/SDS-PAGE(31) , and the reaction mixture was subjected to electrophoresis.

Capillary Column LC/MS

The molecular weight of and the amino acid sequence of its N-terminal fragment were determined by a capillary column LC/MS system. The system consisted of a high performance liquid chromatograph (model 140A, Applied Biosystems), which was connected to a triple-stage quadrupole mass spectrometer equipped with an electrospray interface (ESI/TSQMS, model TSQ-700, Finnigan MAT, San Jose, CA). The protein or peptide samples were injected to a reversed-phase capillary column (0.25 times 100 mm, Biotech Research, Saitama, Japan) packed with Capcell Pak C18 (particle size 3 µm; Shiseido, Tokyo) and eluted with a 40-min gradient of 0-80% acetonitrile in 0.1% trifluoroacetic acid. The pump was operated at 100 µl/min, and the flow was split prior to the sample injector (model 7725, Rheodyne) so that an optimal flow (2.5 µl/min) was delivered to the capillary column without a long delay time. The eluate was mixed at the post-column with a mixture of 2-methoxyethanol/methanol/water/acetic acid (60/20/20/1) to assist ionization and introduced to the electrospray interface of the mass spectrometer. The spectrometer was operated under the following conditions: electrospray voltage, 4.5 kV; heating capillary temperature, 200 °C; electron multiplier voltage, 1,200 V for peptide mapping and 1,600 V for sequencing.

cDNA Cloning of

Bovine spleen poly(A) RNA was used to construct cDNA library in gt11 phage. The spleen cDNAs were also used for polymerase chain reaction templates to amplify DNA fragment. Degenerate oligonucleotide primers, 5`-AC(A/C/G/T)AA(C/T)AA(C/T)AT(A/C/T)GC(A/C/G/T)CA(A/G)GC-3` and 5`-CA(A/G)CA(C/T)TT(C/T)TT(A/G)TC(C/T)TT(A/G)AA(A/C/G/T)GG-3`, were synthesized according to the amino acid residues Thr^7-Ala and Pro-Thr of for the following polymerase chain reactions: the first 5 cycles at 95 °C for 30 s, 45 °C for 30 s, and 72 °C for 1 min; 25 cycles at 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min; and the final cycle at 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 5 min. The polymerase chain reaction products were cloned and sequenced to reveal that four out of six independent clones had a partial sequence of bovine (7)(9) , and two had a partial of . The latter was used as a probe for screening of the bovine spleen cDNA library (4 times 10^5 clones). The cDNA clones that hybridized strongly to the probe were purified and subcloned in pUC119 for sequencing both strands.

Phosphorylation of beta Complexes in Vitro

Phosphorylation of various beta complexes by PKC was carried out as described previously (24) with a minor modification.

For electrophoretic analyses, various beta complexes (0.5 µg each) in a solution containing sodium cholate (final concentration of 0.06-0.12%) were incubated with PKC (0.03 units) in a reaction mixture that contained 20 mM Tris-HCl (pH 7.5), 5 mM magnesium acetate, 10 µM [-P]ATP (500-1,000 cpm/pmol), 0.5 mM CaCl(2), 40 µg/ml phosphatidylserine, and 0.8 µg/ml diolein (total volume 75 µl) at 30 °C for 1 h, unless otherwise specified. After incubation, 3 µl of a solution of 10% SDS was added to each reaction mixture, which was then lyophilized. The residue was incubated in sample buffer for Tricine/SDS-PAGE at 40 °C for 30 min and subjected to Tricine/SDS-PAGE. The gel was stained with silver or Coomassie Blue, dried, and subjected to autoradiography at -80 °C. The amount of phosphate incorporated into was estimated by Cerenkov counting of the appropriate band cut out from the gel.

For functional analyses, beta (25 µg) was treated with PKC (0.15 units) in the presence and in the absence of 100 µM unlabeled ATP (total volume, 150 µl) at 30 °C for 20 min. After incubation, each reaction mixture was dialyzed against 20 mM Hepes-NaOH (pH 8.0), 0.1 mM dithiothreitol, 0.3% sodium cholate at 4 °C for 4 h. The resultant samples are referred to as phosphorylated and unphosphorylated beta, respectively.

Phosphorylation of beta in Cultured Cells

Rat aortic smooth muscle cells were prepared as described previously(32) . Swiss 3T3 fibroblasts and smooth muscle cells were grown in Dulbecco's modified Eagle's medium that contained 10% fetal calf serum at 37 °C in a humidified atmosphere of 5% CO(2), 95% air. Cells at confluence (60-mm dishes) were used for experiments. For labeling with [P]orthophosphate, Swiss 3T3 cells and rat aortic smooth muscle cells were washed with 4 ml of serum- and phosphate-free medium (Eagle's minimum essential medium without sodium phosphate). The washed cells were preincubated for 1 h at 37 °C with 2 ml of the same medium that had been supplemented with 0.4 mCi of carrier-free [P]orthophosphate, and then they were incubated for 1 h further with 0.1 µM 4alpha-PMA, 0.1 µM PMA, 40 mM NaF, 1 µM angiotensin II, or 1 µM arginine vasopressin. After labeling, the cells were washed with ice-cold phosphate-buffered saline, kept frozen at -80 °C for 1 h, and then suspended in 0.6 ml of a solution that contained 20 mM Tris-HCl (pH 8.0), 1 mM EDTA, 1% sodium cholate, 0.1% SDS, 3 mM benzamidine, 10 µM leupeptin, 1 mM phenylmethylsulfonyl fluoride, 2 µg/ml trypsin inhibitor, and 100 mM NaF. This suspension was sonicated in a bath-type sonicator and centrifuged at 55,000 times g for 30 min. The supernatant (400 µg of protein) was incubated at room temperature overnight with 20 µg of affinity-purified antibody against (7), which recognizes both the phosphorylated and unphosphorylated forms of , and then 50 µl of a suspension of protein A-Sepharose beads (0.5 mg/ml) were added to the mixture for further incubation at room temperature for 1 h. The Sepharose beads were washed with 0.5 ml of TEN buffer (20 mM Tris-HCl, pH 8.0, 1 mM EDTA, and 0.1 M NaCl) containing 1% sodium cholate and 0.1% SDS. The beads were then incubated at 40 °C for 30 min with 45 µl of the sample buffer for electrophoresis, and 20 µl of each resultant supernatant were subjected to Tricine/SDS-PAGE.

Binding of beta to Galpha-Agarose

Phosphorylated and unphosphorylated beta were incubated for 30 min at room temperature in G(o)alpha-conjugated Sepharose columns, which had been equilibrated with TEN buffer containing 0.05% Lubrol PX. Then the column was successively washed with TEN buffer containing 0.05% Lubrol PX, with TEN buffer containing 0.4% or 1% Lubrol PX and with TEN buffer containing 0.05% Lubrol PX. The protein bound to the column was eluted with Tris-HCl (pH 8.0), 1 mM EDTA, 0.3 M NaCl, 0.05% Lubrol PX, and 5 µM GDP and AMF (20 µM AlCl(3), 6 mM MgCl(2), and 10 mM NaF), as described previously(33) . The amount of beta in the eluate was quantitated by an enzyme immunoassay(27) .

Other Methods

Tricine/SDS-PAGE was performed by the method of Schägger and von Jagow(31) . Immunoblotting was performed as described previously (17) by the use of 3,3`-diaminobenzidine or chemiluminescence reagent (Renaissance, DuPont NEN). Cholate extracts of various rat tissues were prepared as described(17) . Purified proteins were quantitated by the method of Schaffner and Weissmann(34) , and proteins in cholate extracts from cells were quantitated with a Micro BCA protein assay kit (Pierce) with bovine serum albumin as the standard. The analysis of phosphoamino acid was performed as described previously(35) . Pertussis toxin-catalyzed ADP-ribosylation was monitored by the method of Asano et al.(12) .


RESULTS

Primary Structure of

Although the N terminus of was blocked, most of the sequence was determined by Edman degradation of the three fragments produced by calpain or S. aureus V8 protease (assigned fragment 1-3 in Fig. 1A). To determine the N-terminal sequence including the blocked structure, the Arg-C digest of was analyzed by a capillary LC-ESI/TSQMS system, and one of the fragments (fragment 4 in Fig. 1A) with a mass of 1492 was selected for sequence analysis using collision-activated dissociation (CAD) tandem mass spectrometry. The doubly charged peptide ion [M+2H] with m/z = 746.1 was fragmented by CAD, and the product ions detected in the third quadrupole mass spectrometer were analyzed to obtain the sequence information (Fig. 1B). As expected from the specificity of Arg-C, y" series ion signals were significantly detected on the CAD spectrum of fragment 4 due to the positive charge of the C-terminal Arg. A series of product ions (Fig. 1B) showed a N-terminal sequence, Ac-SSKTASTNN(I/L)AQAR. On the other hand, the LC/MS analysis demonstrated that the intact has a molecular mass of 7,925 daltons (average mass value). This value was accounted for by the sequence shown in Fig. 1A, which predicted that cysteine residues at positions 42 and 68 (C terminus) and that the C-terminal cysteine was geranylgeranylated and carboxylmethylated. These modifications common to many isoforms(2, 11, 16, 36, 37, 38) are consistent with our previous observation that geranylgeranyl (CH) groups were released from by Raney nickel treatment (16) .


Figure 1: Amino acid sequence of bovine protein determined by Edman degradation and mass spectrometry. A, the amino acid sequence of bovine and summary of the sequence analysis. The protein was digested with µ-calpain, S. aureus V8 protease (V8), and Arg-C, and fragments were analyzed with Edman degradation (fragments 1-3) or mass spectrometry (fragment 4). Two cysteine residues at positions 42 and 68 were predicted from a molecular mass of the intact (see text). B, CAD spectrum of an Arg-C fragment of . A doubly charged ion of the fragment (m/z = 746.1) was analyzed by capillary LC/MS/MS. Ions of type y" and b are labeled. The amino acid sequence reconstructed from these product ions is shown in the figure by the single letter code.



The primary structure of thus predicted was perfectly matched with that deduced from the nucleotide sequence of cDNA isolated from bovine spleen cDNA library (data not shown). The cloned cDNA had a 216-base pair open reading frame encoding a 72-amino acid protein. A comparison of the deduced amino acid sequence of and other known sequences of subunits is shown in Fig. 2. The sequence identity between the translated products of and the other subunits ranged from 36% ((1)) to 76% ((7)). The deduced amino acid sequence Cys-Thr-Ile-Leu at the C terminus of coincided with the consensus sequence for geranylgeranylation at the cysteine residue(16, 37, 38) . The C-terminal leucine selects geranylgeranyl like (2), (3), (5), (7), and instead of farnesyl to be linked to (1) and ending with serine(11, 36) . Taken together, we concluded that is composed of 68 amino acid residues having acetylated serine at N terminus and C-terminal cysteine modified with geranylgeranyl and methyl groups.


Figure 2: Comparison of amino acid sequences of various isoforms of subunit of G protein. The amino acid sequence of is aligned with the sequences of (1)(3, 4) , (2)(5, 6) , (3)(7) , (5)(8) , (7)(9) , (localized in olfactory neurons; (10) ), and (localized in cone, (11) ). The sequences of three additional subunits, (4), , and have been recently identified(51) . Amino acid residues conserved among more than six sequences are boxed.



During the sequence analysis, we noticed that µ-calpain caused a limited proteolysis in (see Fig. 5), which resulted in truncation of the N-terminal tripeptide including the site of PKC phosphorylation (see below).


Figure 5: Proteolysis of phosphorylated and unphosphorylated beta by µ-calpain. Unphosphorylated (A) and P-phosphorylated (B and C) beta (0.25 µg) were incubated at 30 °C for 30 min with various amounts (lanes 1-4: 0, 0.5, 1, and 2 µg, respectively) of calpain in the presence of 1 mM CaCl(2). Each reaction was terminated by the addition of 7 mM EGTA, and the samples were subjected to Tricine/SDSPAGE. The gels were stained with silver (A and B) or subjected to autoradiography (C).



Tissue Distribution of

Antisera were raised in rabbits against the synthetic peptide corresponding to Ser^1-Asn^9 of . The affinity-purified antibody specifically reacted with among six isoforms of tested (Fig. 3A). This antibody revealed ubiquitous distribution of in rat tissues (Fig. 3B). The rat showed slightly slower migration than bovine (Fig. 3B), which was used as a standard protein in the Tricine/SDS-PAGE. This is probably due to the species but not due to the modification such as phosphorylation (see below).


Figure 3: Reactivity of the antibody against and tissue distribution of in the rat. A, five forms of the purified beta complex (0.5 µg each) and free (5) (0.1 µg) were subjected to Tricine/SDS-PAGE, and the gel was then stained with Coomassie Blue (beta(1)) or silver (other beta complexes or (5)) or it was immunoblotted with antibody against . Immunoreactive proteins were visualized with 3,3`-diaminobenzidine. B, cholate extracts (30 µg of protein) of various rat tissues and the purified bovine beta (10 ng) were subjected to Tricine/SDS-PAGE and immunoblotted with the antibody against . Immunoreactive proteins were visualized by a chemiluminescence reaction. Seminal v., seminal vesicle.



Phosphorylation of by PKC

We found that, among five forms of beta complex, beta(1), beta(2), beta(3), beta(7), and beta, only was efficiently phosphorylated by PKC purified from rat brain (Fig. 4A). To see which of the enzymes in this PKC preparation was responsible for phosphorylation, various kinds of purified isozyme of PKC were tested. Then, was phosphorylated most efficiently by conventional PKC (cPKC) alpha and beta, modestly by novel PKC (nPKC) and , but not by atypical PKC (aPKC) (Fig. 4B).


Figure 4: Phosphorylation of by PKC. A, phosphorylation of various forms of the beta complex of G proteins by PKC. Various beta complexes containing different subunits (0.5 µg each) were incubated at 30 °C for 1 h with PKC (0.03 units) in a reaction mixture that contained 20 mM Tris-HCl (pH 7.5), 5 mM magnesium acetate, 10 µM [-P]ATP, 0.5 mM CaCl(2), 40 µg/ml phosphatidylserine, and 0.8 µg/ml diolein. The reaction was terminated by the addition of SDS, and samples were subjected to Tricine/SDS-PAGE. Proteins were visualized by staining with Coomassie Blue (beta(1)) or silver (other beta complexes). The gel was dried and subjected to autoradiography at -80 °C. Numbers on the right indicate molecular masses in kDa. B, phosphorylation of by various isozymes of PKC. Purified beta (0.5 µg) was incubated at 30 °C for 1 h with various isozymes of PKC (0.01 units) in a reaction mixture that contained 20 mM Tris-HCl (pH 7.5), 5 mM magnesium acetate, 10 µM [-P]ATP, 0.5 mM CaCl(2), 40 µg/ml phosphatidylserine, and 50 ng/ml PMA. Samples were subjected to Tricine/SDS-PAGE and then to autoradiography. C, time course of phosphorylation of by PKC. Purified beta (3.5 µg) was incubated at 30 °C with PKC (0.21 units) in the reaction mixture described in A, in a total volume of 525 µl. At the indicated times, aliquots (75 µl) were withdrawn for analysis by Tricine/SDS-PAGE.



As shown in Fig. 4A, the PKC-catalyzed phosphorylation gave two protein bands of in the Tricine/SDS-PAGE, and the more slowly migrating band coincided with the phosphorylated one identified by the autoradiogram (Fig. 4A). This was clearly shown by the time course study of the phosphorylation of by PKC (Fig. 4C). The time-dependent increase in density of the upper band paralleled the phosphate incorporation, indicating the phosphorylation slightly slowed down the electrophoretic mobility in the gel. Maximal incorporation of phosphate was about 0.8 mol/mol of , suggesting that a single phosphorylation site in .

To identify the phosphorylated amino acid in , P-labeled was isolated by HPLC, hydrolyzed with 5.7 M HCl, and subjected to two-dimensional thin layer electrophoresis, which identified a phosphoserine (data not shown). The phosphoserine seemed to be in the N-terminal region, since the antibody against the N-terminal peptide of did not react with phosphorylated (see Fig. 6B). Consistently, the phosphorylation site was estimated to be Ser^1 or Ser^2, because P-labeled beta lost radioactivity upon treatment even with a smaller amount of µ-calpain (Fig. 5, B and C), which specifically removed Ser^1-Lys^3 from (Fig. 1A). The N-truncated large fragment of (Fig. 5B) gave no positive signal in the autoradiogram (Fig. 5C). In N-terminal two serines, Ser^1 is more likely to be the phosphorylation site, because Ser^1 fulfills the consensus sequence of PKC (39) .


Figure 6: Phosphorylation of in cultured cells that contain as a major subunit. A, identification of isoforms of G protein in Swiss 3T3 and aortic smooth muscle cells. As standard proteins (lane 1 in each panel), various isoforms of the subunit (or beta complex) were subjected to Tricine/SDS-PAGE (from left to right panels): 10 ng of beta(2), 10 ng of beta(3), 2 ng of (5), 10 ng of beta, and a mixture of 10 ng each of beta (upper band) and beta(7) (lower band). In all of the panels, the cholate extracts (15 µg of protein) of Swiss 3T3 (lane 2) and aortic smooth muscle cells (lane 3) were electrophoresed and immunoblotted with antibodies against (2), (3), (5), , and (7). The bands stained with the antibody against (7) (lanes 2 and 3 in the right end panel) were assigned as because (i) the antibody against (7) cross-reacted with (17) and (ii) the mobility of the positive band in each lane was identical to that stained with the antibody against . B, effect of PMA on the phosphorylation of in Swiss 3T3 cells. Swiss 3T3 cells were incubated with 0.1 µM 4alpha-PMA (lane 3) or 0.1 µM PMA (lane 4) for 1 h. Cholate extracts of cells (15 µg of protein) and standard proteins (10 ng each; lane 1, unphosphorylated beta; lane 2, phosphorylated beta) were subjected to Tricine/SDS-PAGE and immunoblotted with antibodies against and (7). C, effect of PMA, NaF, and various hormones on the phosphorylation of in Swiss 3T3 and aortic smooth muscle cells. Swiss 3T3 (3T3) and aortic smooth muscle cells (SMC) were labeled with [P]orthophosphate for 1 h and then incubated with 0.1 µM 4alpha-PMA (lane 1), 0.1 µM PMA (lane 2), 40 mM NaF (lane 3), 1 µM angiotensin II (lane 4), or 1 µM arginine vasopressin (lane 5) for 1 h. Cholate extracts isolated from the stimulated cells were immunoprecipitated with antibodies against (7) as described under ``Experimental Procedures,'' and an aliquot of the immunoprecipitate was subjected to Tricine/SDS-PAGE with subsequent autoradiography. The position of the phosphorylated form of bovine is indicated by an arrow. Numbers on the right indicate molecular masses in kDa.



Here, we should note the difference in susceptibility of to µ-calpain between phosphorylated and unphosphorylated states. In comparison with unphosphorylated beta (Fig. 5A), higher concentration of µ-calpain was required to cleave the N-terminal part of in phosphorylated beta (Fig. 5B). This observation suggests that the PKC-catalyzed phosphorylation possibly at the N-terminal region of induced a conformational change of beta.

Phosphorylation of in Cultured Cells

By using antibodies specific for each isoform, we found that mouse Swiss 3T3 cells and rat cultured aortic smooth muscle cells contained as a major subunit and less abundant (5) (Fig. 6A). As shown in the forth panel, in mouse Swiss 3T3 cells (lane 2) had the same mobility to bovine (lane 1) during Tricine/SDS-PAGE, but in rat aortic smooth muscle cells (lane 3) showed slightly slower migration than bovine and mouse . The similar result was obtained with antibody against (7), which reacted with as well as (7) and detected only in these cells (Fig. 6A, right end panel). This is consistent with the data of Fig. 3B, which showed the difference in mobility between bovine and that in various rat tissues. beta purified from bovine spleen, as well as in unstimulated cells, seems to have only unphosphorylated as judged from the absence of the upper band corresponding to the phosphorylated (Fig. 4C and Fig. 6B). This may reflect a short life-time (including dephosphorylation) of phosphorylated in vivo and/or in vitro.

In these two cultured cell systems, we tested whether is phosphorylated by PKC in vivo. First, Swiss 3T3 cells were treated with a direct activator of PKC (PMA), and cholate extracts of these cells were analyzed by Western blots with both antibodies against and (7) (Fig. 6B). Although the antibody against did not react with phosphorylated , the results showed that PMA treatment decreased the amount of unphosphorylated in the cells (lane 4 in left panel). By contrast, the antibody against (7) could react both unphosphorylated and phosphorylated , and the extract from PMA-treated cells showed an additional upper band corresponding to phosphorylated , indicating that PMA induced phosphorylation of in the intact cells. Pretreatment of cells with staurosporine, an inhibitor of PKC(40) , completely blocked PMA-stimulated phosphorylation of (data not shown). In addition, down-regulation of PKC by long-term pretreatment of Swiss 3T3 cells with PMA (41) attenuated PMA-induced phosphorylation of (data not shown). These results suggested that phosphorylation was catalyzed by PKC.

To examine whether is indeed phosphorylated in the cells, Swiss 3T3 and aortic smooth muscle cells were prelabeled with [P]orthophosphate and then tested with PMA, NaF, angiotensin II, or vasopressin, which are known to stimulate PKC either directly or indirectly. Cholate extracts of these stimulated cells were immunoprecipitated with antibody against (7), and the precipitated proteins were subjected to Tricine/SDS-PAGE and autoradiography. Exposure of both lines of cells to PMA significantly stimulated the phosphorylation of , while the inactive isomer, 4alpha-PMA, had little effect (Fig. 6C). In the aortic smooth muscle cells, the phosphorylation of was also stimulated by the addition of natural vasoactive agents such as angiotensin II and arginine vasopressin, which are known to activate phospholipase C(42) . In addition, direct activation of G protein(s) in both lines of cells by NaF induced moderate phosphorylation of . All these data strongly suggested that is phosphorylated by PKC during activation of receptors and G proteins in vivo.

Effects of Phosphorylation on the Function of beta

To identify the physiological role of the phosphorylation of , purified beta was maximally phosphorylated by PKC, and the functional difference between phosphorylated and unphosphorylated forms of beta was investigated. We found that betadependent ADP-ribosylation reaction of G(o)alpha catalyzed by pertussis toxin accelerated 10-20% by the phosphorylation of (Fig. 7A). As it is known that the rate of the ADP-ribosylation reflects alpha-beta coupling of G protein, our observation suggests that the phosphorylation increases the affinity of beta for G(o)alpha. We confirmed it by using G(o)alpha-conjugated agarose columns, to which phosphorylated or unphosphorylated beta was applied. The amount of phosphorylated beta (Fig. 7, B and C), which was eluted by increasing the concentration of Lubrol PX, was much less than that of unphosphorylated beta. Both forms of beta bound to the column were eluted with the buffer containing AMF as described previously(33) , indicating that beta was bound to the column through a specific interaction with G(o)alpha. Lower recovery of phosphorylated beta was ascribed to its tighter binding to the column because a larger amount of phosphorylated beta was observed when SDS extracts of G(o)alpha-agarose resin after AMF elution were analyzed on Tricine/SDS-PAGE. Similar results were obtained from Galpha-agarose (data not shown). Thus, it was shown that the phosphorylation of provided a tighter interaction of beta with G(o)alpha and Galpha.


Figure 7: Effect of phosphorylation of on the interaction of beta with G(o)alpha. A, time courses of ADP-ribosylation reaction of G(o)alpha in the presence of phosphorylated and unphosphorylated beta. The ADP-ribosylation of G(o)alpha was performed at 30 °C in a reaction mixture that contained 200 nM G(o)alpha, 1 µM [^3H]NAD, 1 µg/ml preactivated pertussis toxin, and 5 nM (circle, bullet) or 10 nM (up triangle, ) of phosphorylated (bullet, ) or unphosphorylated (circle, up triangle) beta. At an appropriate time of incubation, aliquots were withdrawn from the mixture to measure the amount of [^3H]ADP-ribose incorporated into G(o)alpha. B and C, elution profile of phosphorylated and unphosphorylated beta from G(o)alpha-agarose column. Phosphorylated (bullet) and unphosphorylated beta (circle, 3 µg each) were loaded onto the G(o)alpha-agarose column (0.1 ml), which was pre-equilibrated with TEN containing 0.05% Lubrol PX, and washed with the same buffer (fractions 1-2). Then the column was successively washed with the TEN buffer containing 0.4% Lubrol (B) or 1% Lubrol PX (C) (fractions 3-5), and with the TEN buffer containing 0.05% Lubrol (fractions 6-10). The protein bound to the column was eluted with 20 mM Tris-HCl, pH 7.5, 1 mM EDTA, 0.3 M NaCl, and 5 µM GDP and AMF (fractions 11-14). The amount of beta in each fraction (0.4 ml) was quantitated by an immunoassay. The total recovery of the affinity chromatography was between 60 and 100%.



These results are consistent with the idea we have described (Fig. 5) that the phosphorylation of induces a conformational change of beta. In fact, the alpha-beta interaction domain in beta seemed to involve the phosphorylation site of , since the phosphorylation of beta by PKC was markedly inhibited by G(o)alpha (Fig. 8). This inhibition was relieved by the addition of GTPS or AMF, a reagent converting G(o)alpha into GTP-bound form to be released from beta. These results imply that, upon activation of G protein, the dissociated beta becomes a substrate for PKC and that the phosphorylation in turn raises the affinity of beta for alpha subunit.


Figure 8: Effect of G(o)alpha on the phosphorylation of beta by PKC. Prior to phosphorylation, beta (0.5 µg) was incubated with (lanes 2-4) or without (lane 1) an equimolar amount of G(o)alpha at 0 °C for 10 min, and then it was incubated at 30 °C for 10 min in the absence (lanes 1 and 2) or presence of 6.5 µM GTPS and 10 mM magnesium acetate (lane 3) and AMF (lane 4, magnesium acetate was used instead of magnesium chloride). After incubation, the mixtures were subjected to phosphorylation by PKC at 30 °C for 20 min and then to Tricine/SDS-PAGE with subsequent autoradiography. The position of the phosphorylated is indicated by an arrow.




DISCUSSION

A novel form of subunit, , was originally purified from bovine spleen, partially sequenced, and designated previously(12) . In the present study, we have determined the complete structure of intact protein, which exactly matched with that predicted from the cDNA sequence. The CXXL motif found at the C terminus of also agreed with geranylgeranylation of the C-terminal cysteine in mature (16) . After the prenylation, three amino acids, Thr-Ile-Leu, are known to be removed, and newly exposed cysteine is carboxylmethylated(2) . The subunits other than (1) and (3)(36, 43) have been speculated to be N-acylated, because their N termini is blocked and those of (2) and (7) were able to be truncated by an acylamino acid-releasing enzyme(44) . Here, we have identified N-acetylated serine at the N terminus of . Co- or post-translationally, the N-terminal methionine could be cleaved to expose serine to be N-acetylated.

In contrast to specific localization of various subunits including (1), (3), and two (8)s(3, 4, 7, 9, 10, 11, 17) , showed ubiquitous distribution in rat tissues as well as (5)(9, 17) . Taking our previous study (17) on the tissue distribution of various subunits into consideration, seems to be a major subunit in many tissues other than neural tissues, in contrast with (2) and (3) enriched in the brain. These results suggested that may be involved in the signal transduction common to the various cellular function.

We should emphasize the unique property of being phosphorylated at Ser^1 (or Ser^2) by PKC in vitro. Even in cultured cells having as a major subunit, was phosphorylated by treatment of cells with PMA, a direct activator of PKC. Among various isozymes of PKC tested, was efficiently phosphorylated by diacylglycerol- (or PMA-)dependent PKC such as cPKCalpha, cPKCbeta, and nPKC but not by diacylglycerol-independent aPKC in vitro. This is compatible with PMA-induced phosphorylation of in vivo. Since cPKCalpha, nPKC, nPKC, and aPKC are expressed in both Swiss 3T3 and aortic smooth muscle cells(45, 46, 47) , it is speculated that cPKCalpha and nPKC would participate in the phosphorylation of in these cells. In addition, similar selectivity for isozymes of PKC was observed with a physiological substrate, myristoylated alanine-rich protein kinase C substrate, which has the same phosphorylation site domain (S*XK) as the predicted site of (39) . It should be also stressed that physiological vasoactive ligands such as angiotensin II and vasopressin for aortic smooth muscle cells induced the phosphorylation of endogenous . These extracellular signals are known to activate the cellular phospholipase C (42) possibly via G(q)-type G proteins. These results strongly suggest that is phosphorylated by PKC, which is activated in the cells by physiological agonists.

It is important to identify the subtype of the alpha subunit associating with betain vivo. If the partner is a G(q)-type alpha subunit activating phospholipase C-beta or alternatively beta by itself regulating the phospholipase C-beta, the phosphorylation of stimulated by the product (diacylglycerol) of the signaling pathway may have a feedback role. Alternatively, beta may transduce signal other than the phospholipase C pathway together with alpha subunits such as G(o)alpha and G(i)alpha, and the dual pathways may interact at the site of the phosphorylation of beta. In fact, one of the physiological partners of beta should be G(o)alpha and G(i)alpha capable of interacting with beta in a phosphorylation-sensitive manner. Although we have not examined this, some alpha subunits of G protein may also be phosphorylated in these cells stimulated as shown in other cells (21, 22) and may affect the coupling of alpha-beta subunits. In all cases, identification of alpha/beta-mediated signaling pathway will help to assess the possibilities described above concerning the physiological role of the reinforced alpha-beta interaction due to the phosphorylation.

Like the other beta complexes playing signal-transducing roles by itself, beta was able to inhibit the Ca/calmodulin-stimulated adenylyl cyclase in rat retinal membranes and stimulate the activity of phospholipase C in the cytosol of HL60 cells (data not shown). Thus, beta itself had potency to transmit some signals, although the phosphorylation of beta gave almost no effect on these regulations. These observations suggest that a possible conformational change of beta due to the phosphorylation is localized at a contact site with G(o)alpha without affecting the beta-effector interaction. Comparison of amino acid sequences of various isoforms of revealed diverged residues concentrated at the N-terminal region, which seems to participate in individual functions of beta complexes. The experimental evidence that the N-terminal 15 residues of subunits specifies the interaction with Galphas (33) is in line with the regulatory role of the extreme N-terminal phosphorylation of in the alpha-beta interaction.

Another interesting aspect of the present study is the Ca-dependent cleavage at a specific site (Lys^3-Thr^4) of by calpain. Calpain, a widely distributed enzyme, absolutely requires Ca ion for proteolyzing specific endogenous substrates, such as enzymes, membrane proteins, cytoskeletal proteins, and calmodulin binding proteins(48, 49) . Stimulation of phospholipase C by a specific receptor/G protein system simultaneously induces activation of PKC and mobilization of intracellular Ca ion, which probably lead to activation of calpain(50) . The is a common target of these two enzymes. As shown in Fig. 5, however, the proteolysis of by calpain was noticeably inhibited by the PKC-catalyzed phosphorylation. We speculate that one of the physiological roles or an exclusive one of the phosphorylation of might be the protection of from calpain attack when beta was dissociated from Galpha upon G protein activation. Whether the calpain-induced degradation of occurs in vivo and whether this degradation is protected by PKC-induced phosphorylation are intriguing questions that should be clarified in future experiments.


FOOTNOTES

*
This work was supported in part by grants-in-aid from the Japanese Ministry of Education, Science, and Culture and by grants from Toray Science Foundation and SUNBOR. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U37561[GenBank].

§
To whom correspondence should be addressed: Tel.: 81-568-88-0811; Fax: 81-568-88-0829.

(^1)
The abbreviations used are: G proteins, heterotrimeric guanine nucleotide-binding regulatory proteins; GTPS, guanosine 5`-O-(3-thiotriphosphate); PKC, protein kinase C; cPKC, conventional PKC; nPKC, novel PKC; aPKC, atypical PKC; PMA, phorbol 12-myristate 13-acetate; PAGE, polyacrylamide gel electrophoresis; Arg-C, arginyl endopeptidase; HPLC, high performance liquid chromatography; LC/MS, liquid chromatography-mass spectrometry; ESI/TSQMS, electrospray ionization/triple-stage quadrupole mass spectrometry; CAD, collision-activated dissociation.


REFERENCES

  1. Neer, E. J. (1995) Cell 80, 249-257 [Medline] [Order article via Infotrieve]
  2. Iñiguez-Lluhi, J., Kleuss, C., and Gilman, A. G. (1993) Trends Cell Biol. 3, 230-236 [CrossRef]
  3. Hurley, J. B., Fong, H. K. W., Teplow, D. B., Dreyer, W. J., and Simon, M. I. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 6948-6952 [Abstract]
  4. Yatsunami, K., Pandya, B. V., Oprian, D. D., and Khorana, H. G. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 1936-1940 [Abstract]
  5. Gautam, N., Baetscher, M., Aebersold, R., and Simon, M. I. (1989) Science 244, 971-974 [Medline] [Order article via Infotrieve]
  6. Robishaw, J. D., Kalman, V. K., Moomaw, C. R., and Slaughter, C. A. (1989) J. Biol. Chem. 264, 15758-15761 [Abstract/Free Full Text]
  7. Gautam, N., Northup, J., Tamir, H., and Simon, M. I. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 7973-7977 [Abstract]
  8. Fisher, K. J., and Aronson, N. N., Jr. (1992) Mol. Cell. Biol. 12, 1585-1591 [Abstract]
  9. Cali, J. J., Balcueva, E. A., Rybalkin, I., and Robishaw, J. D. (1992) J. Biol. Chem. 267, 24023-24027 [Abstract/Free Full Text]
  10. Ryba, N. J. P., and Tirindelli, R. (1995) J. Biol. Chem. 270, 6757-6767 [Abstract/Free Full Text]
  11. Ong, O. C., Yamane, H. K., Phan, K. B., Fong, H. K. W., Bok, D., Lee, R. H., and Fung, B. K.-K. (1995) J. Biol. Chem. 270, 8495-8500 [Abstract/Free Full Text]
  12. Asano, T., Morishita, R., Matsuda, T., Fukada, Y., Yoshizawa, T., and Kato, K. (1993) J. Biol. Chem. 268, 20512-20519 [Abstract/Free Full Text]
  13. Iñiguez-Lluhi, J. A., Simon, M. I., Robishaw, J. D., and Gilman, A. G. (1992) J. Biol. Chem. 267, 23409-23417 [Abstract/Free Full Text]
  14. Kisselev, O., and Gautam, N. (1993) J. Biol. Chem. 268, 24519-24522 [Abstract/Free Full Text]
  15. Ueda, N., Iñiguez-Lluhi, J. A., Lee, E., Smrcka, A. V., Robishaw, J. D., and Gilman, A. G. (1994) J. Biol. Chem. 269, 4388-4395 [Abstract/Free Full Text]
  16. Morishita, R., Fukada, Y., Kokame, K., Yoshizawa, T., Masuda, K., Niwa, M., Kato, K., and Asano, T. (1992) Eur. J. Biochem. 210, 1061-1069 [Abstract]
  17. Asano, T., Morishita, R., Ohashi, K., Nagahama, M., Miyake, T., and Kato, K. (1995) J. Neurochem. 64, 1267-1273 [Medline] [Order article via Infotrieve]
  18. Berridge, M. J., and Irvine, R. F. (1989) Nature 341, 197-205 [CrossRef][Medline] [Order article via Infotrieve]
  19. Nishizuka, Y. (1986) Science 233, 305-312 [Medline] [Order article via Infotrieve]
  20. Katada, T., Gilman, A. G., Watanabe, Y., Bauer, S., and Jakobs, K. H. (1985) Eur. J. Biochem. 151, 431-437 [Abstract]
  21. Carlson, K. E., Brass, L. F., and Manning, D. R. (1989) J. Biol. Chem. 264, 13298-13305 [Abstract/Free Full Text]
  22. Bushfield, M., Murphy, G. J., Lavan, B. E., Parker, P. J., Hruby, V. J., Milligan, G., and Houslay, M. D. (1990) Biochem. J. 268, 449-457 [Medline] [Order article via Infotrieve]
  23. Haga, K., Uchiyama, H., Haga, T., Ichiyama, A., Kangawa, K., and Matsuo, H. (1989) Mol. Pharmacol. 35, 286-294 [Abstract]
  24. Asano, T., Morishita, R., Kobayashi, T., and Kato, K. (1990) FEBS Lett. 266, 41-44 [CrossRef][Medline] [Order article via Infotrieve]
  25. Fukada, Y., Matsuda, T., Kokame, K., Takao, T., Shimonishi, Y., Akino, T., and Yoshizawa, T. (1994) J. Biol. Chem. 269, 5163-5170 [Abstract/Free Full Text]
  26. Morishita, R., Masuda, K., Niwa, M., Kato, K., and Asano, T. (1993) Biochem. Biophys. Res. Commun. 194, 1221-1227 [CrossRef][Medline] [Order article via Infotrieve]
  27. Asano, T., Kamiya, N., Morishita, R., and Kato, K. (1988) J. Biochem. (Tokyo) 103, 950-953 [Abstract]
  28. Kitano, T., Go, M., Kikkawa, U., and Nishizuka, Y. (1986) Methods Enzymol. 124, 349-352 [Medline] [Order article via Infotrieve]
  29. Saido, T. C., Mizuno, K., Konno, Y., Osada, S., Ohno, S., and Suzuki, K. (1992) Biochemistry 31, 482-490 [Medline] [Order article via Infotrieve]
  30. Fujise, A., Mizuno, K., Ueda, Y., Osada, S., Hirai, S., Takayanagi, A., Shimizu, N., Owada, M. K., Nakajima, H., and Ohno, S. (1994) J. Biol. Chem. 269, 31642-31648 [Abstract/Free Full Text]
  31. Schägger, H., and von Jagow, G. (1987) Anal. Biochem. 66, 368-379
  32. Ito, Y., Kozawa, O., Tokuda, H., Suzuki, A., Watanabe, Y., Kotoyori, J., and Oiso, Y. (1994) Atherosclerosis 110, 69-76 [Medline] [Order article via Infotrieve]
  33. Rahmatullah, M., Ginnan, R., and Robishaw, J. D. (1995) J. Biol. Chem. 270, 2946-2951 [Abstract/Free Full Text]
  34. Schaffner, W., and Weissmann, C. (1973) Anal. Biochem. 56, 502-514 [Medline] [Order article via Infotrieve]
  35. Santell, L., Bartfeld, N. S., and Levin, E. G. (1992) Biochem. J. 284, 705-710 [Medline] [Order article via Infotrieve]
  36. Fukada, Y., Takao, T., Ohguro, H., Yoshizawa, T., Akino, T., and Shimonishi, Y. (1990) Nature 346, 658-660 [CrossRef][Medline] [Order article via Infotrieve]
  37. Yamane, H. K., Farnsworth, C. C., Xie, H., Howald, W., Fung, B. K.-K., Clarke, S., Gelb, M. H., and Glomset, J. A. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 5868-5872 [Abstract]
  38. Mumby, S. M., Casey, P. J., Gilman, A. G., Gutowski, S., and Sternweis, P. C. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 5873-5877 [Abstract]
  39. Graff, J. M., Stumpo, D. J., and Blackshear, P. J. (1989) J. Biol. Chem. 264, 11912-11919 [Abstract/Free Full Text]
  40. Tamaoki, T., Nomoto, H., Takahashi, I., Kato, Y., Morimoto, M., and Tomita, F. (1986) Biochem. Biophys. Res. Commun. 135, 397-402 [Medline] [Order article via Infotrieve]
  41. Rodriguez-Pena, A., and Rozengurt, E. (1984) Biochem. Biophys. Res. Commun. 120, 1053-1059 [Medline] [Order article via Infotrieve]
  42. Birnbaumer, L., Abramowitz, J., and Brown, A. M. (1990) Biochim. Biophys. Acta 1031, 163-224 [Medline] [Order article via Infotrieve]
  43. Morishita, R., Kato, K., and Asano, T. (1994) FEBS Lett. 337, 23-26 [CrossRef][Medline] [Order article via Infotrieve]
  44. Sohma, H., Hashimoto, H., Hiraike, N., Ohguro, H., and Akino, T. (1993) Biochem. Biophys. Res. Commun. 190, 849-856 [CrossRef][Medline] [Order article via Infotrieve]
  45. Olivier, A. R., and Parker, P. J. (1992) J. Cell. Physiol. 152, 240-244 [Medline] [Order article via Infotrieve]
  46. Sasaguri, T., Kosaka, C., Hirata, M., Sasuda, J., Shimokado, K., Fujishima, M., and Ogata, J. (1993) Exp. Cell Res. 208, 311-320 [CrossRef][Medline] [Order article via Infotrieve]
  47. Assender, J. W., Kontny, E., and Fredholm, B. B. (1994) FEBS Lett. 342, 76-80 [CrossRef][Medline] [Order article via Infotrieve]
  48. Wang, K. K. W., Villalobo, A., and Roufogalis, B. D. (1989) Biochem. J. 262, 693-706 [Medline] [Order article via Infotrieve]
  49. Suzuki, K., and Ohno, S. (1990) Cell Struct. Funct. 15, 1-6 [Medline] [Order article via Infotrieve]
  50. Banno, Y., Nakashima, S., Hachiya, T., and Nozawa, Y. (1995) J. Biol. Chem. 270, 4318-4324 [Abstract/Free Full Text]
  51. Ray, K., Kunsch, C., Bonner, L. M., and Robishaw, J. D. (1995) J. Biol. Chem. 270, 21765-21771 [Abstract/Free Full Text]

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