The Ca2+-dependent Binding of Calmodulin to an N-terminal Motif of the Heterotrimeric G Protein beta  Subunit*

(Received for publication, January 29, 1997, and in revised form, April 29, 1997)

Mingyao Liu , Bo Yu , Osamu Nakanishi , Thomas Wieland and Melvin Simon Dagger

From the Division of Biology, California Institute of Technology, Pasadena, California 91125

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

Ca2+ ion concentration changes are critical events in signal transduction. The Ca2+-dependent interactions of calmodulin (CaM) with its target proteins play an essential role in a variety of cellular functions. In this study, we investigated the interactions of G protein beta gamma subunits with CaM. We found that CaM binds to known beta gamma subunits and these interactions are Ca2+-dependent. The CaM-binding domain in Gbeta gamma subunits is identified as Gbeta residues 40-63. Peptides derived from the Gbeta protein not only produce a Ca2+-dependent gel mobility shifting of CaM but also inhibit the CaM-mediated activation of CaM kinase II. Specific amino acid residues critical for the binding of Gbeta gamma to CaM were also identified. We then investigated the potential function of these interactions and showed that binding of CaM to Gbeta gamma inhibits the pertussis toxin-catalyzed ADP-ribosylation of Galpha o subunits, presumably by inhibiting heterotrimer formation. Furthermore, we demonstrated that interaction with CaM has little effect on the activation of phospholipase C-beta 2 by Gbeta gamma subunits, supporting the notion that different domains of Gbeta gamma are responsible for the interactions of different effectors. These findings shed light on the molecular basis for the interactions of Gbeta gamma with Ca2+-CaM and point to the potential physiological significance of these interactions in cellular functions.


INTRODUCTION

In response to external stimuli or changes in ligand concentration, the activated seven-transmembrane receptors of the cell interact with heterotrimeric G proteins. These in turn activate or inhibit a variety of intracellular proteins through either the Galpha subunits with GTP bound or the free Gbeta gamma subunits or both, leading to the generation of second-messenger molecules and changes in the patterns of cellular metabolic activity and growth. Whereas direct interaction of G protein alpha  subunits with effectors has been known for a long time, recently it has been demonstrated that Gbeta gamma subunits also play critical roles in effector activation, in modulating the interaction among various G protein pathways, and in regulating cellular functions. For example, the Gbeta gamma dimers have been shown to participate in interactions with adenylyl cyclases (1), phospholipases (2, 3), phosducin (4), beta -adrenergic receptor kinases (5-7), Bruton tyrosine kinase (8), inositol trisphosphate kinases (9, 10), mitogen-activated protein kinase (11), and a number of ion channels (12-16). The activation of phospholipase C-beta 2 and beta 3 by Gbeta gamma has been suggested to account for the PTX1-sensitive increase in intracellular Ca2+ in response to a number of chemoattractants (17, 18) and other ligands, thus regulating intracellular Ca2+ concentration.

Calmodulin (CaM) has been known to act as an intracellular calcium sensor protein. When the intracellular Ca2+ concentration increases, CaM can bind up to four Ca2+ ions, changing its conformation and regulating cellular functions such as activation or inhibition of a large number of enzymes (19, 20), ion channels (21), and receptors (22). These Ca2+-dependent interactions of CaM with its target proteins have played an important role in intracellular Ca2+ signaling and in various cellular functions including cell growth and differentiation. There are reports demonstrating that G protein beta gamma complex can bind to CaM when passed through the CaM-agarose column (23, 24); however, the precise nature of the interaction is not clear.

In this report, we investigated the interaction of Ca2+-CaM with Gbeta gamma subunits and the potential physiological significance of this interaction. We found that CaM not only binds to Gbeta gamma subunits purified from brain but also to the most diverse Gbeta 5Lgamma complex from retina. We then identified and characterized the CaM-binding domain of the Gbeta subunit by using synthetic peptides and site-specific mutation. Furthermore, we showed that binding of CaM to beta gamma inhibits the beta gamma -dependent PTX-catalyzed ADP-ribosylation of Galpha subunits. Using both brain Gbeta gamma subunits and the CaM-binding peptide derived from the beta  subunit, we demonstrated that the Ca2+-CaM-dependent activation of CaM kinase II could be inhibited at a molar ratio of 1 (peptide/CaM). These studies provide insight into the molecular basis for the interactions of beta gamma with Ca2+-CaM and the potential functions of these interactions in modulating cellular signaling and other functions.


EXPERIMENTAL PROCEDURES

Materials

Synthetic peptides were obtained from the Peptide Synthesis Facility (Beckman Institute, California Institute of Technology). Peptides were purified by high performance liquid chromatography and verified by mass spectrometry. Bovine brain CaM was purchased from Calbiochem. Phosphatidylethanolamine and phosphatidylinositol-4,5-diphosphate (PtdInsP2) were purchased from Avanti Polar Lipids (Alabaster, AL) and Boehringer Manheim, respectively. [3H]PtdInsP2, myo-[2-3H]inositol, and [32P]NAD were obtained from DuPont NEN. Pertussis toxin was purchased from List Biological Laboratories Inc.

CaM Binding Assay of G Protein beta 5Lgamma Subunits

Bleached bovine rod outer segment membranes were prepared from bovine retina as described (25). Transducin was removed from the membranes by 6 × hypotonic elution with 100 µM GTPgamma S in the presence of 3 mM MgCl2 (26). The remaining membranes were solubilized for 12 h at 4 °C in 20 ml of a buffer containing 50 mM Hepes, pH 8.0, 100 mM NaCl, 3 mM MgCl2, 10 mM 2-mercaptoethanol, and 1% (by mass) sodium cholate. Unsolubilized material was pelleted for 40 min at 100,000 × g, and the supernatant was diluted with 30 ml of a buffer containing 50 mM Hepes, pH 8.0, 100 mM NaCl, 3 mM MgCl2, 10 mM 2-mercaptoethanol, and 1.67 mM CaCl2 (final concentration: 1 mM CaCl2, 0.4% sodium cholate). The diluted supernatant was then applied at a flow rate of 0.3 ml/min to a CaM-Sepharose column (10-ml bed volume, Pharmacia Biotech Inc.) equilibrated with the above described buffer. The column was washed with 50 ml of the above buffer. Proteins bound to the CaM-Sepharose column were then isocratically eluted with a buffer containing 50 mM Hepes, pH 8.0, 100 mM NaCl, 3 mM MgCl2, 10 mM 2-mercaptoethanol, 10 mM EDTA, and 0.4% sodium cholate. Fractions of 600 µl were collected. 20 µl of the indicated fractions were separated by 10% SDS-PAGE and immunoblotted using the Gbeta 5-specific antiserum CT215 as described (27).

Gel Mobility Shifting and Gel Overlay Assays

High affinity binding of G protein beta  peptides to CaM was demonstrated by the gel mobility shifts of CaM in 12.5% nondenaturing polyacrylamide gels in the presence or absence of 4 M urea. Different concentrations of Gbeta peptide were incubated with 1 µM CaM at room temperature for 30 min. To determine the Ca2+ dependence and binding stoichiometry, gels were run in the presence of either 0.5 mM CaCl2 or 2 mM EGTA. Proteins were visualized by Coomassie Brilliant Blue staining.

Fluorescence Measurements and Determination of Dissociation Constants

Fluorescence emission spectra were obtained using SLM 4800 spectrofluorimeter. Excitation was at 295 nm. Excitation and emission band-passes were both 10 nm. Total fluorescence was determined by integration of emission spectra. The fluorescence titration data was used to determine the dissociation constant as described previously (28). By plotting the fraction of bound peptide as a function of free CaM concentrations, we obtained the peptide-CaM titration curve. The dissociation constant for the peptide-CaM binding was obtained from the curve fitting.

Site-specific Mutagenesis

Mutations in the putative CaM-binding site of Gbeta 1 were generated by polymerase chain reaction with the high fidelity DNA polymerase, Pfu (Stratagene). The mutations were confirmed by DNA sequencing done in the DNA sequencing facility at Caltech. The beta  constructs were subcloned into pCDNA3.1 vector (Invitrogen, San Diego, CA).

In Vitro Translation and Binding Assays

Transcription and translation were carried out using a rabbit reticulocyte lysate system from Promega at 30 °C for 90 min with amino acid mixtures without methionine. 5 µCi of [35S]methionine (>1000 Ci/mmol, DuPont NEN) was added to the reaction mixture to monitor the synthesis of new proteins. 1-2 µg of cDNAs were used in each translation. 2.5 µl aliquots of the translation mixture were separated on 12% SDS-PAGE. The newly synthesized proteins were identified by autoradiography. After in vitro translation, the protein mixture was incubated with CaM-agarose beads in the presence of Ca2+ for 30 min at room temperature. Then the CaM-agarose beads were washed extensively to get rid of nonspecific binding. Proteins bound to the beads were eluted by a buffer containing 10 mM EGTA. The eluted Gbeta proteins were separated by SDS-PAGE and visualized by autoradiography.

ADP-ribosylation of Galpha o Subunits by PTX

Pertussis toxin-catalyzed ADP-ribosylation of Galpha o was performed as described previously (29). Briefly, 0.1 µg of recombinant Galpha o was mixed with 0.1 µg of purified rat brain beta gamma subunits in the absence or presence of Ca2+-CaM and incubated for 10 min at room temperature before the addition of the reaction mixture (20 mM Tris-HCl, pH 8.0, 1 mM EDTA, 2 mM MgCl2, 2 mM dithiothreitol, 0.5 µM 32P-labeled NAD (20,000 cpm/pmol), and 10 µg/ml pertussis toxin). Reactions were incubated at room temperature for 30 min and terminated by adding 5 × SDS-PAGE sample buffer. Samples were resolved on SDS-polyacrylamide gels and stained with Coomassie Blue. Labeling of proteins was detected by autoradiography.

Enzymatic Activity Assays of CaM Kinase II

The Ca2+-CaM-dependent activity of CaM kinase II was assayed essentially as described previously (30). Briefly, different concentrations of Gbeta peptide were preincubated with Ca2+-CaM for 30 min at room temperature. The phosphorylation reactions contained 50 mM Hepes buffer, pH 7.5, 10 mM magnesium acetate, 1 mM CaCl2, 0.1 mM [gamma -32P]ATP, 1 µM CaM, 5-10 µg of CaM kinase II peptide substrate (Chemicon International, Inc., Temecula, CA), and purified rat brain CaM kinase II (Calbiochem). All assays were initiated by the addition of kinase.

COS-7 Cell Transfection and Phosphoinositol Phospholipase C Activity Assays

In Vivo Transfection Assay

cDNAs encoding PLC-beta 2, Gbeta gamma , and CaM were cotransfected into COS-7 cells with LipofectAMINE (Life Technologies, Inc.). Then the cells were labeled with 10 µCi/ml myo-[2-3H]inositol the following day. 48 h after transfection, the activity of PLC-beta 2 was assayed by determining the levels of inositol phosphates as described previously (31, 37).

In Vitro Reconstitution Assay of PLC-beta 2 Activity

Phospholipid vesicles containing 50 µM [3H]PtdInsP2 and 500 µM phophatidylethanolamine were prepared by mixing with chloroform solution, drying under a stream of N2, then sonicating with 88 mM Hepes buffer, pH 7.5, and 18 mM LiCl. Assays were performed in a 70-µl reaction mixture containing 20 ng of PLC-beta 2, 1.7 µM Gbeta gamma from bovine retina, 50 µM CaCl2, 10 mM LiCl, and phospholipid vesicles. The reaction mixtures were incubated at 30 °C for 10 min in the presence of different concentrations of CaM. The reactions were stopped by adding 0.35 ml of chloroform/methanol/HCl (500:500:3). The released Ins-1,4,5-P3 was extracted by adding 0.1 ml of 1 M HCl with vigorous vortexing. The aqueous phase separated after centrifugation was subjected to scintillation counting.


RESULTS

Binding of Gbeta gamma Subunits to Ca2+-CaM

To demonstrate the direct interaction of Gbeta gamma subunits with CaM, purified bovine brain beta gamma subunits were incubated with CaM-agarose gel in the presence of 1 mM CaCl2. After extensively washing with Ca2+-containing buffer, the bound beta gamma subunits were eluted by buffer containing EGTA. As shown in Fig. 1A, brain beta gamma binds to CaM, and this binding is Ca2+-dependent. Direct binding was also observed in a gel overlay assay with 125I-labeled CaM (data not shown). There are five different G protein beta  subunits that have been characterized in mammalian systems. Among them, Gbeta 5 is the most diverse form and is expressed predominately in neuronal cells (27). A splice variant of Gbeta 5, beta 5L, is found only in rod outer segment membrane. To examine whether CaM binding is a common feature in different beta gamma subunits, Gbeta 5Lgamma extracts were obtained from bovine retina and passed through a CaM-agarose fast protein liquid chromatography column in the presence of Ca2+. Proteins bound to the CaM-agarose column were eluted with buffer containing 10 mM EDTA and detected by specific antibodies against the Gbeta 5 subunit. Like other beta gamma subunits, beta 5Lgamma also binds to Ca2+-CaM (Fig. 1B), suggesting that the CaM-binding domain is conserved in all Gbeta gamma subunits.


Fig. 1. Binding of G protein beta gamma subunits to Ca2+-CaM. A, interaction of rat brain Gbeta gamma subunits with Ca2+-CaM. Purified rat brain beta gamma subunits were incubated with CaM-agarose in the presence of 1 mM CaCl2 for 30 min at room temperature. Then, the CaM-agarose complexes were washed extensively with phosphate-buffered saline containing CaCl2. Proteins bound to CaM-agarose were eluted with buffer containing EGTA. Eluted Gbeta protein was visualized by Western blot using specific antibodies against Gbeta subunits. Con, control. B, binding of Gbeta 5Lgamma subunits to Ca2+-CaM. Gbeta 5Lgamma extracts were obtained from bovine retina and were then applied to a CaM-Sepharose fast protein liquid chromatography column (Pharmacia) equilibrated with Ca2+-containing buffer. Proteins bound to the CaM-Sepharose column were then eluted with an EDTA-containing buffer. 20 µl of the indicated fractions were separated by 10% SDS-PAGE and immunoblotted using the Gbeta 5-specific antiserum CT215.
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Identification of a CaM-binding Domain of Gbeta gamma Subunits

To identify the CaM-binding domain on Gbeta gamma subunits, the amino acid sequences of beta gamma were compared with those of known high affinity CaM-binding proteins. We found that the N terminus of all Gbeta subunits contains a putative CaM-binding domain that exhibits the characteristics typical of CaM-binding peptides (32). Fig. 2 shows the alignment of the N-terminal domain sequences of Gbeta subunits with well known CaM-binding domains of rat olfactory cyclic nucleotide-gated channel (RAT OCNC), skeletal muscle myosin light chain kinase (SK-MLCK), Ca2+ pump, calcineurin, Ras-like GTPase Kir/Gem, murine inducible nitric oxide synthase (iNOS), and CaM kinase II. To demonstrate that the small domain from amino acid residues Val-40-Trp-63 is indeed the CaM-binding domain of Gbeta subunits, synthetic peptides representing the putative CaM-binding domain were prepared and tested for their abilities to bind CaM in the presence of Ca2+ using both gel mobility shifting assay and tryptophan fluorescence assays.


Fig. 2. Alignment of the putative CaM-binding domain in Gbeta subunit with known CaM-binding proteins. The putative CaM-binding sequences of Gbeta subunits are compared with the known CaM-binding domains of rat olfactory cyclic nucleotide-gated channel (OCNC) (21), skeletal muscle myosin light chain kinase (skMLCK) (40), plasma membrane Ca2+-ATPase (Ca2+ pump) (41), calcineurin (42), Ras-like GTPase Kir/Gem (43), murine-inducible nitric oxide synthase (Murin iNOS residues 501-532) (44), and CaM kinase II (45). The conserved hydrophobic residues are boxed and may play an important role in the interaction with CaM.
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Binding of peptides to CaM was first assayed by nondenaturing polyacrylamide gel shifting assay. Depending on the charges and hydrophobicity of the peptide, high affinity binding of the peptide to CaM was detected as a complex band with increased or decreased mobility compared with the unbound CaM band. In the presence of Ca2+, the mobility of CaM was decreased by the peptide corresponding to Gbeta residues 40-63 (Fig. 3A). Several ratios of the peptide to CaM were used. In the absence of peptide, there is a single, fast-moving band reflecting pure Ca2+-CaM (Fig. 3A, CaM alone). At ratios of 0.25 and 0.75 of peptide to CaM, two bands were visible on the gel, the fast-moving CaM band and the lower mobility peptide-Ca2+-CaM complex. At a 1:1 molar ratio of peptide to CaM, all of the CaM was gel-shifted, and the intensity of the peptide-Ca2+-CaM band was increased as the result of complex formation (Fig. 3A). At still higher molar ratios, no new band was detected on the gel nor did the peptide-CaM complex band change in intensity, indicating a 1:1 binding stoichiometry of peptide to CaM and the absence of multivalent peptide-CaM complexes (Fig. 3A). Similar results were obtained when gel shift assays were performed in the presence of 4 M urea. However, in the presence of Ca2+ chelator, EGTA, only the pure CaM bands were detected on the gel and no peptide-CaM complex band was visible (Fig. 3B), indicating the complex formation is Ca2+-dependent. Peptides from other regions of the Gbeta subunit had no effect on the mobility of CaM (data not shown).


Fig. 3. Gel mobility shifting assay of CaM by synthetic peptide. CaM (10 µg) was incubated with the indicated amount of peptide derived from Gbeta subunit in the presence of 1 mM CaCl2 (A) or 2 mM EGTA (B). Samples were then separated on 12% nondenaturing polyacrylamide gels containing either CaCl2 or EGTA. The relative mobility of CaM and CaM-peptide complex was visualized by Coomassie Blue staining. The molar ratios of peptide to CaM are shown as indicated. The results are representative of three separated experiments.
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Since the synthetic peptide contains a tryptophan residue, whereas CaM contains none, binding of Gbeta peptide to CaM was directly studied by monitoring the changes in tryptophan fluorescence (Fig. 4). When the peptide was excited at 295 nm, it exhibited an intrinsic tryptophan fluorescence emission peak at 353 nm. Addition of CaM in the presence of Ca2+ not only caused a blue shift of the fluorescence peak but also increased the maximal fluorescence intensity (Fig. 4A). Since Ca2+ alone has no effect on the emission spectrum, the observed change must have resulted from the binding of Ca2+-CaM to the peptide. The blue shift in fluorescence emission and the increase in total intensity indicate that the environment of the Gbeta peptide became hydrophobic, presumably due to interactions with the hydrophobic domains of CaM upon formation of complex, further confirming the CaM binding property of the peptide. The changes in fluorescence property upon formation of the peptide-CaM complex were fully reversed by the addition of EGTA (Fig. 4A), indicating that the binding of CaM to the peptide is Ca2+-dependent. Titration of fixed amounts of peptide with different concentrations of CaM showed a saturation pattern. As shown in Fig. 4B, when a 0.1 µM concentration of peptide was used, the fluorescence intensity increased almost linearly with increasing CaM concentrations and reached a plateau at 0.1 µM CaM, confirming a 1:1 tight binding between the peptide and CaM. When the fraction of bound peptide was plotted as a function of free CaM concentration, the dissociation constant (Kd) obtained was ~40 nM (Fig. 4C), indicating high affinity binding to Ca2+-CaM. The actual binding affinity could be higher than 40 nM; however, the limitation of this fluorescence measurement does not allow us to test low peptide concentration.


Fig. 4.

Direct interaction of calmodulin with Gbeta peptide assayed by measuring the changes in tryptophan fluorescence spectrum. A, Ca2+-dependent interactions of the Gbeta peptide with CaM, measured by the emission spectrum of the peptide and peptide-CaM complex. When the peptide (0.1 µM) was excited at 295 nm, the peptide exhibited a fluorescence emission spectrum with a maximal emission peak at 354 nm (peptide w/Ca2+). In the presence of Ca2+, addition of CaM not only caused a blue shift of the fluorescence peak but also increased the total fluorescence intensity (peptide + Ca2+-CaM). Addition of EGTA completely reversed these fluorescence changes (peptide + Ca2+-CaM + EGTA), indicating that the interactions between the Gbeta peptide and CaM is Ca2+-dependent. B, the Gbeta peptide was titrated with different concentrations of CaM in the presence of 0.5 mM CaCl2. C, determination of the affinity constant for the interaction between CaM and Gbeta peptide. The fraction of bound peptide was calculated and plotted against free CaM concentration. The curve was fitted by a ligand binding equation with a dissociation constant of ~40 nm.


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Mutation Analysis of the Putative CaM-binding Domain of Gbeta Subunits

To identify the key amino acids essential for CaM binding, two types of mutations were carried out in the putative CaM-binding domain by site-directed mutagenesis. One set contains a number of basic amino acid residues (Arg-42, Arg-48, Arg-49), and the other group involves hydrophobic residues (Ile-43, Leu-51, Leu-55). These specific amino acids were individually replaced by alanine residues. Mutant proteins were then compared with wild type protein for their ability to bind Ca2+-CaM. Both wild type and mutant cDNA were translated in vitro together with gamma 2 subunit in rabbit reticulocyte lysates in the presence of 35S-labeled methionine. Fig. 5A shows the in vitro translated wild type and mutant Gbeta proteins separated by SDS-gel and detected by autoradiography. The amount of wild type protein and mutant proteins synthesized are similar under these assay conditions. The in vitro translated protein mixtures were incubated with CaM-agarose beads to assess the CaM binding ability. After extensive washing with Ca2+-containing buffer (100-200 bead volume), the bound protein was eluted with EGTA buffer. As shown in Fig. 5B, like the wild type Gbeta protein, one of the mutants (R42A) retained its ability to bind CaM. However, the binding affinities of other mutants (I43A, L55A, R48A/R49A, and L51A) for CaM were reduced by varying degrees under the same assay conditions. For instance, mutations at residues Arg-48, Arg-49, and Leu-51 dramatically reduced the CaM binding ability of the protein (Fig. 5B), suggesting that these residues are critical in the interactions. To further probe the conformation and activity of mutant Gbeta subunits, we analyzed the formation of beta gamma dimers using in vitro translated proteins and coimmunoprecipitation with specific anti-Ggamma 2 antibodies. The wild type Gbeta and its mutant proteins can be precipitated by gamma 2 antibodies, and there is little difference in their ability to interact with gamma  subunits (data not shown), indicating that mutant beta  subunits could still fold into the native conformation and retain affinity for gamma  subunits.


Fig. 5. Mutation analysis of putative CaM-binding domain of Gbeta subunit. A, similar amounts of wild type (wt) and mutant Gbeta 1 proteins were obtained by in vitro translation in rabbit reticulocyte lysates (Promega). B, CaM binding of in vitro translated Gbeta protein and its mutants. Compared with wild type Gbeta protein, the mutant protein (R42A) retained its binding ability to Ca2+-CaM; however, other mutant proteins (R48,49A, L51A, I43A, and L55A) showed weak binding ability to Ca2+-CaM, especially mutations at residues Arg-48, Arg-49, and Leu-51.
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Effects of CaM Binding on ADP-ribosylation of Galpha o Subunits

To explore the possible physiological functions of CaM binding to Gbeta gamma subunits, we first examined the effects of CaM binding on beta gamma -dependent pertussis toxin-catalyzed ADP-ribosylation of Galpha o subunit. PTX is a bacterial toxin that catalyzes the ribosylation of the C-terminal cysteine residues of Galpha o, Galpha i, and Galpha t subunits. PTX modification blocks the interactions of Galpha subunits with receptors and thus blocks the ligand-mediated signal transduction. The labeling of Galpha o by PTX requires the Galpha obeta gamma heterotrimer rather than the free Galpha o subunit (33). Thus, ADP-ribosylation of alpha o can be used as a sensitive indicator of formation of alpha obeta gamma heterotrimers. Incubation of CaM with bovine brain Gbeta gamma subunits in the presence of 0.1 mM Ca2+ inhibited the ribosylation reaction, as shown in Fig. 6. The reduction of labeling is concentration-dependent. At equal molar concentrations of CaM and beta gamma subunits, the PTX-catalyzed ribosylation of Galpha o was almost completely blocked by Ca2+-CaM. Since Ca2+ alone had no effect on the reaction, the simplest interpretation of this observation is that Ca2+-CaM formed a complex with Gbeta gamma subunits and this complex lost its ability to interact with Galpha o subunit; therefore, CaM inhibited the beta gamma -dependent ADP-ribosylation of Galpha o.


Fig. 6. CaM inhibited Gbeta gamma -dependent PTX-catalyzed ADP-ribosylation of Galpha o. 0.1 µg of recombinant Galpha o was mixed with 0.1 µg of purified bovine brain beta gamma complex in the presence of indicated molar ratios of CaM. The reaction mixture was incubated for 10 min at room temperature.
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Binding of CaM to Gbeta gamma Has Little Effect on beta gamma -activated PLC-beta 2 Activity

To further investigate the nature of CaM binding on Gbeta gamma subunits, we examined its effects on beta gamma -stimulated PLC-beta 2 activity. As shown in Fig. 7A, CaM has little effect on PLC-beta 2 activity stimulated by beta gamma subunits in an in vitro reconstituted system. To confirm the observations obtained in the reconstitution experiments, cDNAs encoding PLC-beta 2, Gbeta gamma , and CaM were transfected into COS-7 cells. Coexpression of PLC-beta 2 and beta gamma subunits increased inositol 1,4,5-trisphosphate release 3-5 fold (Fig. 7B, column 3). However, cotransfection of CaM expression plasmids had little effect on the beta gamma -stimulated PLC-beta 2 activity (Fig. 7B, column 4). These results indicated that in the Gbeta subunit, the domain responsible for PLC-beta 2 activation is distinct from the domain binding to CaM, suggesting that different domains of the beta  subunit are involved in interactions with different effector proteins.


Fig. 7. Calmodulin has little effect on Gbeta gamma -stimulated PLC-beta 2 activity. A, in vitro reconstitution assay of PLC-beta 2 activity. The Gbeta gamma -activated PLC-beta 2 activity was assayed in a reaction mixture containing PLC-beta 2, brain or retinal Gbeta gamma , 50 µM CaCl2, 10 mM LiCl, phospholipid vesicles, and different concentrations of CaM. The released inositol 1,4,5-trisphosphate (IP3) was extracted, separated, and subjected to scintillation counting. B, in vivo transfection assays of PLC-beta 2 activity. Accumulation of inositol phosphates was measured in COS-7 cells transfected with expression vectors carrying cDNAs corresponding to beta -galactosidase (lacZ), PLC-beta 2, Gbeta 1, Ggamma 2, and CaM. The cDNAs transfected are indicated beneath each column.
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Inhibition of Ca2+-CaM-dependent Enzyme, CaM Kinase II, by Gbeta gamma Subunits and Its Peptide

The putative CaM-binding domain of Gbeta subunits was further characterized by using the synthetic peptide derived from Gbeta to inhibit Ca2+-CaM-dependent activation of CaM kinase II. Concentration-dependent effects of the peptide on CaM kinase II activity were examined. As shown in Fig. 8A, the peptide was a potent inhibitor of CaM kinase II activity. At a 1:1 binding ratio of Gbeta peptide to CaM, the peptide totally inhibited the Ca2+-CaM-dependent activation of CaM kinase II, suggesting that the binding affinity of Gbeta peptide is comparable to the affinity of CaM kinase II toward Ca2+-CaM and should be in the nM range. Other peptides from the N-terminal regions had no effect on the Ca2+-CaM-stimulated enzymatic activity (data not shown). To examine whether Gbeta gamma subunits can also inhibit the Ca2+-CaM-dependent CaM kinase II activity, Ca2+-CaM was incubated with brain Gbeta gamma subunits for 30 min at room temperature. Then the Ca2+-CaM-stimulated CaM kinase II activity was assayed. Fig. 8B shows that brain Gbeta gamma subunits inhibited 70-80% of Ca2+-CaM-stimulated CaM kinase II activity, indicating that beta gamma can competitively bind to Ca2+-CaM.


Fig. 8. Inhibition of Ca2+-CaM stimulated CaM kinase II activity by Gbeta gamma and its CaM-binding peptide. A, the putative CaM-binding peptides derived from Gbeta inhibited the Ca2+-CaM-dependent CaM kinase II activity. Different molar ratios of Gbeta peptide were incubated with CaM, then added to the kinase assay mixture. The final concentration of CaM was kept the same in all the reactions. B, inhibitory effects of brain Gbeta gamma on CaM kinase II activity. At equal molar ratios, Gbeta gamma can compete for CaM, inhibiting the Ca2+-CaM-dependent CaM kinase II activity.
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DISCUSSION

Previous studies have demonstrated that G protein subunits can act as potent inhibitors of the Ca2+-CaM-stimulated phosphodiesterase activity (23, 24), probably through interactions of Gbeta gamma with CaM. In this report, we show direct binding of Gbeta gamma subunits with CaM. This CaM-binding property of Gbeta is Ca2+-dependent and conserved in known Gbeta subunits, including the most diverse beta 5 subunit. We also identified and characterized the CaM-binding domain in beta gamma subunits using three different methods. In the gel mobility shifting assays and tryptophan fluorescence assay, a conserved 25-amino acid peptide in the N-terminal region of Gbeta subunits was found to interact with CaM. By modifying specific amino acids in this region, we identified some key residues (Arg-48, Arg-49, Leu-51, Ile-43, Leu-55) that play an important role in the binding of beta gamma to CaM.

The interaction between Gbeta gamma subunits and CaM is Ca2+-dependent. Ca2+ is an important intracellular messenger in many cellular functions including cell growth and development (34, 35). Many seven-transmembrane receptors activate the G protein subunits, which in turn activate PLC-beta , resulting in the production of inositol 1,4,5-trisphosphate and diacyglycerol from PtdInsP2. Inositol 1,4,5-trisphosphate acts as an intracellular second messenger by binding to the inositol 1,4,5-trisphosphate receptors in the endoplasmic reticular membrane, triggering the release of Ca2+ from the endoplasmic reticulum and therefore increasing the intracellular Ca2+ concentration. Ca2+ is also believed to organize and stabilize CaM domain structure in a conformational state that can bind target proteins (32, 36). The calmodulin concentration in some cells, e.g. neuronal cells, is in the order of micromolar. Calcium release can thus convert 10-50% of these molecules to the Ca2+-bound form. The affinity of Gbeta gamma for Ca2+-CaM is sufficiently high so that under these conditions much of the free beta gamma in the cells should be in the CaM-bound form. Therefore, an increase in intracellular Ca2+ concentration could selectively regulate the interactions of Gbeta gamma with a number of other proteins through binding with CaM.

The interaction of Gbeta gamma with Ca2+-CaM could play an important role in the cross-talk mechanism between different G protein pathways (37). Ca2+-CaM has been shown to regulate the formation and hydrolysis of cAMP. In the Galpha s-coupled pathway, both adenylyl cyclases and phosphodiesterases are Ca2+-CaM-dependent, which could serve as the convergence point for Ca2+-dependent and Galpha s-dependent stimuli.

The primary structure of the identified CaM-binding domain of Gbeta gamma shows features similar to some other CaM-binding proteins and inhibitors (21, 28, 38-45). For instance, the identified beta  CaM-binding domain contains a high percentage of hydrophobic residues and an excess of positively charged residues, a property found in all CaM-binding peptides. It is believed that the basic residues contribute to CaM binding via electrostatic interactions with acidic residues in CaM, whereas the hydrophobic amino acids seem to play a more important role in CaM binding through interactions with the hydrophobic patches of the globular domains of CaM (38, 39). From x-ray structural analysis, 80% of all contacts are van der Waals interactions, and the interaction appears to involve two key hydrophobic amino acids separated by eight residues, although flexibility does exist in the interaction because of the flexible central helix of CaM (36). CaM contains two globular domains, and these two domains are connected by a long alpha helical segment (32, 36). When different CaM binding targets are recognized, this long central segment allows different relative positioning of the two lobes of CaM. Therefore, although the secondary structure of the Gbeta CaM-binding domain is not a typical amphipathic alpha  helix (46), CaM could still position itself to bind to the Gbeta protein, possibly by stabilizing a change in the conformation of the N-terminal domain of Gbeta protein. On the other hand, a number of CaM-binding proteins have been identified and shown to contain sites that are not typical amphipathic helices but consist of basic amino acids interspersed with nonpolar amino acids (52-54).

The binding of CaM to Gbeta gamma interfered with the formation of Galpha beta gamma trimers as assayed by the inhibition of PTX-catalyzed ADP-ribosylation of Galpha o. Based on the secondary structure of the beta  subunit, the N-terminal region of the beta  subunit is in close proximity to the N-terminal portion of Galpha subunit (47-49). It has been reported that effector activation by the beta gamma subunits is blocked upon the addition of the Galpha subunit presumably by heterotrimerization (1, 13). Thus, by interacting with the N-terminal domain of beta  subunit, the Ca2+-CaM complex could affect the heterotrimer formation of Galpha beta gamma .

Interaction of CaM with beta gamma has little effect on the G protein beta gamma subunit-activated PLC-beta 2 activity. These results support the notion that interaction of different beta gamma -responsive effectors is mediated by distinct domains of Gbeta gamma (50, 51). By using a series of chimeras between Dictyostelium and mammalian beta  subunits, a small C-terminal segment of Gbeta was identified as responsible for the activation of PLC-beta 2 (50). The CaM-binding domain of Gbeta identified in this report is located in the N-terminal region. This region of Gbeta was suggested to play a role in the interaction of adenylyl cyclase type 2, the muscarinic receptor-gated atrial inwardly rectifying potassium channel (GIRK1), and in the activation of mitogen-activated protein kinase pathways (50, 51). It will be of great interest to determine whether binding of CaM to Gbeta gamma can affect the activation of the potassium channels (GIRK) and mitogen-activated protein kinase pathways.


FOOTNOTES

*   This work was supported by a grant from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    To whom correspondence should be addressed: Division of Biology, 147-75, California Institute of Technology, Pasadena, CA 91125. Tel.: 818-395-3944; Fax: 818-796-7066; E-mail: simonm{at}starbase1.caltech.edu.
1   The abbreviations used are: PTX, pertussis toxin; CaM, calmodulin; PtdInsP2, phosphatidylinositol-4,5-diphosphate; GTPgamma S, guanosine 5'-3-O-(thio)triphosphate; PAGE, polyacrylamide gel electrophoresis; PLC-beta 2, phospholipase C-beta 2.

ACKNOWLEDGEMENTS

We thanks Dr. Silvia Cavagnero (Division of Chemistry, Caltech) for her help and discussion with the fluorescence measurements of the peptide-protein interactions and Dr. A.R. Means (Duke University) for cDNA clones of calmodulin.


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