The Ca2+-dependent Binding of Calmodulin to an N-terminal Motif of the Heterotrimeric G Protein β Subunit
1997; Elsevier BV; Volume: 272; Issue: 30 Linguagem: Inglês
10.1074/jbc.272.30.18801
ISSN1083-351X
AutoresMingyao Liu, Bo Yu, Osamu Nakanishi, Thomas Wieland, Melvin I. Simon,
Tópico(s)Protein Structure and Dynamics
ResumoCa2+ 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 βγ subunits with CaM. We found that CaM binds to known βγ subunits and these interactions are Ca2+-dependent. The CaM-binding domain in Gβγ subunits is identified as Gβ residues 40–63. Peptides derived from the Gβ 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 Gβγ to CaM were also identified. We then investigated the potential function of these interactions and showed that binding of CaM to Gβγ inhibits the pertussis toxin-catalyzed ADP-ribosylation of Gαo subunits, presumably by inhibiting heterotrimer formation. Furthermore, we demonstrated that interaction with CaM has little effect on the activation of phospholipase C-β2 by Gβγ subunits, supporting the notion that different domains of Gβγ are responsible for the interactions of different effectors. These findings shed light on the molecular basis for the interactions of Gβγ with Ca2+-CaM and point to the potential physiological significance of these interactions in cellular functions. 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 βγ subunits with CaM. We found that CaM binds to known βγ subunits and these interactions are Ca2+-dependent. The CaM-binding domain in Gβγ subunits is identified as Gβ residues 40–63. Peptides derived from the Gβ 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 Gβγ to CaM were also identified. We then investigated the potential function of these interactions and showed that binding of CaM to Gβγ inhibits the pertussis toxin-catalyzed ADP-ribosylation of Gαo subunits, presumably by inhibiting heterotrimer formation. Furthermore, we demonstrated that interaction with CaM has little effect on the activation of phospholipase C-β2 by Gβγ subunits, supporting the notion that different domains of Gβγ are responsible for the interactions of different effectors. These findings shed light on the molecular basis for the interactions of Gβγ with Ca2+-CaM and point to the potential physiological significance of these interactions in cellular functions. 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 Gα subunits with GTP bound or the free Gβγ 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 α subunits with effectors has been known for a long time, recently it has been demonstrated that Gβγ 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 Gβγ dimers have been shown to participate in interactions with adenylyl cyclases (1Tang W.J. Gilman A.G. Science. 1991; 254: 1500-1503Crossref PubMed Scopus (741) Google Scholar), phospholipases (2Camps M. Carozzi A. Schnabel P. Scheer A. Parker P.J. Gierschik P. Nature. 1992; 360: 684-686Crossref PubMed Scopus (513) Google Scholar, 3Katz A. Wu D. Simon M.I. Nature. 1992; 360: 686-689Crossref PubMed Scopus (413) Google Scholar), phosducin (4Xu J. Wu D. Slepak V.Z. Simon M.I. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2086-2090Crossref PubMed Scopus (66) Google Scholar), β-adrenergic receptor kinases (5Inglese J. Koch W.J. Caron M.G. Lefkowitz R.J. Nature. 1992; 359: 147-150Crossref PubMed Scopus (230) Google Scholar, 6Pitcher J.A. Inglese J. Higgins J.B. Arriza J.L. Casey P.J. Kim C. Benovic J.L. Kwatra M.M. Caron M.G. Lefkowitz R.J. Science. 1992; 257: 1264-1267Crossref PubMed Scopus (562) Google Scholar, 7Koch W.J. Hawes B.E. Allen L.F. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12706-12710Crossref PubMed Scopus (404) Google Scholar), Bruton tyrosine kinase (8Tsukada S. Simon M.I. Witte O.N. Katz A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11256-11260Crossref PubMed Scopus (236) Google Scholar), inositol trisphosphate kinases (9Stephens L. Eguinoa A. Corey S. Jackson T. Hawkins P.T. EMBO J. 1993; 12: 2265-2273Crossref PubMed Scopus (136) Google Scholar, 10Stephens L. Smrcka A. Cook F.T. Jackson T.R. Sternweis P.C. Hawkins P.T. Cell. 1994; 77: 83-93Abstract Full Text PDF PubMed Scopus (515) Google Scholar), mitogen-activated protein kinase (11Crespo P. Xu N. Simonds W.F. Gutkind J.S. Nature. 1994; 396: 418-420Crossref Scopus (758) Google Scholar), and a number of ion channels (12Reuveny E. Slesinger P.A. Inglese J. Morales J.M. Iniguez-Lluhi J.A. Lefkowitz R.J. Bourne H.R. Jan Y.N. Jan L.Y. Nature. 1994; 370: 143-146Crossref PubMed Scopus (409) Google Scholar, 13Wickman K.D. Iniguez-Lluhi J.A. Davenport P.A. Taussig R. Krapivinsky G.B. Linder M.E. Gilman A.G. Clapham D.E. Nature. 1994; 368: 255-257Crossref PubMed Scopus (377) Google Scholar, 14Annu. Rev. Neurosci. 17, 441–464Clapham, D. Annu. Rev. Neurosci. , 17, 441–464.Google Scholar, 15Ma J. Li M. Catterall W. Scheuer T. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12351-12355Crossref PubMed Scopus (65) Google Scholar, 16Ikeda S. Nature. 1996; 380: 255-258Crossref PubMed Scopus (705) Google Scholar). The activation of phospholipase C-β2 and β3 by Gβγ has been suggested to account for the PTX 1The abbreviations used are: PTX, pertussis toxin; CaM, calmodulin; PtdInsP2, phosphatidylinositol-4,5-diphosphate; GTPγS, guanosine 5′-3-O-(thio)triphosphate; PAGE, polyacrylamide gel electrophoresis; PLC-β2, phospholipase C-β2. 1The abbreviations used are: PTX, pertussis toxin; CaM, calmodulin; PtdInsP2, phosphatidylinositol-4,5-diphosphate; GTPγS, guanosine 5′-3-O-(thio)triphosphate; PAGE, polyacrylamide gel electrophoresis; PLC-β2, phospholipase C-β2.-sensitive increase in intracellular Ca2+ in response to a number of chemoattractants (17Wu D. LaRosa G.J. Simon M.I. Science. 1993; 261: 101-103Crossref PubMed Scopus (332) Google Scholar, 18Jiang H. Kuang Y. Wu Y. Smrcka A. Simon M.I. Wu D. J. Biol. Chem. 1996; 271: 13430-13434Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar) 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 (19Klee C.B. Vanaman T.C. Adv. Protein Chem. 1982; 35: 213-321Crossref PubMed Scopus (732) Google Scholar, 20Means A.R. Van Berkum M.F. Bagchi I. Lu K.P. Rasmussen C.D. Pharmacol. Ther. 1991; 50: 255-270Crossref PubMed Scopus (196) Google Scholar), ion channels (21Liu M. Chen T.-Y. Ahamed B. Li J. Yau K.-Y. Science. 1994; 266: 1348-1354Crossref PubMed Scopus (240) Google Scholar), and receptors (22Ehlers M. Zhang S. Bernhardt J.P. Huganir R.L. Cell. 1996; 84: 745-755Abstract Full Text Full Text PDF PubMed Scopus (474) Google Scholar). 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 βγ complex can bind to CaM when passed through the CaM-agarose column (23Asano T. Ogasawara N. Kitajima S. Sano M. FEBS Lett. 1986; 205: 135-138Crossref Scopus (28) Google Scholar, 24Katada T. Kusakabe K. Oinuma M. Ui M. J. Biol. Chem. 1987; 262: 11897-11900Abstract Full Text PDF PubMed Google Scholar); however, the precise nature of the interaction is not clear. In this report, we investigated the interaction of Ca2+-CaM with Gβγ subunits and the potential physiological significance of this interaction. We found that CaM not only binds to Gβγ subunits purified from brain but also to the most diverse Gβ5Lγ complex from retina. We then identified and characterized the CaM-binding domain of the Gβ subunit by using synthetic peptides and site-specific mutation. Furthermore, we showed that binding of CaM to βγ inhibits the βγ-dependent PTX-catalyzed ADP-ribosylation of Gα subunits. Using both brain Gβγ subunits and the CaM-binding peptide derived from the β 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 βγ with Ca2+-CaM and the potential functions of these interactions in modulating cellular signaling and other functions. 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. Bleached bovine rod outer segment membranes were prepared from bovine retina as described (25Papermaster D.S. Dreyer W.J. Biochemistry. 1974; 13: 2438-2444Crossref PubMed Scopus (579) Google Scholar). Transducin was removed from the membranes by 6 × hypotonic elution with 100 μmGTPγS in the presence of 3 mm MgCl2 (26Wieland T. Ulibarri I. Gierschik P. Jakobs K.H. Eur. J. Biochem. 1991; 196: 707-716Crossref PubMed Scopus (42) Google Scholar). 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 mmMgCl2, 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 Gβ5-specific antiserum CT215 as described (27Watson A.J. Katz A. Simon M.I. J. Biol. Chem. 1994; 269: 22150-22156Abstract Full Text PDF PubMed Google Scholar). High affinity binding of G protein β 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 Gβ 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 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 (28DeGrado W.F. Erickson-Viitanen S. Wolfe Jr., H.R. O'Neil K.T. Proteins Struct. Funct. Genet. 1987; 2: 20-33Crossref PubMed Scopus (28) Google Scholar). 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. Mutations in the putative CaM-binding site of Gβ1were 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 β constructs were subcloned into pCDNA3.1 vector (Invitrogen, San Diego, CA). 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 Gβ proteins were separated by SDS-PAGE and visualized by autoradiography. Pertussis toxin-catalyzed ADP-ribosylation of Gαo was performed as described previously (29Slepak V.Z. Wilkie T.M. Simon M.I. J. Biol. Chem. 1993; 268: 1414-1423Abstract Full Text PDF PubMed Google Scholar). Briefly, 0.1 μg of recombinant Gαo was mixed with 0.1 μg of purified rat brain βγ 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 mmEDTA, 2 mm MgCl2, 2 mmdithiothreitol, 0.5 μm32P-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. The Ca2+-CaM-dependent activity of CaM kinase II was assayed essentially as described previously (30Colbran R.J. Soderling T.R. J. Biol. Chem. 1990; 265: 11213-11219Abstract Full Text PDF PubMed Google Scholar). Briefly, different concentrations of Gβ 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 mmCaCl2, 0.1 mm [γ-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. cDNAs encoding PLC-β2, Gβγ, and CaM were cotransfected into COS-7 cells with LipofectAMINE (Life Technologies, Inc.). Then the cells were labeled with 10 μCi/mlmyo-[2-3H]inositol the following day. 48 h after transfection, the activity of PLC-β2 was assayed by determining the levels of inositol phosphates as described previously (31Wu D. Lee C-H. Rhee S.G. Simon M.I. J. Biol. Chem. 1992; 267: 1811-1817Abstract Full Text PDF PubMed Google Scholar, 37Liu M. Simon M.I. Nature. 1996; 382: 83-87Crossref PubMed Scopus (189) Google Scholar). Phospholipid vesicles containing 50 μm[3H]PtdInsP2 and 500 μmphophatidylethanolamine 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-β2, 1.7 μm Gβγ 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. To demonstrate the direct interaction of Gβγ subunits with CaM, purified bovine brain βγ subunits were incubated with CaM-agarose gel in the presence of 1 mm CaCl2. After extensively washing with Ca2+-containing buffer, the bound βγ subunits were eluted by buffer containing EGTA. As shown in Fig.1 A, brain βγ 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 β subunits that have been characterized in mammalian systems. Among them, Gβ5 is the most diverse form and is expressed predominately in neuronal cells (27Watson A.J. Katz A. Simon M.I. J. Biol. Chem. 1994; 269: 22150-22156Abstract Full Text PDF PubMed Google Scholar). A splice variant of Gβ5, β5L, is found only in rod outer segment membrane. To examine whether CaM binding is a common feature in different βγ subunits, Gβ5Lγ 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 Gβ5 subunit. Like other βγ subunits, β5Lγ also binds to Ca2+-CaM (Fig.1 B), suggesting that the CaM-binding domain is conserved in all Gβγ subunits. To identify the CaM-binding domain on Gβγ subunits, the amino acid sequences of βγ were compared with those of known high affinity CaM-binding proteins. We found that the N terminus of all Gβ subunits contains a putative CaM-binding domain that exhibits the characteristics typical of CaM-binding peptides (32O'Neil K.T. DeGrado W.F. Trends Biochem. Sci. 1990; 15: 59-64Abstract Full Text PDF PubMed Scopus (710) Google Scholar). Fig.2 shows the alignment of the N-terminal domain sequences of Gβ 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 Gβ 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. 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 Gβ residues 40–63 (Fig.3 A). 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. 3 A, 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. 3 A). 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. 3 A). 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. 3 B), indicating the complex formation is Ca2+-dependent. Peptides from other regions of the Gβ subunit had no effect on the mobility of CaM (data not shown). Since the synthetic peptide contains a tryptophan residue, whereas CaM contains none, binding of Gβ 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. 4 A). 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 Gβ 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.4 A), 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. 4 B, when a 0.1 μmconcentration 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 (K d) obtained was ∼40 nm(Fig. 4 C), 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. 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 γ2 subunit in rabbit reticulocyte lysates in the presence of35S-labeled methionine. Fig. 5 Ashows the in vitro translated wild type and mutant Gβ 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. 5 B, like the wild type Gβ 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. 5 B), suggesting that these residues are critical in the interactions. To further probe the conformation and activity of mutant Gβ subunits, we analyzed the formation of βγ dimers using in vitro translated proteins and coimmunoprecipitation with specific anti-Gγ2antibodies. The wild type Gβ and its mutant proteins can be precipitated by γ2 antibodies, and there is little difference in their ability to interact with γ subunits (data not shown), indicating that mutant β subunits could still fold into the native conformation and retain affinity for γ subunits. To explore the possible physiological functions of CaM binding to Gβγ subunits, we first examined the effects of CaM binding on βγ-dependent pertussis toxin-catalyzed ADP-ribosylation of Gαo subunit. PTX is a bacterial toxin that catalyzes the ribosylation of the C-terminal cysteine residues of Gαo, Gαi, and Gαt subunits. PTX modification blocks the interactions of Gα subunits with receptors and thus blocks the ligand-mediated signal transduction. The labeling of Gαo by PTX requires the Gαoβγ heterotrimer rather than the free Gαo subunit (33Casey P.J. Graziano M.P. Gilman A.G. Biochemistry. 1989; 28: 611-616Crossref PubMed Scopus (91) Google Scholar). Thus, ADP-ribosylation of αo can be used as a sensitive indicator of formation of αoβγ heterotrimers. Incubation of CaM with bovine brain Gβγ 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 βγ subunits, the PTX-catalyzed ribosylation of Gαo 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 Gβγ subunits and this complex lost its ability to interact with Gαo subunit; therefore, CaM inhibited the βγ-dependent ADP-ribosylation of Gαo. To further investigate the nature of CaM binding on Gβγ subunits, we examined its effects on βγ-stimulated PLC-β2 activity. As shown in Fig.7 A, CaM has little effect on PLC-β2 activity stimulated by βγ subunits in an in vitroreconstituted system. To confirm the observations obtained in the reconstitution experiments, cDNAs encoding PLC-β2, Gβγ, and CaM were transfected into COS-7 cells. Coexpression of PLC-β2 and βγ subunits increased inositol 1,4,5-trisphosphate release 3–5 fold (Fig. 7 B, column 3). However, cotransfection of CaM expression plasmids had little effect on the βγ-stimulated PLC-β2 activity (Fig. 7 B, column 4). These results indicated that in the Gβ subunit, the domain responsible for PLC-β2 activation is distinct from the domain binding to CaM, suggesting that different domains of the β subunit are involved in interactions with different effector proteins. The putative CaM-binding domain of Gβ subunits was further characterized by using the synthetic peptide derived from Gβ 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.8 A, the peptide was a potent inhibitor of CaM kinase II activity. At a 1:1 binding ratio of Gβ peptide to CaM, the peptide totally inhibited the Ca2+-CaM-dependent activation of CaM kinase II, suggesting that the binding affinity of Gβ 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 Gβγ subunits can also inhibit the Ca2+-CaM-dependent CaM kinase II activity, Ca2+-CaM was incubated with brain Gβγ subunits for 30 min at room temperature. Then the Ca2+-CaM-stimulated CaM kinase II activity was assayed. Fig. 8 B shows that brain Gβγ subunits inhibited 70–80% of Ca2+-CaM-stimulated CaM kinase II activity, indicating that βγ can competitively bind to Ca2+-CaM. Previous studies have demonstrated that G protein subunits can act as potent inhibitors of the Ca2+-CaM-stimulated phosphodiesterase activity (23Asano T. Ogasawara N. Kitajima S. Sano M. FEBS Lett. 1986; 205: 135-138Crossref Scopus (28) Google Scholar, 24Katada T. Kusakabe K. Oinuma M. Ui M. J. Biol. Chem. 1987; 262: 11897-11900Abstract Full Text PDF PubMed Google Scholar), probably through interactions of Gβγ with CaM. In this report, we show direct binding of Gβγ subunits with CaM. This CaM-binding property of Gβ is Ca2+-dependent and conserved in known Gβ subunits, including the most diverse β5 subunit. We also identified and characterized the CaM-binding domain in βγ 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 Gβ 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 βγ to CaM. The interaction between Gβγ subunits and CaM is Ca2+-dependent. Ca2+ is an important intracellular messenger in many cellular functions including cell growth and development (34Berridge M.J. Nature. 1993; 361: 315-325Crossref PubMed Scopus (6136) Google Scholar, 35Clapham D.E. Cell. 1995; 80: 259-268Abstract Full Text PDF PubMed Scopus (2250) Google Scholar). Many seven-transmembrane receptors activate the G protein subunits, which in turn activate PLC-β, 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 (32O'Neil K.T. DeGrado W.F. Trends Biochem. Sci. 1990; 15: 59-64Abstract Full Text PDF PubMed Scopus (710) Google Scholar,36Meador W.E. Means A.R. Quicho F.A. Science. 1993; 262: 1718-1721Crossref PubMed Scopus (610) Google Scholar). 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 Gβγ for Ca2+-CaM is sufficiently high so that under these conditions much of the free βγ in the cells should be in the CaM-bound form. Therefore, an increase in intracellular Ca2+ concentration could selectively regulate the interactions of Gβγ with a number of other proteins through binding with CaM. The interaction of Gβγ with Ca2+-CaM could play an important role in the cross-talk mechanism between different G protein pathways (37Liu M. Simon M.I. Nature. 1996; 382: 83-87Crossref PubMed Scopus (189) Google Scholar). Ca2+-CaM has been shown to regulate the formation and hydrolysis of cAMP. In the Gαs-coupled pathway, both adenylyl cyclases and phosphodiesterases are Ca2+-CaM-dependent, which could serve as the convergence point for Ca2+-dependent and Gαs-dependent stimuli. The primary structure of the identified CaM-binding domain of Gβγ shows features similar to some other CaM-binding proteins and inhibitors (21Liu M. Chen T.-Y. Ahamed B. Li J. Yau K.-Y. Science. 1994; 266: 1348-1354Crossref PubMed Scopus (240) Google Scholar, 28DeGrado W.F. Erickson-Viitanen S. Wolfe Jr., H.R. O'Neil K.T. Proteins Struct. Funct. Genet. 1987; 2: 20-33Crossref PubMed Scopus (28) Google Scholar, 38Ikura M. Clore G.M. Gronenborn A.M. Zhou G. Klee C.B. Bax A. Science. 1992; 256: 632-638Crossref PubMed Scopus (1173) Google Scholar, 39Meador W.E. Means A.R. Quicho F.A. Science. 1992; 257: 1251-1255Crossref PubMed Scopus (935) Google Scholar, 40Blumenthal D.K. Takio K. Edelman A.M. Charbonneau H. Titani K. Walsh K.A. Krebs E.G. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 3187-3191Crossref PubMed Scopus (200) Google Scholar, 41Verma A.K. Filoteo A.G. Stanford D.R. Wieben E.D. Penniston J.T. Strehler E.E. Fischer R. Heim R. Vogel G. Mathews S. Strehler-Page M.A. James P. Vorherr T. Krebs J. Carafoli E. J. Biol. Chem. 1988; 263: 14152-14159Abstract Full Text PDF PubMed Google Scholar, 42Kincaid R.L. Nightingale M.S. Martin B.R. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 8983-8987Crossref PubMed Scopus (100) Google Scholar, 43Fischer R. Yu W. Anagli J. Berchtold M.W. J. Biol. Chem. 1996; 271: 25067-25070Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 44Venema R.C. Sayegh H.S. Kent J.D. Harrison D.G. J. Biol. Chem. 1996; 271: 6435-6440Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 45Payne M.E. Fong Y.-L. Ono T. Colbran R.J. Kemp B.E. Soderling T.R. Means A.R. J. Biol. Chem. 1988; 263: 7190-7195Abstract Full Text PDF PubMed Google Scholar). For instance, the identified β 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 (38Ikura M. Clore G.M. Gronenborn A.M. Zhou G. Klee C.B. Bax A. Science. 1992; 256: 632-638Crossref PubMed Scopus (1173) Google Scholar, 39Meador W.E. Means A.R. Quicho F.A. Science. 1992; 257: 1251-1255Crossref PubMed Scopus (935) Google Scholar). 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 (36Meador W.E. Means A.R. Quicho F.A. Science. 1993; 262: 1718-1721Crossref PubMed Scopus (610) Google Scholar). CaM contains two globular domains, and these two domains are connected by a long alpha helical segment (32O'Neil K.T. DeGrado W.F. Trends Biochem. Sci. 1990; 15: 59-64Abstract Full Text PDF PubMed Scopus (710) Google Scholar, 36Meador W.E. Means A.R. Quicho F.A. Science. 1993; 262: 1718-1721Crossref PubMed Scopus (610) Google Scholar). 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 Gβ CaM-binding domain is not a typical amphipathic α helix (46Sondek J. Bohm A. Lambright D.G. Hamm H.E. Sigler P.B. Nature. 1996; 379: 369-374Crossref PubMed Scopus (705) Google Scholar), CaM could still position itself to bind to the Gβ protein, possibly by stabilizing a change in the conformation of the N-terminal domain of Gβ 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 (52Warr C.G. Kelly L.E. Biochem. J. 1996; 314: 497-503Crossref PubMed Scopus (81) Google Scholar, 53Wes P.D. Yu M. Montell C. EMBO J. 1996; 15: 5839-5848Crossref PubMed Scopus (72) Google Scholar, 54Chevesich J. Kreuz A.J. Montell C. Neuron. 1997; 18: 95-105Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar). The binding of CaM to Gβγ interfered with the formation of Gαβγ trimers as assayed by the inhibition of PTX-catalyzed ADP-ribosylation of Gαo. Based on the secondary structure of the β subunit, the N-terminal region of the β subunit is in close proximity to the N-terminal portion of Gα subunit (47Lambright D.G. Sondek J. Bohm A. Skiba N.P. Hamm H.E. Sigler P.B. Nature. 1996; 379: 311-319Crossref PubMed Scopus (1042) Google Scholar, 48Wall M.A. Coleman D.E. Lee E. Iniguez-Lluhi J.A. Posner B.A. Gilman A.G. Sprang S.R. Cell. 1995; 83: 1047-1058Abstract Full Text PDF PubMed Scopus (1002) Google Scholar, 49Neer E.J. Smith T.F. Cell. 1996; 84: 175-176Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). It has been reported that effector activation by the βγ subunits is blocked upon the addition of the Gα subunit presumably by heterotrimerization (1Tang W.J. Gilman A.G. Science. 1991; 254: 1500-1503Crossref PubMed Scopus (741) Google Scholar, 13Wickman K.D. Iniguez-Lluhi J.A. Davenport P.A. Taussig R. Krapivinsky G.B. Linder M.E. Gilman A.G. Clapham D.E. Nature. 1994; 368: 255-257Crossref PubMed Scopus (377) Google Scholar). Thus, by interacting with the N-terminal domain of β subunit, the Ca2+-CaM complex could affect the heterotrimer formation of Gαβγ. Interaction of CaM with βγ has little effect on the G protein βγ subunit-activated PLC-β2 activity. These results support the notion that interaction of different βγ-responsive effectors is mediated by distinct domains of Gβγ (50Zhang S. Coso O.A. Collins R. Gutkind J.S. Simonds W.F. J. Biol. Chem. 1996; 271: 20208-20212Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 51Yan K. Gautam N. J. Biol. Chem. 1996; 271: 17597-17600Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). By using a series of chimeras between Dictyostelium and mammalian β subunits, a small C-terminal segment of Gβ was identified as responsible for the activation of PLC-β2 (50Zhang S. Coso O.A. Collins R. Gutkind J.S. Simonds W.F. J. Biol. Chem. 1996; 271: 20208-20212Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). The CaM-binding domain of Gβ identified in this report is located in the N-terminal region. This region of Gβ 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 (50Zhang S. Coso O.A. Collins R. Gutkind J.S. Simonds W.F. J. Biol. Chem. 1996; 271: 20208-20212Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 51Yan K. Gautam N. J. Biol. Chem. 1996; 271: 17597-17600Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). It will be of great interest to determine whether binding of CaM to Gβγ can affect the activation of the potassium channels (GIRK) and mitogen-activated protein kinase pathways. 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.
Referência(s)