Characterization of a Phospholipase C beta 2-Binding Site Near the Amino-terminal Coiled-coil of G Protein beta gamma Subunits*

Daniel M. Yoshikawa, Karen Bresciano, Mamata Hatwar, and Alan V. SmrckaDagger

From the Department of Pharmacology and Physiology, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642

Received for publication, July 10, 2000, and in revised form, January 4, 2001



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In previous work (Sankaran, B., Osterhout, J., Wu, D., and Smrcka, A. V. (1998) J. Biol. Chem. 273, 7148-7154), we showed that overlapping peptides, N20K (Asn564-Lys583) and E20K (Glu574-Lys593), from the catalytic domain of phospholipase C (PLC) beta 2 block Gbeta gamma -dependent activation of PLC beta 2. The peptides could also be directly cross-linked to beta gamma subunits with a heterobifunctional cross-linker succinimidyl 4-[N-maleimidomethyl]-cyclohexane-1-carboxylate. Cross-linking of peptides to Gbeta 1 was inhibited by PLC beta 2 but not by alpha i1(GDP), indicating that the peptide-binding site on beta 1 represents a binding site for PLC beta 2 that does not overlap with the alpha i1-binding site. Here we identify the site of peptide cross-linking and thereby define a site for PLC beta 2 interaction with beta  subunits. Each of the 14 cysteine residues in beta 1 were altered to alanine. The ability of the PLC beta 2-derived peptide to cross-link to each beta gamma mutant was then analyzed to identify the reactive sulfhydryl moiety on the beta  subunit required for the cross-linking reaction. We find that C25A was the only mutation that significantly affected peptide cross-linking. This indicates that the peptide is specifically binding to a region near cysteine 25 of beta 1 which is located in the amino-terminal coiled-coil region of beta 1 and identifies a PLC-binding site distinct from the alpha  subunit interaction site.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Guanine nucleotide-binding proteins (G proteins)1 are a large group of structurally similar proteins consisting of three subunits (alpha , beta , and gamma ) that are central molecules coupling seven-transmembrane domain-spanning receptors to downstream effector molecules. Activation of G proteins begins with a ligand-induced conformational change of the receptor which catalyzes the release of GDP from the alpha  subunit in exchange for GTP (1, 2). In the GDP-bound heterotrimeric state, alpha (GDP)·beta gamma , neither alpha (GDP) nor beta gamma can regulate effector activity. Upon receptor-catalyzed G protein activation, the heterotrimer dissociates into free alpha (GTP) and free beta gamma subunits. It is well understood that both alpha (GTP) and beta gamma subunits can interact with a variety of downstream effector molecules including enzymes and ion channels. GTP is hydrolyzed to GDP, and reassociation of alpha (GDP) with beta gamma results in deactivation of beta gamma -dependent signaling. Despite detailed knowledge of alpha - and beta gamma subunit functions, the mechanism for how beta gamma subunits activate its variety of effectors is not entirely understood.

Effector-binding sites on the surface of beta gamma are beginning to be mapped. The putative competition between alpha (GDP) and effectors for beta gamma forms the premise for recent studies to map effector-binding sites at the alpha  subunit-binding interface on beta . The three-dimensional structure of the G protein heterotrimer reveals that the beta  subunit is a beta -propeller with seven "blades" and an amino-terminal alpha -helix (3, 4). The alpha  subunit binds to a portion of the top of the beta -propeller and along side one of the blades of the propeller. Two groups have shown that alanine substitution of alpha -contacting residues on the top surface of the beta -propeller differentially affected the ability of beta gamma to regulate various target molecules (5, 6). One of these groups tested whether the sides of the beta -propeller may be important for effector interactions. Residues along the outer strand of each of the 7 blades of the beta -propeller were altered, and each beta gamma mutant was tested for its ability to stimulate phospholipase C (PLC) beta 2 and regulate adenylyl cyclases (7). Mutations in three of the blades eliminated the ability of beta gamma to activate PLC beta 2, whereas these same mutations did not affect regulation of adenylyl cyclase isoforms 1 and 2. Consistent with these studies, synthetic peptides from discrete blade regions of beta  were used in competition experiments to define a region located near the interface between alpha  and beta gamma as being important for activation of PLC beta 2 (8).

A screen for dominant-negative yeast Gbeta mutants that could interfere with mating factor signaling identified different regions involved in beta  subunit-effector interactions (9). In particular, one set of mutations maps to an amino-terminal region involved in a coiled-coil interaction with gamma  subunits on the opposite side of the beta  subunit from the alpha  subunit-binding site. Taken together, these studies show that the top surface of beta , where many alpha -contact points are located, is a region critical for effector binding and regulation. However, effector-binding sites are being mapped to a variety of regions on the surface of beta gamma . Multiple interaction regions are reported even for the same effector. It has been suggested that distinct sets of contacts may define the specificity of the interaction of beta gamma with various effectors (i.e. each effector will have a characteristic footprint along the sides of the molecule).

In previous work (10) we showed that overlapping peptides, N20K (Asn564-Lys583) and E20K (Glu574-Lys593) from the catalytic domain of phospholipase C (PLC) beta 2, block PLC activation by beta gamma and could be directly cross-linked to beta  subunits with a heterobifunctional cross-linker (SMCC). In the study presented here we have identified the site of cross-linking of N20K to the beta  subunit thereby defining a binding site for PLC beta 2. This approach identified a site distinct from those mapped by site-directed mutagenesis but consistent with the region identified in yeast beta  subunits as being important for beta  subunit-effector interactions (9).


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Peptides were purchased from Biosynthesis (Lewisville, TX) and had a purity of greater than 90% based on high pressure liquid chromatography analysis, and identities were confirmed by mass spectrometry. Biotin-labeled peptide (B-N20K) was purchased from Alpha Diagnostic International (San Antonio, TX). Succinimidyl 4-[N-maleimidomethyl]-cyclohexane-1-carboxylate (SMCC) and horseradish peroxidase-conjugated NeutrAvidin were from Pierce. Trypsin and 2-aminoethanol (monoethanolamine) were from Sigma. PVDF membrane was from PerkinElmer Life Sciences. Nitrocellulose membrane was from Schleicher & Schuell.

beta 1-Cysteine mutants in pALTER (11) were kindly provided by Dr. Eva J. Neer.

Construction of Recombinant Baculoviruses and Sf9 Culture-- Each beta 1-cysteine mutant was subcloned into the baculovirus transfer vector pFASTBAC (Life Technologies, Inc.). Recombinant baculoviruses were generated using standard manufacturer's procedures. Sf9 cells were grown at 27 °C in Sf900 medium (Life Technologies, Inc.).

Purification of Phospholipase C beta 2-- Baculovirus constructs directing expression of recombinant His6 PLC beta 2 were used to infect 800 ml of Sf9 cells at a density of 2.5 × 106 cells/ml. PLC beta 2 was purified according to published procedures (12).

Purification of Wild Type beta 1gamma 2 Cysteine-mutated beta 1gamma 2 and Sf9 beta gamma Subunits-- For purification of wild type beta 1gamma 2, baculovirus constructs encoding beta 1, gamma 2, and His6-tagged alpha i1 were obtained from Alfred Gilman's laboratory. 800 ml of Sf9 cells at 2.5 × 106 cells/ml were simultaneously infected with the three constructs, and the beta gamma subunits were purified according to published procedures (13) and modified as in Romoser et al. (12). The protein was concentrated on a 0.3-ml macro-prep ceramic hydroxyapatite (Bio-Rad) column and eluted into beta gamma vehicle (20 mM HEPES, pH 8.0, 100 mM NaCl, 1% octyl glucoside, 200 mM potassium phosphate, pH 8.0).

For purification of beta 1gamma 2-cysteine (C) mutants, 200 ml of Sf9 cells at 2.5 × 106 cells/ml were simultaneously infected with baculovirus constructs encoding each beta 1-cysteine mutant, gamma 2, and His6-tagged alpha i1. Each beta gamma -cysteine mutant was purified according to the above procedures but modified to batch style with 0.5 ml of nickel-nitrilotriacetic-agarose. Peak fractions were pooled and concentrated on a 0.3-ml hydroxyapatite column, and protein was eluted into beta gamma vehicle.

For purification of endogenous Sf9 beta , 1 liter of Sf9 cells at 2.5 × 106 cells/ml was simultaneously infected with baculovirus constructs encoding His6-tagged alpha i1 and gamma 2. Sf9 beta gamma subunits were purified according to the procedures for the purification of wild type beta gamma as described above.

Purification of alpha  Subunits-- Recombinant myristoylated alpha i1 and alpha o were purified from Escherichia coli coexpressing alpha i1 or alpha o with N-myristoyltransferase according to published procedures (14) with some modification. Briefly, alpha i1 or alpha o were expressed in 1 liter of E. coli containing the N-myristoyltransferase plasmid for 16 h. After preparation of the lysate, the protein was applied to a 50-ml column of Q Sepharose fast flow (Amersham Pharmacia Biotech) that had been equilibrated with 50 mM Tris, pH 8.0, 2 mM dithiothreitol, 2 µM GDP. The column was washed with 150 ml of equilibration buffer followed by elution of the protein with a 400-ml linear gradient to 250 mM NaCl in equilibration buffer. Fractions containing alpha  subunits were further purified by fast protein liquid chromatography phenyl-Superose chromatography resulting in two peaks of alpha  subunits eluting from the column. The second peak corresponding to myristoylated alpha  subunit was pooled and concentrated; 50 µM GDP was added, frozen in aliquots in liquid N2, and stored at -80 °C. Functionality of the alpha  subunits was confirmed by the ability to be ADP-ribosylated by pertussis toxin and the ability to inhibit beta gamma -mediated activation of PLC beta 2.

Chemical Cross-linking-- We used two different cross-linking protocols in this study. Protocol 1, used for the experiments described in Figs. 1 and 7, has been described previously (10). Here, beta gamma subunits at the indicated concentrations were mixed with N20K (NRSYVISSFTELKAYDLLSK) peptide followed by addition of SMCC to 200 µM from a 2 mM stock solution. The reaction was allowed to proceed for 5 min. For protocol 2, N20K was reacted with SMCC for 10 min followed by addition of 500 mM ethanolamine, pH 7.3, to inactivate the unreacted succinimide moiety. beta gamma subunits were then added, and the reaction was allowed to proceed at room temperature for 1.5 min. For both protocols reactions were quenched with SDS sample buffer (32 mM Tris, pH 6.8, 5% glycerol, 1% SDS, 350 mM 2-mercaptoethanol, bromphenol blue (final concentrations)), resolved on SDS-PAGE, and transferred overnight onto a PVDF membrane (PerkinElmer Life Sciences). For some experiments (as indicated) cross-linked species were detected with horseradish peroxidase (HRP)-conjugated NeutrAvidin (Pierce). In other experiments cross-linked beta  subunits were detected with the carboxyl-terminal anti-beta subunit antibody B600 and/or amino-terminal specific beta  subunit antibody followed by secondary antibody linked to HRP and development with Amersham Pharmacia Biotech chemiluminescence reagents.

Cross-linking to Trypsin Digestion of beta gamma Subunits-- Wild type or cysteine mutant beta 1gamma 2 was digested with trypsin at a beta gamma :trypsin ratio of 75:1, for 30 min at 30 °C. The digestion was stopped by addition of 62.5 µM phenylmethylsulfonyl fluoride. Digested beta gamma (80 nM), 100 µM N20K, and cross-linker were mixed as in cross-linking protocol 1 and analyzed by immunoblotting with an amino-terminal specific beta  subunit antibody.

Phospholipase C Assay-- PLC assays were performed as described previously (12). Briefly, purified PLC beta 2 was mixed with sonicated phospholipid vesicles containing 50 µM phosphatidylinositol 4,5-bisphosphate, 200 µM phosphatidylethanolamine, and [3H]phosphatidylinositol 4,5-bisphosphate (6000-8000 cpm/assay), with or without 100 nM purified beta 1gamma 2 or mutants and/or peptides. Reactions were allowed to proceed from 3 to 5 min at 30 °C. Intact lipids and proteins were precipitated with bovine serum albumin and 10% trichloroacetic acid and removed by centrifugation. Supernatant containing soluble [3H]inositol 1,4,5-trisphosphate was analyzed by liquid scintillation counting.


    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

PLC beta 2 Inhibits Peptide Cross-linking to beta gamma Subunits but alpha  Subunits Do Not-- We have previously shown that two peptides derived from PLC beta 2 (N20K and E20K) are specifically and chemically cross-linked to both G protein beta  and gamma  subunits using a heterobifunctional cross-linking reagent (SMCC) (10). SMCC contains maleimide- and succinimide-reactive groups, which react with SH- and NH- moieties, respectively. Since the peptides have no cysteine residues, cross-linking must be through a primary amine on the peptide and a sulfhydryl group in the beta  subunit. The cross-linking reaction is equally effective if the amino terminus of the peptide is acetylated indicating that one of the two lysine residues within the N20K peptide sequence is involved in the reaction.

Previous data suggest that cross-linking of N20K to beta gamma subunits could be blocked by PLC beta 2 but not alpha (GDP). To analyze this in greater detail, the concentrations of alpha  subunits and PLC beta 2 required to inhibit N20K cross-linking to the beta  subunit were examined (Fig. 1). Cross-linking of beta  subunit to the peptide is indicated by the increase in apparent molecular weight of the immunoreactive beta  subunit after incubation with peptide and SMCC. The uppermost band is beta  cross-linked to gamma  (this band reacts with gamma  subunit antibodies (not shown)); the bottom band is uncross-linked beta , and the two bands in between are beta  cross-linked to the peptide. There is one prominent peptide cross-linked species and a second minor peptide cross-linked species. PLC beta 2 (90 nM) significantly inhibited cross-linking of 10 µM peptide (N20K) to 30 nM beta gamma and completely inhibited the cross-linking at 150 nM PLC beta 2. This concentration dependence is consistent with the EC50 of ~50-100 nM for PLC activation by beta gamma subunits (15). On the other hand alpha i1(GDP) did not inhibit cross-linking of the peptide up to 250 nM. This same preparation of alpha  subunit was able to block beta gamma -mediated activation of PLC beta 2 by 95% at a 2:1 ratio of alpha  to beta gamma (not shown). These results indicate that the peptide and alpha  subunit-binding sites on beta gamma are distinct and that the peptide-binding site is within the binding site for PLC beta 2 on beta gamma .



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Fig. 1.   Effects of PLC beta 2 and alpha i1(GDP) on peptide cross-linking to Gbeta 1. beta 1gamma 2 (30 nM) was incubated with the indicated concentrations of PLC beta 2 or alpha i1 in the presence of 10 µM GDP and 0.1% C12E10 for 5 min at 23 °C. The N20K peptide (10 µM) was added followed by 200 µM SMCC according to cross-linking protocol 1 under "Experimental Procedures." beta  subunit and cross-linked species of beta  subunit were detected with a carboxyl-terminal specific antibody (B600). Arrows indicate the positions of molecular mass markers in kDa. This experiment was performed multiple times with similar results.

Recombinant Myristoylated alpha  Subunits Do Not Prevent PLC from Inhibiting Cross-linking of the Peptide-- As discussed, alpha  subunits have been suggested to inhibit beta gamma subunit-mediated PLC beta 2 activation by competing for binding of the PLC beta 2 to the beta gamma subunits. To test whether alpha (GDP) inhibits the binding of PLC beta 2 to beta gamma subunits, we tested whether excess alpha o(GDP) (250 nM) altered the ability of PLC beta 2 to block peptide cross-linking to beta gamma (Fig. 2). For this experiment the cross-linking protocol was modified to eliminate intermolecular beta  cross-linking to gamma  and reduce the number of cross-linked species (see "Experimental Procedures" cross-linking protocol 2 and the figure legend). Although PLC beta 2 did not completely inhibit peptide cross-linking to beta  subunits in the absence of alpha o in this experiment (compared with Fig. 1), cross-linking was still significantly inhibited when PLC beta 2 was added. In the presence of the alpha  subunit, PLC beta 2 also inhibited cross-linking, but a higher concentration of PLC was required to observe the inhibition. Similar results were observed with alpha i(GDP). This suggests that PLC beta 2 can bind to beta  subunits at the peptide interaction site even in the presence of alpha  subunits, but alpha  subunits may alter the affinity of PLC for beta gamma .



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Fig. 2.   alpha o(GDP) does not alter the ability of PLC beta 2 to block peptide cross-linking. Peptide N20K at 10 µM was cross-linked to beta 1gamma 2 (30 nM) at the indicated concentrations of PLC beta 2 in the presence or absence of 250 nM alpha o. Incubations, reactions, and electrophoresis are as described in cross-linking protocol 2 under "Experimental Procedures." Arrows indicate the positions of molecular mass markers in kDa. This experiment was performed five times with similar results.

Purification of Cysteine-mutated beta 1 Subunits-- To identify the location of peptide cross-linking and thus map a region for PLC beta 2 binding that does not coincide with alpha  subunit binding, we used a sulfhydryl mutagenesis strategy similar to that designed by Garcia-Higuera et al. (11). Since cross-linking can only occur between a cysteine residue from beta  and lysine from the peptide, we characterized cross-linking of the peptide to a series of beta  subunit mutants where each cysteine residue had been individually mutated to alanine.

We obtained the beta 1-cysteine mutant cDNAs from Dr. Eva J. Neer and created baculovirus constructs for expression and purification of each cysteine mutant from Sf9 insect cells. Fig. 3A displays a Coomassie-stained panel of 11 of the 14 purified beta 1gamma 2-cysteine mutants. The numbers indicate the positions of the cysteine residue in the beta 1 primary sequence. The Cys166, Cys149, and Cys317 proteins are not shown but are of similar purity. beta 1 Cys103, Cys114, Cys121, Cys148, and Cys271 are doublets in which the top band of each doublet is Sf9 beta  that copurifies. It will be shown in later experiments that Sf9 beta  does not cross-link to N20K, and therefore, any cross-linking that is observed must be to the expressed mutant beta  subunit. The concentration of each beta 1-cysteine mutant was estimated by immunoblot analysis because the preparations contained Sf9 beta , and we wanted to base the analysis on equal amounts of the expressed beta  mutants. The concentration of each beta 1-cysteine mutant (lower band in the case of doublets) was estimated by comparing the band intensities to a range of intensities produced by known amounts of wild type beta 1 protein (an example of such an estimation for Cys114 is shown in Fig. 3B).



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Fig. 3.   beta 1gamma 2 cysteine mutants purified on nickel-nitrilotriacetic column. A, each beta 1gamma 2 cysteine mutant was run on SDS-PAGE (12%) followed by staining with Coomassie Brilliant Blue. wt, wild type; numbers, positions of the mutated cysteines in the beta 1 sequence. Arrows indicate the positions of molecular mass markers in kDa. B, immunoblot quantitation of Cys114. The position of the expressed beta 1 subunit is indicated by the arrow on the right. The 38-kDa molecular mass marker is indicated by the arrow on the left.

Each of the mutants was able to bind to His-tagged alpha i and elute with addition of a G protein activator (AlF<UP><SUB>4</SUB><SUP>−</SUP></UP>), suggesting that they were properly folded and active. To be sure that the mutants were able to activate phospholipase C, 13 of the 14 mutants were tested for their ability to activate phospholipase C beta 2 in an in vitro phospholipase C assay (Fig. 4). All of the mutants that were tested were able to activate PLC beta 2 between 3- and 5-fold compared with 7-fold for wild type beta 1gamma 2. Thus, while their efficacy is slightly diminished (by ~1/2) relative to wild type beta 1gamma 2, in general these mutants are properly folded and capable of interacting with alpha  subunits and PLC beta 2. It is not clear that why these mutants have a reduced ability to activate PLC beta 2, but we suspect that it is not a result of the mutations themselves but rather is a consequence of the difficulties in quantitating the mutants as well as repeated freezing and thawing of the samples. Regardless, this cannot be responsible for any differences observed in the cross-linking experiments since there is no correlation between the activities and the degree of cross-linking.



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Fig. 4.   Ability of the purified cysteine mutants to activate PLC beta 2. Each beta gamma cysteine mutant (100 nM) was assayed in the presence of 1 ng of PLC beta 2. CaCl2 was 2.8 mM in the presence of 3 mM EGTA at pH 7.2, and reactions were for 3 min at 30 °C. Each point represents duplicate determinations, and the experiment was repeated twice.

Cross-linking of Biotin-labeled Peptide to Wild Type beta 1gamma 2 Dimer-- For the survey of the ability of the peptide to cross-link to the mutants, we modified cross-linking protocol 2 (see "Experimental Procedures") to use an amino-terminal biotin-modified form of the N20K peptide (B-N20K) which we could detect with horseradish peroxidase-conjugated avidin (HRP-NeutrAvidin from Pierce). With this modified peptide our procedure for monitoring cross-linking to beta gamma subunits allows only the peptide cross-linked species of beta  to be detected.

To confirm that the biotin modification did not affect the activity of the peptide, we tested whether the B-N20K would block PLC activation by beta gamma . Consistent with our previous report (10), B-N20K inhibited beta gamma -stimulated PLC activity with an IC50 of ~50 µM with 88% inhibition of activity occurring at 100 µM (Fig. 5A). B-N20K was cross-linked to wild type beta 1gamma 2 and immunoblotted with beta  subunit antibodies to confirm that this peptide behaved like unmodified N20K in the cross-linking reaction (Fig. 5B). A shift to higher molecular weight was observed as has been seen for unmodified N20K. One major cross-linked species was observed, and one very minor species appeared with longer reaction times, confirming that the procedure could be used to monitor specific reaction of the peptide with the beta  subunit at a single site.



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Fig. 5.   Effects of biotin modification on peptide-binding properties. A, PLC beta 2 (2 ng) activity was measured in the presence () or absence (open circle ) of beta 1gamma 2 (100 nM). beta gamma subunits and biotin-labeled peptide (concentrations as indicated) were preincubated together at 1.5 times the final concentrations in the presence of 0.15% octyl glucoside. The beta gamma /peptide preincubation mixture was then diluted 1.5-fold into a PLC assay such that the final concentration of beta gamma subunits was 100 nM, the peptide concentrations as indicated, and octyl glucoside was 0.1%. CaCl2 was 2.8 mM in the presence of 3 mM EGTA at pH 7.2, and reactions were for 5 min at 30 °C. Each point represents duplicate determinations. B, cross-linking of biotin-labeled peptide to wild type beta 1gamma 2 dimer. 50 µM biotin-N20K was reacted with 200 µM cross-linker and beta gamma as in Fig. 2, and the beta  subunit was detected by Western blotting (cross-linking protocol 2). The 38-kDa molecular mass marker and the bands representing beta 1 subunit cross-linked to peptide are indicated with arrows. C, biotin-labeled peptide and SMCC were reacted and quenched as in B. Wild type beta 1gamma 2 subunits (30 nM) were preincubated with the indicated concentrations of PLC beta 2. The cross-linking reactions were initiated by addition of the beta 1gamma 2/PLC beta 2 mixtures as in cross-linking protocol 2 for 1.5 min, quenched by addition of SDS sample buffer, and resolved on SDS-PAGE (12% gel). Proteins were then transferred overnight onto PVDF membrane and detected with HRP-NeutrAvidin. The 38-kDa molecular mass marker and the bands representing beta 1 subunit cross-linked to biotin-labeled peptide are indicated with arrows.

We went on to determine whether we could detect the cross-linked B-N20K cross-linked to beta  using HRP-NeutrAvidin. This method detected only one cross-linked species at a molecular weight corresponding to the cross-linked species detected with beta  subunit-specific antibody (Fig. 5C, indicated by the arrow on the right). Consistent with our previous report (10), PLC beta 2 inhibits cross-linking of B-N20K peptide to beta 1, further demonstrating that the biotin modification does not affect the binding properties of the peptide and that the site of peptide cross-linking represents a binding site for PLC beta 2 on beta gamma subunits.

Panel of Biotin-labeled Peptide Cross-linked to Wild Type and Cysteine Mutant beta 1gamma 2 Dimers-- Fig. 6A displays the results of a comprehensive cross-linking screen of B-N20K to wild type beta 1gamma 2 and all 14 cysteine mutants. Only the cysteine 25 alteration eliminated peptide cross-linking, whereas the remaining 13 cysteine mutations did not significantly affect peptide cross-linking to beta 1, indicating that the major peptide cross-linking site is cysteine 25 of beta 1. Whereas the other mutations may have had a minor effect on cross-linking, it is difficult to make conclusions as to their significance with this method since it is not strictly quantitative. Clearly, however, in multiply repeated experiments the only mutation that consistently resulted in a clearly significant reduction in cross-linking of the peptide was Cys25. As discussed, Cys103, Cys114, Cys121, Cys148, and Cys271 show double bands with the upper band corresponding to Sf9 beta  that copurifies with the mutant beta  subunit (Fig. 3). We purified Sf9 beta  (see under "Experimental Procedures") to determine whether the peptide cross-links to this beta  subunit. We were unable to detect cross-linking of B-N20K to Sf9 beta  subunit (Fig. 6B). This indicates that it is not Sf9 beta  contamination of the preparation that is responsible for the cross-linking observed with any of the cysteine mutants.



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Fig. 6.   Panel of biotin-labeled peptide cross-linked to wild type and cysteine mutant beta 1gamma 2 dimers. A, cross-linking to cysteine mutants. B, cross-linking to Sf9 beta gamma . Cross-linking reactions and membrane blotting are as in Fig. 5C. The 38-kDa molecular mass marker is indicated with an arrow. The numbers indicate the positions of the mutated cysteines in the beta 1 sequence.

Cross-linking of Peptide to Trypsin-digested Wild Type and C Mutant beta 1gamma 2 Dimers-- To confirm further that peptide cross-linking to the Cys25 mutant is eliminated, we performed trypsin cleavage to isolate the amino-terminal fragment of beta 1 where cysteine 25 is located and assayed for a shift in the molecular weight of this fragment in the presence or absence of N20K (not biotinylated) and cross-linker. In the native state only one of the 32 potential trypsin cleavage sites in beta 1 (arginine at position 129) is accessible to trypsin. Thus trypsin cleavage of the native beta gamma complex produces two fragments of 14- and 24-kDa (amino and carboxyl termini, respectively) (16). Fig. 7 shows immunoblots from a cross-linking experiment of trypsin-digested wild type and cysteine mutant beta 1gamma 2 complexes in the presence and absence of N20K, detected with an amino-terminal specific anti-beta 1 antibody. The cross-linking reaction resulted in appearance of an immunoreactive species at ~17 kDa (top arrow), only in the presence of peptide, for wild type beta 1gamma 2, Cys103, and Cys114. The apparent molecular weight of the cross-linked band corresponds to what would be expected if one peptide of ~2.5 kDa is covalently attached to the amino-terminal fragment of 14 kDa. No cross-linking to the amino-terminal fragment was observed for Cys25 confirming that Cys25 is a site of N20K cross-linking.



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Fig. 7.   Cross-linking of peptide to trypsin-digested wild type and cysteine mutant beta 1gamma 2 dimers. Wild type or C mutant beta 1gamma 2 were digested with trypsin prior to incubation with N20K peptide (pep) and cross-linker as described under "Experimental Procedures." After transfer to nitrocellulose the proteins were immunoblotted with Gbeta 1 amino-terminal (N-term) specific antibodies. Molecular mass markers are in kDa. The Gbeta 1 amino-terminal fragment (14 kDa) and peptide cross-linked fragment bands are indicated by arrows. wt, wild type beta 1gamma 2; numbers, positions of the mutated cysteines in the beta 1 amino acid sequence.



    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Studies to determine effector-binding sites on beta gamma suggest that effectors bind to various regions along the beta gamma surface and that each effector will have its own characteristic set of contact points or footprints along the sides of the beta gamma complex. These contact points may serve to define the specificity of interactions between beta gamma and its effectors. All of the mapping studies to define these footprints are based on measurement of beta gamma -mediated stimulation of effector activity to monitor the effects of site-directed mutation within the beta  subunit at sites predicted to be important for effector binding (5-7). Here, we take an alternative approach that does not require prediction of the binding site but rather directly assesses the location of a specific cross-linking site. Although we used site-directed mutagenesis, the mutations in the beta 1 subunit were not intended and did not disrupt the function of the beta gamma complex. Our mutations were used only as a tool to monitor direct protein-protein interactions while retaining the function of the protein. That the Cys25 beta gamma mutant stimulated PLC beta 2 activity and supported PTX-catalyzed alpha i subunit ADP-ribosylation similarly to wild type beta gamma indicates that the complex is likely to be properly folded and that the PLC interaction site is intact.

This study clearly defines the Cys25 as a site of peptide cross-linking. Cysteine 25 is located in the beta gamma amino-terminal coiled-coil region (Fig. 8). The cross-linker SMCC can link the sulfhydryl groups on cysteine residues to epsilon -amino groups on lysine that are separated by a maximum of 11.6 Å (Pierce (17)) (for perspective, the distance between Cys25 to Leu30 of 15 Å is shown in Fig. 8). This defines a surface area on the beta gamma complex with cysteine 25 situated in the center of 450 A2. The beta  subunit has approximate dimensions of 40 Å in diameter by 30 Å high. Given these dimensions, if the beta  subunit is modeled as a cylinder with a smooth surface, it would have a surface area of 6248 Å2. Thus we can restrict the area that can be reached by this cross-linker to less than 10% of the beta  subunit surface that includes part of the amino-terminal coiled coil as well as the sides of propeller blade 5 and possibly blade 4. Our previously published work (10) has shown that the region of the peptide involved in binding to the beta gamma subunits is within the carboxyl-terminal 10 amino acids (ELKAYDLLSK) of the peptide where the lysines that are involved in cross-linking are located. Thus the peptide-binding site must be within or very near to the area that can be reached by the cross-linker.



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Fig. 8.   Location of cysteine 25. This representation is a "top" view of the structure of beta 1gamma 2 generated from coordinates deposited at the Brookhaven protein data bank (4) using Protein Explorer (Eric Martz, University of Massachusetts). The beta  subunit is displayed as a ribbon, and gamma 2 is in yellow. The blades in the beta -propeller are numbered according the nomenclature of Sprang and co-workers (4). beta Cys25 and Leu40 are shown in a space-filling representation, and the distance between the Cys25 sulfur atom and an Leu30 methyl carbon is indicated. The amino-terminal helix of beta  involved in a coiled-coil interaction with gamma  is in red.

Cross-linking of the peptide to cysteine 25 is not consistent with its binding to three previously identified PLC-binding sites, blades 2, 6, and 7. The closest of these three blades to Cys25 is blade 6. The most proximate amino acid to Cys25 on propeller blade 6 is 25 Å away. Given a lysine side chain length of 5 Å and the 11.6-Å cross-linker length, the peptide could not be binding to this region and cross-linking to cysteine 25. The closest amino acid on the surface of another putative PLC-binding region, blade 2, is 40 Å away if measured in a direct line from cysteine 25 through the protein. The distance along the surface of the protein is of course even greater. Thus the peptide cannot be binding to blade 2 and cross-link to cysteine 25. A similar argument can be made for blade 7, which is 50 Å away from Cys25. Given the length of the cross-linker the most reasonable sites for binding are the amino-terminal coiled-coil region and the sides of blade 5 or 4. We do not conclude that the results from our study and the results from site-directed mutagenesis are mutually exclusive. Given the size of PLC beta 2, it is possible that the enzyme can interact with multiple regions of the beta  subunit. Our study simply identifies an interaction site that was not implicated in other studies of mammalian beta  subunits.

Supporting these ideas is the observation that none of the other cysteine mutations significantly affect cross-linking. In the cross-linking study by Garcia-Higuera et al. (11) that utilized this same approach to identify alpha  subunit interaction sites, Cys204 and Cys271 were both found to be responsible for cross-linking of alpha o to beta 1 by bismaleimidohexane (16-Å cross-linker). The mutation at cysteine 25 had no affect on alpha  subunit cross-linking but did prevent cross-linking to gamma 2 in their study. Thus two highly accessible cysteine residues are present at the alpha  subunit interaction interface, but apparently they are not the major site of cross-linking of the N20K peptide. In particular Cys271 is in the loop region connecting blades 5 and 6. If the peptide were binding to blade 6 one might expect this cysteine to be a cross-linking site. This also suggests that if the peptide were binding in the alpha  subunit interaction region it would be detected. Within or near blade 2 of the beta -propeller are cysteines 114, 121, 148, 149, and 166, none of which appear to be critical for cross-linking of the peptide. However, these amino acids may not be accessible to the cross-linker since they are mostly buried and not clearly accessible to solvent. The critical data supporting a binding site other than blades 2 and 6 is that a cross-linking site at Cys25 has been identified that is not within range of these regions. The lack of a major effect of mutation of cysteine residues that are within range of these regions supports this result and speaks to the specificity of the cross-linking of the peptide.

This general location is in contrast with what has been found in some mutagenic studies but correlates well with an effector interaction domain identified in a screen for dominant negative yeast Gbeta mutants (9). Recent studies show that mutation of some of the residues in the amino terminus of yeast beta  subunits responsible for the dominant negative phenotype eliminates binding to Ste20p, the yeast homologue of mammalian PAK (18). Other mutants in the coiled-coil region of yeast beta  subunits (Leu65 and Leu49) are unable bind to the scaffolding protein Ste5p (19). These data strongly suggest that the amino-terminal coiled-coil region is important for effector binding in yeast. Evidence for involvement of this region in interactions of mammalian beta  subunits with effectors is lacking. One study (20) of this region with a series of single point mutations found that some of the mutations had dramatic effects on assembly with gamma  subunits, whereas those mutants that did assemble had little effect on the ability of beta gamma subunits to activate JNK in a COS cell transfection assay. A possible explanation for this is that mammalian effectors may have contacts with other regions of the beta  subunit, and as such single point mutations in this region are not sufficient to disrupt effector interactions in overexpression assays. Our data support the idea that this region is important for binding of effectors to mammalian beta  subunits and that this interaction is important for regulation since binding of a peptide to this region blocks the ability of beta gamma to activate effectors.

The amino-terminal coiled-coil region of the beta  subunit is far from the alpha -interacting region located at the top of the beta  subunit complex making contact with blades 2 and 7 of the propeller. This general location for peptide cross-linking is consistent with our previous finding that alpha  subunit does not block cross-linking of peptide to beta gamma (10), and our data show that alpha (GDP) subunits do not completely prevent PLC from binding to beta  and thereby preventing cross-linking of peptide to beta . This latter finding suggests that perhaps PLC could bind near the amino-terminal region of beta  even in the presence of the alpha  subunit. This idea is supported by the results of another group (21) who propose alpha (GDP) can bind to beta gamma ·PLC beta 2 complex to form alpha (GDP)·beta gamma ·PLC beta 2. Thus, the site we have mapped may represent a region where PLC can remain bound to beta gamma even in the presence of alpha . We can envision a model in which effector and G protein heterotrimer are precomplexed and poised for immediate activation. The top surface of the beta gamma complex may be critical for effector activation and may become available to the effector upon activated receptor-induced activation of alpha  subunits without a requirement for a potentially rate-limiting dissociation of alpha  subunits or association of PLC beta 2. Deactivation could involve occlusion of the effector-binding site on beta  by alpha (GDP) subunits with the beta gamma ·PLCbeta 2 complex without dissociation of PLC beta 2 from beta gamma . Identification of a binding site for PLC that does not overlap with alpha  subunits supports this hypothesis and provides a starting point for understanding molecular interactions that could allow PLC to remain associated with beta gamma subunits in the presence of bound alpha  subunits.


    FOOTNOTES

* This work was supported by Grant GM 53536 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: Dept. of Pharmacology and Physiology, University of Rochester School of Medicine and Dentistry, 601 Elmwood Ave., Rochester, NY 14642. Tel.: 716-275-0892; Fax: 716-273-2652; E-mail: Alan_Smrcka@URMC.rochester.edu.

Published, JBC Papers in Press, January 5, 2001, DOI 10.1074/jbc.M006073200


    ABBREVIATIONS

The abbreviations used are: G protein, GTP-binding protein; PLC, phospholipase C; SMCC, succinimidyl 4-[N-maleimidomethyl]-cyclohexane-1-carboxylate; PVDF, polyvinylidene difluoride; PAGE, polyacrylamide gel electrophoresis; HRP, horseradish peroxidase.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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