Identification of a Structural Element in Phospholipase C beta 2 That Interacts with G Protein beta gamma Subunits*

Banumathi Sankaran, James Osterhout, Dianqing Wu, and Alan V. SmrckaDagger

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

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References

To delineate the specific regions of phospholipase C beta 2 (PLC beta 2) involved in binding and activation by G protein beta gamma subunits, we synthesized peptides corresponding to segments of PLC beta 2. Two overlapping peptides corresponding to Asn-564-Lys-583 (N20K) and Glu-574-Lys-593 (E20K) inhibited the activation of PLC beta 2 by beta gamma subunits (IC50 50 and 150 µM, respectively), whereas two control peptides did not. N20K and E20K, but not the control peptides, inhibited beta gamma -dependent ADP-ribosylation of Galpha i1 by pertussis toxin and beta gamma -dependent activation of phosphoinositide 3-kinase. To demonstrate direct binding of the peptides to beta gamma subunits, the peptides were chemically cross-linked to purified beta 1gamma 2. N20K and E20K cross-linked to both beta 1 and gamma 2 subunits, whereas the control peptides did not. Cross-linking to beta  and gamma  was inhibited by incubation with excess PLC beta 2 or PLC beta 3, whereas cross-linking to gamma  but not beta  was inhibited by r-myr-alpha i1. These data together demonstrate specificity of N20K and E20K for G beta gamma binding and inhibition of effector activation by beta gamma subunits. The results suggest that an overlapping region of the two active peptides, Glu-574-Lys-583, mimics a region of PLC beta 2 that is involved in binding to beta gamma subunits. Changing a tyrosine to a glutamine in this overlapping region of the peptides inhibited binding of the peptide to beta gamma subunits. Alignment of these peptides with the three-dimensional structure from PLC delta 1 identifies a putative alpha  helical region on the surface of the catalytic domain of PLC beta 2 that could interact with beta gamma subunits.

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Many transmembrane receptors coupled to heterotrimeric G proteins can initiate the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2)1 to produce inositol trisphosphate (IP3) and diacylglycerol. Multiple experimental paradigms provide convincing evidence that increased PIP2 hydrolysis occurs as a result of enzymatic activation of phosphatidylinositol-specific phospholipase C (PLC) due to direct interactions of PLC with activated G protein alpha  or beta gamma subunits (1-3). G protein alpha  subunits of the Gq class are thought to be responsible for receptor-mediated activation of PLC that is not inhibited by treatment with the toxin from Bordatella pertussis (PTX). beta gamma subunits released during activation of Gi/o proteins are thought to activate PLC in a manner that is inhibited by PTX, since this toxin blocks interaction of the Gi/o heterotrimer with receptors.

PLC enzymes consist of at least nine different isoforms that have been classified into three groups; beta , gamma , and delta  (2, 4). All of these enzymes require Ca2+ for activity. PLC beta  isoforms are the primary enzymes that hydrolyze PIP2 in response to activation by G proteins. Four isozymes of the PLC beta  class have been identified and designated PLCbeta 1, beta 2, beta 3, and beta 4. Each isoform is regulated differently by G protein beta gamma or alpha  subunits. In in vitro enzyme assays and in co-transfection assays there are some conflicting results, but in general PLC beta 1 and beta 4 are regulated primarily by Galpha q, PLC beta 2 is regulated primarily by beta gamma subunits, and PLC beta 3 is regulated by both beta gamma and alpha q subunits.

Based on sequence alignments between PLC isoforms and other proteins, some domain structure has been predicted. The first 100 amino acids are predicted to form a pleckstrin homology domain (5). This domain in PLC delta 1, when expressed in isolation, binds to PIP2 and IP3 and when removed from PLC delta 1, inhibits anchoring to PIP2-containing membranes but does not inhibit catalysis (6-8). The role of the pleckstrin homology domain in the other PLC isoforms is unclear but is likely to be distinct from PLC delta 1, since PLC beta 2 membrane binding is unaffected by IP3 (9). Two highly conserved regions have been identified in all mammalian PLCs and designated X and Y. In PLC beta  and delta  isoforms, these regions are adjacent in the primary sequence, whereas in PLC gamma , the X and Y are separated by intervening src-homology SH2 and SH3 domains. PLC beta  isoforms are unique in that there is an extended (40 kDa) C-terminal domain that extends beyond the Y domain (4).

A three-dimensional crystal structure has been solved for PLC delta 1 that contains the X and Y domains but is missing the N-terminal pleckstrin homology domain (10). The structure shows that the N-terminal region between the pleckstrin homology domain and the X domain has a structural fold that is very similar to the EF-hand domain found in Ca2+-binding proteins, including calmodulin. The entire X domain and two-thirds of the N terminus of the Y domain fold to form a catalytic core similar in structure to the beta  barrel found in triose phosphate isomerase (TIM barrel). The C-terminal one-third of the Y domain forms a domain similar to the C2 domains found in PKC and PLA2 where they function as calcium-dependent phospholipid binding domains. In PLC delta 1, the C2 domain primarily interacts with the N-terminal EF-hand domain, and its function is unclear.

Some progress has been made mapping the regions in the overall sequences of the PLCs that are involved in interaction with G proteins. Removal of the C-terminal third of PLC beta 1 abolishes activation by Galpha q, but Ca2+-dependent activity remains intact (11, 12). Further analysis of this region has served to define important regions for interaction with alpha  subunits (13). Removal of the C-terminal third of PLC beta 2 does not affect its ability to be stimulated by beta gamma subunits (14, 15). In a series of experiments by Kuang et al. (16), segments of the PLC beta 2 X and Y domains when coexpressed with PLC beta 2 in COS-7 cells blocked activation by beta gamma subunits. When expressed as GST fusion proteins, a 116-amino acid polypeptide from this region bound tightly to G protein beta gamma subunits, whereas a 60-amino acid sequence of this same region bound weakly to beta gamma subunits (16).

In this paper we further define the regions of PLC beta 2 that interact with G protein beta gamma subunits. Synthetic peptides corresponding to sections of the 116-amino acid region previously identified (16) were tested for their ability to inhibit activation of PLC beta 2 by beta gamma subunits in a reconstituted, purified system. The peptides were designed based on sequence alignment of PLC beta 2 and PLC delta 1 and referral to the crystal structure of PLC delta 1. This allowed us to identify regions within Gln-526-Val-641 that were on the surface and potentially accessible to beta gamma subunit binding. Based on these studies we propose a model for the structural features of PLC beta 2 involved in beta gamma -PLC interactions.

    EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Materials

Peptides were purchased from Coast Scientific or from Biosynthesis and had a purity of greater than 90% based on high performance liquid chromatography analysis and mass spectrometry. Phosphatidylethanolamine (bovine liver) and phosphatidylinositol (bovine liver) (PI) were from Avanti Polar Lipids. PIP2 was prepared from bovine brain lipids (Sigma) according to the method of Schacht (17) or obtained from Sigma. Pertussis toxin was obtained from List Biologicals. Succinimidyl 4-[N-maleimidomethyl]-cyclohexane-1-carboxylate (SMCC) was from Pierce.

Baculovirus for expression of the PI 3-kinase gamma  110-kDa subunit and 101-kDa subunit were kindly provided by Len Stephens and Phil Hawkins (Inositide Laboratory, The Babraham Institute, Babraham, Cambridge, UK). A cDNA encoding full-length PLC beta 3 was provided by Dr. Gunther Weber (Karolinska Institute, Stockholm, Sweden).

Methods

Plasmid Construction and Cloning of Recombinant Baculoviruses-- Purified, recombinant PLC beta 2, beta 3, and PI 3-kinase proteins were prepared using a baculovirus expression system. Construction of baculovirus for expression of PLC beta 2 has been previously described (9). 6-His-tagged PLC beta 3 was prepared as follows. The cDNA for PLC beta 3 in bluescript SK- was cut with BamHI at base pairs 636 and 2556, and the remaining sequence of PLC beta 3 was religated. This removed multiple NcoI sites from the coding region of PLC beta 3. Oligonucleotides encoding an EcoRI site (at the 5'-end) followed by the coding sequence for an initiator methionine, an alanine, a histidine tag (6 histidines), and an NcoI site were ligated at the 5' end of the PLC beta 3 sequence at the NcoI site at base pair 1 of the cDNA coding sequence and EcoRI of bluescript. A 250-base pair fragment was excised from this construct with EcoRI and BstEII and ligated with the original PLC beta 3 cDNA cut with BstEII and EcoRI after removal of the insert. This fragment was then excised with EcoRI and HindIII and inserted into the EcoRI-HindIII site of Fastbac (Life Technologies, Inc.). Recombinant, clonal baculoviruses were generated according to the protocol described by Life Technologies, Inc.

Sf9 Cell Culture and Purification of Phospholipase C and PI 3-Kinase-- Sf9 cells were grown at 27 °C in IPL-41 medium containing 10% fetal bovine serum, 0.1% pluronic acid, and 50 mg/ml gentamycin. For large scale cultures (1 liter and above), the cells were switched into medium containing 1% fetal bovine serum and 1% lipid concentrate (Life Technologies, Inc.). Baculoviruses directing expression of recombinant 6-His PLC beta 2 and 6-His PLC beta 3 were used to infect 1 liter of Sf9 cells at a density of 1.5 × 106 cells/ml. The proteins were purified according to (9).

For PI 3-kinase, the Sf9 cells were coinfected with viruses coding for expression of 6-His-tagged p110gamma with EE-tagged p101, and the protein was purified on a column containing immobilized anti-EE antibody (18). The protein prepared in this way is predominantly heterodimeric p110gamma and p101.

Expression and Purification of G Protein alpha i1 and beta 1gamma 2 Subunits-- For purification of beta 1gamma 2, baculoviruses encoding beta 1, gamma 2, and 6 × His-tagged alpha i1 were obtained from Alfred Gilman's laboratory. 1 liter of Sf9 cells at 1 × 106 cells/ml was simultaneously infected with the three baculovirus constructs, and the beta gamma subunits were purified according to the published procedures (19), which were modified as in Romoser et al. (9).

Recombinant myristoylated alpha i1 was purified from Escherichia coli coexpressing alpha i1 and N-myristoyl transferase according to published procedures (20). Protein concentrations were determined with an amido black protein assay (21).

Phospholipase C and PI 3-Kinase Assays-- Phospholipase C assays were conducted as described previously (9) and in the figure legends. PI 3-kinase activity was assayed using sonicated micelles containing 600 µM phosphatidylethanolamine and 300 µM bovine liver PI as in Parish et al. (22).

ADP-ribosylation of Galpha i1-- ADP-ribosylation assays were performed as described by Casey et al. (23). Briefly, 0.4 pmol of beta 1gamma 2 was mixed with 20 pmol of alpha i1 followed by the addition of various concentrations of peptide. Reactions were initiated by the addition of pertussis toxin (5 µg/ml final concentration), NAD (2.5 µM final concentration), and [32P]NAD (750,000 cpm/assay) in a total reaction volume of 40 µl. No phospholipids were used in the reaction. Reactions were terminated after 20 min, and proteins were precipitated in 15% trichloroacetic acid and collected on nitrocellulose filters. After extensive washing with 6% trichloroacetic acid, the filters were dried and analyzed by liquid scintillation counting.

Cross-linking and Immunoblot Analysis-- Peptides (30 or 150 µM) were added to beta 1gamma 2 (100 nM) at room temperature in a solution containing 1 mM MgCl2, 80 mM KCl, 50 mM HEPES, pH 7.4, and 0.1% C12E10 (decaethylene glycol dodecyl ether) in a total volume of 100 µl. The cross-linker, SMCC, was dissolved in Me2SO and diluted to 2 mM in 50 mM Pi buffer, pH 5.0, before dilution into the reaction buffer (final concentration 200 µM). The cross-linking reaction was carried out as described in the figure legends and quenched with 10 mM Tris, pH 8.6, and 10 mM 2-mercaptoethanol. When PLC beta 2, PLCbeta 3, or alpha i1 (250 or 500 nM) were added, they were incubated with beta gamma subunits at room temperature for 10 min before the addition of peptides and cross-linker. The cross-linked proteins were resolved on a 12% SDS-acrylamide gel or a 17% SDS-acrylamide gel containing 4 M urea. The proteins were then transferred to nitrocellulose and analyzed by Western blotting with antibodies against beta 1 or gamma 2. gamma  subunit antibody X-263 has been previously described and recognize gamma 2, gamma 3, and gamma 7 (24). The beta  subunit antibody B600 was raised against a synthetic peptide corresponding to the C terminus of beta 1, beta 2, and beta 3 (MAVATGSWDSFLKIWN) and could recognize beta 4 as well. Enhanced chemiluminescence reagents ECL (Amersham Pharmacia Biotech) were used to visualize the proteins.

    RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Peptide Design-- There is significant homology between PLC beta 2 and PLC delta 1 in the 116-amino acid region of PLC beta 2 that was found to bind to beta gamma subunits (16): 33% identity and 47% similarity overall with a 70-amino acid region having 54% identity and 67% similarity (Fig. 1). Based on this high level of homology, we predicted that PLC beta 2 might have a very similar fold as PLC delta l in this region. Using this assumption, we examined the structure of PLC delta 1 to determine which regions of PLC beta 2 would be likely to be on the surface of the protein and accessible to G protein beta gamma subunits. Three peptides were chosen for our initial studies, corresponding to amino acids 564-583 (N20K), 584-609 (A20G), and 575-594 (E20K) of PLC beta 2 shown in (Fig. 1B). Also tested as a control was a peptide composed of the same amino acids as N20K, except the primary sequence was randomized (YVLSKNRSDLFTKAYISSEL).


View larger version (31K):
[in this window]
[in a new window]
 
Fig. 1.   Primary structure and alignments of PLC beta 2 with PLC delta 1. A, linear representation of the overall domain structures of PLC beta 2 and PLC delta 1. The black bar above shows the location of the region defined by Kuang et al. (16) as being involved in activation by beta gamma subunits. B, alignment of the sequence of the beta gamma binding region of PLC beta 2 with the similar region from PLC delta 1. *, identical; |, conservatively substituted amino acids. The peptides used for this study are identified as N20K, A20G, and E20K. PH, pleckstrin homology.

Inhibition of beta gamma Stimulated PLC beta 2 Activity-- The peptides were tested for their ability to inhibit the stimulation of PLC beta 2 activity by beta gamma subunits (Fig. 2). In the absence of peptide, beta gamma subunits stimulated PLC beta 2 approximately 10-fold over basal, Ca2+-dependent activity. The addition of N20K inhibited beta gamma -stimulated activity with an IC50 of 50 µM with 95% inhibition of activity occurring at 200 µM. The adjacent peptide, A20G, and the scrambled peptide had no effect on stimulation of PLC activity by beta gamma subunits at concentrations up to 300 µM. The peptide that overlaps the C terminus of N20K and the N terminus of A20G-E20K (E20K) inhibited beta gamma -stimulated activity with an IC50 of 150 µM. Both peptides inhibited the basal Ca2+-stimulated activity of PLC beta 2, with N20K inhibiting activity by 58 ± 10% and E20K inhibiting activity by 43 ± 10% (n = 4 experiments). Importantly, beta gamma -stimulated activity was inhibited to a greater extent than was basal activity. To quantitate this effect, data were normalized by determining the fold stimulation of activity over basal activity by beta gamma subunits (activity with beta gamma divided by activity without beta gamma ). The percent inhibition of the fold stimulation by beta gamma subunits in the presence of 200 µM peptides was then calculated. N20K inhibited fold stimulation over basal by 67 ± 2%, whereas E20K inhibited fold stimulation by 42 ± 4%. We also tested a peptide corresponding to N20K where Tyr-15 in the region that overlaps with E20K was changed to Gln (N20K(Y15Q)). This peptide stimulated PLC beta 2 activity in the absence of beta gamma subunits (data not shown). The significance of this is unclear, but it did not allow us to measure effects of N20K(Y15Q) peptide on beta gamma -stimulated PLC beta 2 activity. To prove that the inhibition of beta gamma -stimulated PLC activity was attributable, at least in part, to binding of the peptides to beta gamma subunits, several other assays were performed to demonstrate binding of the peptides to beta gamma subunits.


View larger version (26K):
[in this window]
[in a new window]
 
Fig. 2.   Inhibition of beta gamma -dependent activity of PLC beta 2 by N20K and E20K but not A20G or scrambled peptide. A, N20K and scrambled (Scram) peptides. B, E20K and A20G. PLC beta 2 activity was measured in the presence or absence of 100 nM beta 1gamma 2. The beta gamma subunits were added in 1% octylglucoside to yield a final concentration of 0.1%. PLC beta 2 was 0.06 nM. CaCl2 was 2.8 mM in the presence of 3 mM EGTA at pH 8.0 (free Ca2+ ~ 100 nM), and reactions were for 30 min at 30 °C. Each point represents duplicate determinations. This experiment was repeated at least five times. Three different batches of N20K and A20G from different syntheses and two manufacturers were tested. All experiments yielded similar results.

Inhibition of ADP-ribosylation-- G protein alpha  subunits block the ability of beta gamma subunits to activate PLC by sequestering the beta gamma subunits in the heterotrimeric form. This is thought to work because the alpha  subunits sterically hinder interaction between the beta gamma subunits and the PLC. This predicts that peptides mimicking PLC beta 2 binding to beta gamma subunits could block interaction between alpha  and beta gamma subunits. One way to measure this is to measure the beta gamma -dependent enhancement of ADP-ribosylation of alpha  subunits by pertussis toxin. Since beta gamma binding to alpha  subunits is required for ADP-ribosylation of alpha , peptides that block interaction between alpha  and beta gamma subunits will inhibit ADP-ribosylation.

N20K or E20K inhibited incorporation of ADP-ribose into purified recombinant myristoylated alpha i1 in the presence of beta gamma subunits. beta gamma -stimulated ADP-ribosylation was inhibited by close to 100% by 100 µM N20K or E20K with an IC50 of 8 µM (Fig. 3A). Neither A20G nor the scrambled peptide (100 µM each) had an effect on ADP-ribosylation. This indicates that N20K and E20K are binding to the beta gamma subunits at a site that may overlap the binding sites for alpha i1 or that the peptides are binding at a site on beta gamma that prevents binding of PTX. We also tested the N20K(Y15Q) peptide. This peptide was much less potent in inhibiting beta gamma -dependent ADP-ribosylation (Fig. 3B), indicating that this tyrosine 15 in the peptide is important for beta gamma binding.


View larger version (21K):
[in this window]
[in a new window]
 
Fig. 3.   Peptide effects on beta gamma -dependent ADP-ribosylation of Galpha i1. A, comparison of N20K E20K, A20G, and scrambled (Scram) peptides. B, comparison of N20K with N20K(Y15Q). Assays contained 20 pmol of recombinant myristoylated alpha i1 with 0.4 pmol of beta 1gamma 2 subunits. Each point represents duplicate determinations. This experiment was repeated five times for A and three times for B with similar results.

Effects of Peptides on Stimulation of PI 3-Kinase by beta gamma Subunits-- To further confirm that these peptides inhibit various effectors by binding to beta gamma subunits, we tested the effects of these peptides on the stimulation of PI 3-kinase by beta gamma subunits. We utilized a p110gamma /p101 heterodimer purified from sf9 cells as described by Stephens et al. (18). The activity of the p110/p101 heterodimer was increased 5-fold by 150 nM beta gamma subunits. N20K or E20K inhibited the stimulation of this enzyme by 150 nM beta gamma subunits, with an IC50 of 50-100 µM (Fig. 4). A20G and scrambled peptide had no effect. The peptides did not have a significant effect on basal PI 3-kinase activity.


View larger version (23K):
[in this window]
[in a new window]
 
Fig. 4.   Peptide effects on PI 3-kinase p110gamma /p101 heterodimer by beta gamma subunits. Peptides were added at the indicated concentrations to reactions containing 200 ng of purified PI 3-kinase in the presence or absence of 150 nM beta gamma subunits. Each point represents duplicate determinations. This experiment was repeated at least five times with similar results. Scram, scrambled peptides.

Effects of Peptides on Stimulation of PLC beta 3 by beta gamma Subunits-- PLC beta 3 is activated by G protein beta gamma subunits at a similar potency as PLC beta 2 (25). Activation of PLC beta 3 by beta gamma subunits and Ca2+ was measured in the presence of E20K and N20K. Surprisingly, E20K had little effect on the activation of PLC beta 3 by G protein beta gamma subunits, whereas N20K inhibited activity but not to the same extent as for PLC beta 2 (Fig. 5). Basal, Ca2+-dependent activity of PLC beta 3 was not measurable in these assays. We have reported previously that basal activity of PLC beta 3 is much lower that for PLC beta 2 under these assay conditions (25).


View larger version (23K):
[in this window]
[in a new window]
 
Fig. 5.   Effects of peptides on PLC beta 3 activation by beta gamma subunits. Assays were performed exactly as in Fig. 2 with 0.06 nM purified recombinant PLC beta 3. This experiment was repeated four times with similar results. Each point represents duplicate determinations.

Cross-linking of Peptides to beta gamma Subunits-- To directly demonstrate that these peptides bind to beta gamma subunits, we developed a cross-linking assay that uses a heterobifunctional cross-linker to covalently link the peptides to purified beta gamma subunits. The cross-linker (SMCC) has a succinimide ester group that reacts with primary amines and a maleimido group that reacts with SH moieties. Since the peptides have no cysteine residues, the only way to cross-link the peptides to beta  or gamma  is via a primary amine on the peptide and SH groups on either beta  or gamma  subunits. Peptide cross-linking was monitored by immunoblotting for beta  or gamma  subunits after electrophoresis to resolve cross-linked subunits from the unmodified subunits.

The specificity for peptide cross-linking to beta gamma was tested by incubating purified beta gamma subunits with N20K, E20K, N20K(Y15Q), A20G, or scrambled peptides and cross-linker. Only in the presence of N20K or E20K (30 or 150 µM each) was there an increase in the apparent molecular weight of gamma  after incubating with cross-linker (Fig. 6A). Some cross-linking of N20K(Y15Q) to gamma  is observed at 150 µM peptide, consistent with the data demonstrating that N20K(Y15Q) is less potent in inhibiting ADP-ribosylation of alpha i1.


View larger version (60K):
[in this window]
[in a new window]
 
Fig. 6.   Cross-linking of peptides to gamma 2. A, specificity of cross-linking to gamma 2. beta 1gamma 2 was mixed with no peptide (lane 1) or N20K (lane 2), N20K(Y15Q) (lane 3), E20K (lane 4), A20G (lane 5), or scrambled peptide (lane 6) (30 or 150 µM each) at room temperature for 1 min. B, effect of various competitors on cross-linking of E20K and N20K peptides to gamma 2. beta 1gamma 2 was incubated alone (lanes 1 and 2), with 250 (for 30 µM peptide treatments), or with 500 nM (for 150 µM peptide treatments) PLC beta 2 (lane 3), PLC beta 3 (lane 4), boiled PLC beta 2 (lane 5), boiled PLC beta 3 (lane 6), or alpha i1 (lane 7) for 10 min room temperature. 30 or 150 µM peptide was then added to the samples (lanes 2-7). The mixtures were all treated with 200 µM SMCC for 30 min at room temperature. Reactions were stopped by the addition of 10 mM Tris, pH 8.6, and 10 mM 2-mercaptoethanol. Proteins were then resolved by SDS-polyacrylamide gel electrophoresis in 17% acrylamide gels containing 6 M urea. The gels were run at a constant current of 35 mA for 2.5 h. The proteins were transferred to a nitrocellulose membrane and blotted with anti-gamma 2 antibody. The positions of gamma 2 and peptides cross-linked to gamma 2 (gamma 2 xlink) are shown. Each experiment was repeated three times and yielded similar results.

N20K and E20K also cross-linked to the beta  subunit (Fig. 7A). Only in the presence of N20K or E20K and cross-linker does a prominent cross-linked species appear. A number of minor higher molecular weight species are also seen only in the presence of cross-linker and peptide. It is unclear what the higher molecular weight bands correspond to, although all can be visualized with beta  subunit antibodies, and some are visualized with gamma  subunit antibodies. These could represent more than one peptide cross-linked to each beta  subunit or peptide cross-linked to beta  that is cross-linked to gamma . The higher molecular weight band observed in the absence of peptides (Fig. 7B, lane 1) corresponds to beta  cross-linked to gamma , because the upper band could be visualized with a gamma  subunit antibody (data not shown).


View larger version (62K):
[in this window]
[in a new window]
 
Fig. 7.   Cross-linking of peptides to beta 1. A, specificity of peptide cross-linking. beta 1gamma 2 was incubated without any peptide (lane 1) or mixed with 30 µM N20K (lane 2), N20K(Y15Q) (lane 3), E20K (lane 4), A20G (lane 5), or scrambled (lane 6) at room temperature. B, effect of competitors on cross-linking of E20K and N20K peptides to beta 1. beta 1gamma 2 was incubated alone (lanes 1 and 2) or with 250 nM PLC beta 2 (lane 3), PLC beta 3 (lane 4), boiled PLC beta 2 (lane 5), boiled PLC beta 3 (lane 6), or alpha i1 (lane 7) for 10 min at room temperature. 30 µM peptide was then added to the samples (lanes 2-7). Cross-linking reactions were performed as in Fig. 6 except only for 5 min. Proteins were then resolved by SDS-polyacrylamide gel electrophoresis in 12% acrylamide mini-gels. The gels were run at a constant voltage of 150 volts for 1 h. The proteins were transferred to a nitrocellulose membrane and blotted with anti-beta 1 antibody. Positions of uncross-linked beta 1, and molecular weight markers in kDa are shown. Each experiment was repeated three times and yielded similar results.

To further demonstrate the relevance of the cross-linking, the beta gamma subunits were incubated with excess PLC beta 2, PLC beta 3, or alpha i1 before incubation with peptide and cross-linker to determine if they could compete for peptide cross-linking. PLC beta 2, PLC beta 3, and alpha i1 all inhibited cross-linking of N20K and E20K to the gamma subunit, although PLC beta 3 was less effective than PLC beta 2 (Fig. 6B). PLC beta 2 and PLC beta 3 both inhibited cross-linking of the peptides to the beta  subunit, although PLC beta 3 was again consistently less effective than PLC beta 2 (Fig. 7B). Interestingly, GDP-liganded alpha i1 was unable to inhibit cross-linking of peptides to the beta  subunit.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Two synthetic 20-amino acid overlapping peptides that mimic a region of PLC beta 2 bind specifically to G protein beta gamma subunits and prevent interaction with different biochemical partners. An adjacent peptide and scrambled N20K had no effect in any of the assays we have examined. All the peptides are very similar with respect to amino acid chemistry. Although the peptides have some effect on the basal activities of PLC beta 2, this cannot explain the extent of the inhibition of beta gamma stimulation of PLC beta 2 activity. It is clear based on the other assays involving beta gamma subunits that these peptides bind to the beta gamma subunits and that this is responsible, at least in part, for the observed inhibition of beta gamma stimulated PLC beta 2.

The N20K and E20K peptides inhibit the beta gamma -dependent activation of PLC beta 2 with IC50s of 50 and 150 µM, respectively. These IC50s are similar to what has been reported for a 27-amino acid peptide derived from adenylyl cyclase type II (QEHA peptide) for inhibiting a number of beta gamma subunit-regulated effectors (26). N20K and E20K do not contain the beta gamma binding consensus sequence identified in the QEHA peptide. However, in the common 10 amino acids of the N20K and E20K there is a short stretch of seven amino acids where 4 amino acids are identical to a sequence at the C terminus of QEHA and a 5th amino acid is conservatively substituted. When we changed one of these amino acids in N20K, binding to beta gamma subunits was dramatically inhibited (Figs. 3B, 6A, and 7A).

The N20K and E20K peptides are more potent in inhibiting beta gamma -dependent ADP-ribosylation of alpha i1 (IC50 8 µM) than for inhibition of PLC beta 2. This is surprising if we assume that the peptides block ADP-ribosylation of alpha i1 by blocking alpha i1 interactions with beta gamma because the affinity of beta gamma for alpha i1 (Kd ~ 1 nM) is much greater than the affinity for PLC beta 2 (Kd ~ 100 nM). The exact mechanism for how beta gamma subunits stimulate ADP-ribosylation of alpha subunits is unclear but is known to involve a process where beta gamma subunits must cycle catalytically among a stoichiometric excess of alpha  subunits. Since the mechanism is not entirely understood, it is possible that the peptides interfere with this catalytic ADP-ribosylation in a way that is not directly related to the known high affinity of beta gamma subunits for alpha  subunits. One possibility is that the peptides are not blocking alpha  binding to beta gamma but are occupying a binding site on beta gamma required for PTX interaction. This possibility is supported by the cross-linking data that shows that cross-linking of the peptides to the beta  subunit is not blocked by alpha i1-GDP.

Both N20K and E20K must cross-link directly to cysteine residues in either the beta  and gamma  subunits due to the nature of the cross-linker. The site of gamma  cross-linking is clearly cysteine 41, since this is the only cysteine in gamma 2. The site(s) of cross-linking of the peptide to the beta  subunit is unclear, but there are 14 cysteine residues in the beta  subunit where cross-linking could have occurred. Cross-linking of E20K and N20K to both beta  and gamma  was prevented by incubation with PLC beta 2. PLC beta 3 also prevented cross-linking of N20K and E20K but was not as effective as PLC beta 2, suggesting that the binding sites for these two enzymes on beta gamma subunits do not entirely overlap. This is consistent with the observation that the peptides were not as effective or as potent at inhibiting PLC beta 3 activation by beta gamma subunits.

The cross-linking of the peptides to the beta  subunit was not prevented by preincubation of the alpha  subunit with the beta  subunit. This suggests that the peptide binding site on beta gamma does not entirely overlap with the alpha  subunit. Although it is known that alpha -GDP blocks activation of PLC beta  by beta gamma subunits, it is probable that the binding sites for PLC beta  and alpha  subunits overlap but do not match. Thus the binding site represented by the peptide may lie outside the overlap region. This idea is supported by a recent study by Bluml et al. (27) where it was demonstrated that a peptide from phosducin binds to beta gamma subunits. Visualization of the location of this peptide in the crystal structure of beta gamma complexed with phosducin (28) shows that this peptide would bind at a region on the beta  subunit that does not overlap the alpha  subunit binding site, whereas the N-terminal domain of phosducin does overlap the alpha  subunit binding site. That alpha -GDP inhibits cross-linking to the gamma  subunit may result from steric interference with the cross-linking reaction without interfering directly with peptide binding.

The data we have presented allows us to narrow down in the primary sequence of PLC beta 2 a region of 20 amino acids that may be involved in beta gamma subunit binding. The N terminus of the E20K peptide overlaps the C terminus of N20K peptide by 10 amino acids. The C-terminal 10 amino acids of E20K overlaps with the N-terminal 10 amino acids of A20G, which does not inhibit beta gamma activation in any assay. This suggests that the potential region that is binding to beta gamma subunits can be narrowed down to 10 amino acids, Glu-574-Lys-583. We have tested a 10-amino acid peptide corresponding to this region and found no evidence for inhibition. A possible explanation is that this 10-amino acid peptide may be too short to adopt the appropriate secondary structure. This has been seen for other peptides including the peptide from adenylyl cyclase type II (26).

Visualization of the regions homologous to our peptides in the PLC delta 1 structure supports the idea that the 10-amino acid overlap region is the critical region of the peptides involved in binding to beta gamma subunits. The catalytic domain of PLC delta  is composed of parts of the conserved X and Y domains that form a TIM barrel constructed from of a series of beta  sheet alpha  helix repeats. The beta  strands line the inside of the barrel, and side chains from these strands project into the core of the structure to provide the chemistry for substrate binding and catalysis. The alpha  helices are on the outside of this barrel structure and in some cases are exposed to solvent. When the sequence of the N20K is aligned with the PLC delta 1 sequence, the N-terminal 10 amino acids align with a small amount of linker sequence and the Tbeta 6 strand of the barrel (nomenclature of Essen et al. (10)). The sequence in Tbeta 6 is very conserved between the various PLC isoforms with two amino acids, serine 571 and phenylalanine 572 (beta 2 sequence), being conserved in all known PLC isoforms. For this reason we predict that this sequence in PLC beta 2 will occupy a similar position to that observed in the PLC delta  sequence. Since this region is on the inside of the TIM barrel in PLC delta  and is directly involved in substrate binding, it would be unlikely to be accessible to interaction with beta gamma subunits. The region homologous to Glu-574-Lys-583 of PLC beta 2 (overlap region between N20K and E20K) forms an alpha  helix on the surface of PLC delta 1, and because of the significant sequence homology in this region, would likely form the same type of structure on PLC beta 2. The surface exposure of just this 10-amino acid region of N20K further suggests that this overlap region corresponds to a portion of PLC beta 2 that is important for interaction with and regulation by G protein beta gamma subunits. The location of this binding site directly adjacent to structures that contribute amino acids to substrate binding and catalysis suggests a mechanism by which beta gamma subunits could activate PLC beta . If the binding of beta gamma subunits to the helix on the surface caused movement of the adjacent beta  strands, this could position amino acids so they are more favorable for catalysis there by increasing enzymatic activity.

The region we have defined only overlaps the portion of PLC beta 2 (Leu-580-Val-641) originally defined by Kuang et al. (16) as a beta gamma binding domain by 4 amino acids (580-583). Using two-hybrid analysis, Yan and Gautam (29) used the N-terminal 100-amino acid region of the beta  subunit to demonstrate interactions with this 62-amino acid domain. One peptide (A584-G604) overlaps extensively with this domain yet had no effect in any of our assays for beta gamma subunit interactions. This suggests that either only a very short 4-amino acid region is necessary for some beta gamma binding or that other regions within a 62-amino acid region are also involved in PLC beta 2-beta gamma interactions.

    ACKNOWLEDGEMENTS

We would like to thank Monica Schertler for technical assistance. We would also like to thank Dr. Roger Williams for providing the coordinates for PLC delta 1.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant 1-R01-GM53536-01 (to A. V. S.) and GM53162 (to D. W.).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: 716-244-9283; E-mail: Smrcka{at}pharmacol.rochester.edu.

1 The abbreviations used are: PIP2, phosphatidylinositol 4,5-bisphosphate; PI, phosphatidylinositol; PLC phospholipase C; G protein, GTP-binding protein; PTX, pertussis toxin; IP3, inositol 1,4,5-trisphosphate; SMCC, succinimidyl 4-[N-maleimidomethyl]-cyclohexane-1- carboxylate.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

  1. Sternweis, P. C., and Smrcka, A. V. (1992) Trends Biochem. Sci. 17, 502-506[CrossRef][Medline] [Order article via Infotrieve]
  2. Singer, W. D., Brown, H. A., and Sternweis, P. C. (1997) Annu. Rev. Biochem. 66, 475-509[CrossRef][Medline] [Order article via Infotrieve]
  3. Kim, C. G., Park, D., and Rhee, S. G. (1996) J. Biol. Chem. 271, 21187-21192[Abstract/Free Full Text]
  4. Rhee, S. G., and Choi, K. D. (1992) J. Biol. Chem. 267, 12393-12396[Free Full Text]
  5. Parker, P. J., Hemmings, B. A., and Gierschik, P. (1994) Trends Biochem. Sci. 19, 54-55[CrossRef][Medline] [Order article via Infotrieve]
  6. Lemmon, M. A., Ferguson, K. M., O'Brien, R., Sigler, P. B., Schlessinger, J. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 10472-10476[Abstract]
  7. Cifuentes, M. E., Honkanen, L., and Rebecchi, M. J. (1993) J. Biol. Chem. 268, 11586-11593[Abstract/Free Full Text]
  8. Paterson, H. F., Savopoulos, J. W., Perisic, O., Cheung, R., Ellis, M. V., Williams, R. L., Katan, M. (1995) Biochem. J. 312, 661-666[Medline] [Order article via Infotrieve]
  9. Romoser, V., Ball, R., and Smrcka, A. V. (1996) J. Biol. Chem. 271, 25071-25078[Abstract/Free Full Text]
  10. Essen, L. O., Perisic, O., Cheung, R., Katan, M., and Williams, R. L. (1996) Nature 380, 595-602[CrossRef][Medline] [Order article via Infotrieve]
  11. Wu, D., Jiang, H., Katz, A., and Simon, M. I. (1993) J. Biol. Chem. 268, 3704-3709[Abstract/Free Full Text]
  12. Park, D., Jhon, D.-Y., Lee, C.-W., Ryu, S. H., Rhee, S. G. (1993) J. Biol. Chem. 268, 3710-3714[Abstract/Free Full Text]
  13. Paulssen, R. H., Woodson, J., Liu, Z., and Ross, E. M. (1996) J. Biol. Chem. 271, 26622-26629[Abstract/Free Full Text]
  14. Wu, D., Katz, A., and Simon, M. I. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 5297-5301[Abstract]
  15. Lee, S. B., Shin, S. H., Hepler, J. R., Gilman, A. G., Rhee, S. G. (1993) J. Biol. Chem. 268, 25952-25957[Abstract/Free Full Text]
  16. Kuang, Y., Wu, Y., Smrcka, A., Jiang, H., and Wu, D. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 2964-2968[Abstract/Free Full Text]
  17. Schacht, J. (1978) J. Lipid Res. 19, 1063-1067[Abstract]
  18. Stephens, L. R., Erdjument-Bromage, H., Lui, M., Cooke, F., Coadwell, J., Smrcka, A. V., Thelen, M., Cadwallader, K., Tempst, P., Hawkins, P. T. (1997) Cell 89, 105-114[Medline] [Order article via Infotrieve]
  19. Kozasa, T., and Gilman, A. G. (1995) J. Biol. Chem. 270, 1734-1741[Abstract/Free Full Text]
  20. Taussig, R., Iñiguez-Lluhi, J. A., Gilman, A. G. (1993) Science 261, 218-221[Medline] [Order article via Infotrieve]
  21. Schaffner, W., and Weissmann, C. (1973) Anal. Biochem. 56, 502-514[Medline] [Order article via Infotrieve]
  22. Parish, C. A., Smrcka, A. V., and Rando, R. R. (1995) Biochemistry 34, 7722-7727[Medline] [Order article via Infotrieve]
  23. Casey, P. J., Pang, I., and Gilman, A. G. (1991) Methods Enzymol. 195, 315-321[Medline] [Order article via Infotrieve]
  24. Ueda, N., Iñiguez-Lluhi, J. A., Lee, E., Smrcka, A. V., Robishaw, J. D., Gilman, A. G. (1994) J. Biol. Chem. 269, 4388-4395[Abstract/Free Full Text]
  25. Smrcka, A. V., and Sternweis, P. C. (1993) J. Biol. Chem. 268, 9667-9674[Abstract/Free Full Text]
  26. Chen, J., DeVivo, M., Dingus, J., Harry, A., Li, J., Sui, J., Carty, D. J., Blank, J. L., Exton, J. H., Stoffel, R. H., Inglese, J., Lefkowitz, R. J., Logothetis, D. E., Hildebrandt, J. D., Iyengar, R. (1995) Science 268, 1166-1169[Medline] [Order article via Infotrieve]
  27. Bluml, K., Schnepp, W., Schroder, S., Beyermann, M., Macias, M., Oschkinat, H., and Lohse, M. J. (1997) EMBO J. 16, 4908-4915[Abstract/Free Full Text]
  28. Gaudet, R., Bohm, A., and Sigler, P. B. (1996) Cell 87, 577-588[Medline] [Order article via Infotrieve]
  29. Yan, K., and Gautam, N. (1997) J. Biol. Chem. 272, 2056-2059[Abstract/Free Full Text]


Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.