©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification of Determinants in the -Subunit of G Required for Phospholipase C Activation (*)

(Received for publication, October 19, 1995; and in revised form, December 20, 1995)

Gita Venkatakrishnan John H. Exton (§)

From the Howard Hughes Medical Institute and the Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0295

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

A series of chimeras between a constitutively active mutant of the alpha-subunit of G(q) and the alpha-subunit of G(s) was constructed to identify the domains in alpha(q) specifically involved in interaction with its effector phosphoinositide phospholipase C (PLC). Transient expression of the chimeric proteins and measurement of the production of inositol phosphates and cAMP in HEK-293 cells revealed that the Ile-Lys sequence of alpha(q) contained the PLC interaction sites, whereas the residues for activation of adenylyl cyclase were in the Ile-Leu sequence of alpha(s). Alanine scanning mutagenesis of the Ile-Lys region of alpha(q) further identified two clusters of amino acids (Asp,Asn,Glu and Arg,Thr) that were specifically required for interaction with PLC.

Comparison of the sequences of alpha(q), alpha(s), and alpha(t) showed that the PLC-interacting residues identified in alpha(q) are different from the corresponding residues in alpha(s) and alpha(t) that are involved in effector activation. Alignment of the sequences of alpha(q) and alpha(t), based on the crystal structure of alpha(t) (Noel, J. P., Hamm, H. E., and Sigler, P. D.(1993) Nature 366, 654-663), indicated that the PLC-activating residues of alpha(q) are located in alpha-helix 3 and its linker to beta-sheet 4, which are adjacent to a switch region whose conformation changes with activation. It is proposed that the selectivity of alpha(q) for PLC involves relatively few amino acids, but that the effector may interact with other nonselective sequences in the alpha-subunit.


INTRODUCTION

Heterotrimeric GTP-binding proteins (G-proteins) transduce signals from certain cell surface receptors to various intracellular effectors (1, 2, 3, 4) . Upon agonist binding, the receptors promote the exchange of GTP for GDP on the G-protein alpha-subunit. As a result, the subunit undergoes conformational changes which promote the dissociation of the beta complex. The GTP-liganded alpha-subunit and the free beta complex can then modulate the activity of various intracellular enzymes and ion channels(1, 2, 3, 4) .

Biochemical and mutational analyses of G-protein alpha-subunits have made it possible to assign specific functions to different domains in the polypeptide. Regions involved in GTP binding and hydrolysis, receptor and beta recognition, and guanine nucleotide-induced conformational changes have been identified by expression of mutant and chimeric proteins(5) . By employing chimeric proteins in which regions of alpha(s) were replaced with cognate regions of alpha(i), Osawa et al.(6) and Berlot and Bourne (7) revealed that 121 amino acids between Gln and Arg of alpha(s) contained the adenylyl cyclase activating region. Using the complementary approaches of homolog and alanine scanning mutagenesis, Berlot and Bourne (7) further identified four noncontiguous stretches of amino acids within this region that were critical for activation of adenylyl cyclase. Studies by Hamm and associates (8) using synthetic peptides also demonstrated that a peptide corresponding to alpha(t) residues Glu to Glu stimulated cGMP phosphodiesterase directly. Furthermore, site-directed mutagenesis of Trp of alpha(t) abolishes the interaction with the -subunit of the phosphodiesterase(9) . However, until very recently, there were no reports of the domains of alpha(q) involved in the interactions with PLC. (^1)Then, Arkinstall et al.(10) mapped the regions of alpha(q) that interact with recombinant PLCbeta1 using multiple overlapping synthetic peptides. With this approach, they identified peptides corresponding to two distinct regions of alpha(q) (Ser-Gln and Ala-Asp) that inhibited G(q)-mediated PLC activation. Based on the crystal structure of alpha(11) and findings with chimeras between human and Xenopus alpha(s)(12) , it has been proposed that the helical domain of these alpha-subunits encodes yet another effector-interacting domain.

In the present study, we attempted to define more precisely the domain and amino acid residues in alpha(q) that are responsible for regulating PLC activity. Taking advantage of the similarity in the primary structures of alpha(s) and alpha(q) (43% identical) and the completely different second messenger pathways that they regulate, we constructed a series of chimeras between these alpha-subunits and transiently expressed them in HEK-293 cells. The results indicated that the sequence Ile-Lys of alpha(q) encoded the PLC recognition site. Alanine scanning mutagenesis further identified two clusters of amino acids (Asp,Asn,Glu) and (Arg,Thr) as being specifically involved in PLC interaction.


MATERIALS AND METHODS

Construction of Chimeras

Chimeras were constructed from cDNAs for the entire translated sequence of alpha(q), PCR amplified from CHO cell RNA (gift from Dr. Gary Johnson, National Jewish Center, Denver, CO), and rat liver alpha(s) cDNA (gift from Dr. Randall Reed, Johns Hopkins University, Baltimore, MD). Full-length alpha(s) (residues 1-394) was amplified using as primers A957 sense oligonucleotide 5`-GG GGT ACC ATG GGC TGC CTC GGC AAC and B689 antisense oligonucleotide 3`-GCT CTA GAT TAG AGC AGC TCG TAT TGG and rat liver alpha(s) cDNA as template. To generate chimera 1, the alpha(q) fragment (residues 1-328) was generated by PCR using alpha(q) 5` (sense) oligonucleotide primer A1670, 5`-GG GGT ACC ATG ACT CTG GAG TCC A and alpha(q) 3` (antisense) primer C640, 3`-GAA GTG CGA GTA GAT AAT. The alpha(s) (residues 364-394) fragment was generated with primers B115, 5`-ACC TGC GCC GTG GAC ACT, and B689. The product of the third PCR reaction using equal amounts of the two fragments as templates and primers A1670 and B689 resulted in an alpha(q) (residues 1-328)/alpha(s) (residues 364-394) chimera. Chimeras 2, 3, and 4 were generated by taking advantage of the conserved internal restriction sites. The fragments corresponding to residues 342-394, 295-394, and 235-394 obtained by digesting alpha(s) with the restriction enzymes XhoI, BglII, and BamHI were ligated with N-terminal fragments of alpha(q) residues 1-309, 1-276, and 1-216 obtained by digestion with the same enzymes to produce chimeras 2, 3, and 4. Ligation of the KpnI/BamHI fragment of alpha(q) (residues 1-216), the BamHI/BglII fragment of alpha(s) (residues 235-294), and the BglII/HindIII fragment of alpha(q) (residues 277-359) resulted in chimera 5.

Site-directed Mutagenesis

The alpha(q) and alpha(s) cDNAs were subcloned at the KpnI/HindIII and KpnI/XbaI sites of M13mp18. Mutations were introduced by oligonucleotide-directed in vitro mutagenesis (13) using a Mutagene In Vitro Mutagenesis Kit Version 2 (Bio-Rad). All mutations and chimeras were verified by restriction enzyme digestion and DNA sequencing using Sequenase Version 2 kit (U. S. Biochemical Corp.). All constitutively active full-length chimeras and mutants were subcloned either at the HindIII, KpnI/HindIII, or KpnI/XbaI sites of the transient expression vector pCMV-4 (gift from Dr. Lee Limbird, Vanderbilt University, Nashville, TN).

Cell Culture and Transient Transfection

HEK-293 (human embryonic kidney) cells (ATCC, CRL 1537) were maintained in alpha-minimum essential medium (Eagle's) containing 10% fetal bovine serum, 2 mM glutamine, and 100 µg/ml gentamycin. Cells (2.5 to 3.0 times 10^6) per 60-mm dish were transiently transfected with 2.5 µg per dish of plasmid DNA and 10 µg of Transfectam reagent (Promega) in serum-free medium. Two h after incubation, cells were washed and maintained in complete medium.

Inositol Phosphate (IP) Formation Assay

Twenty-four h after transfection, cells were washed with inositol-free Dulbecco's modified Eagle's Medium (DMEM) and incubated for 20-22 h in inositol-free medium containing 10% dialyzed fetal bovine serum and 2 µCi/ml myo-[^3H] inositol (DuPont NEN). Cells were later washed with assay medium (25 mM HEPES-buffered DMEM without bicarbonate) and incubated at 37 °C for 1 h in medium containing 5 mM LiCl. The assay was terminated by the addition of 1.5 ml of 20 mM formic acid, and the plates were chilled on ice for 30 min before the addition of 0.2 ml of 0.7 M NH(4)OH to each dish. The IP formation was measured as described by Wedegaertner et al.(14) , but with slight modifications. The cells were scraped from the plates and spun in an Eppendorf microcentrifuge for 10 min. The supernatant was loaded on AGI-8X Dowex (Bio-Rad) columns which were then washed with 4 ml of H(2)O. This fraction together with the flow-through contained free [^3H]inositol. The columns were then washed with 4 ml of 40 mM ammonium formate, 0.1 M formic acid, and the IP fraction was eluted with 5 ml of 1 M ammonium formate, 0.1 M formic acid. Aliquots (0.5 ml) of the fractions were counted in 15 ml of Ready Safe (Beckman) scintillation fluid. Combined radioactivity in the two fractions correlated with the number of cells in each dish. Data are presented as the quotient of [^3H]IP/[^3H]inositol + [^3H]IP.

cAMP Accumulation Assay

Twenty four h after transfection, cells were washed with DMEM and labeled with [^3H]adenine (Amersham Life Sciences) at a concentration of 5 µCi/ml for 24 h. Cells were then washed once with HEPES-buffered DMEM and incubated at 37 °C for 30 min in the same medium containing 1 mM 1-methyl-3-isobutylxanthine. Reactions were terminated by the addition of ice-cold trichloroacetic acid (5%) plus 1 mM ATP and 1 mM cAMP. Nucleotides were separated on ion exchange columns as described by Solomon et al.(15) . cAMP accumulation was expressed as: [^3H]cAMP/([^3H]cAMP + [^3H]ATP) times 10^3.

Membrane Preparation

Transfected cells were released from the plates with Hanks' balanced salt solution containing 1 mM EDTA and washed twice with Dulbecco's phosphate-buffered saline. Membranes were prepared by a modification of the method of Wu et al.(16) . Cells were suspended in 1.0 ml of lysis buffer (50 mM HEPES, pH 7.5, 5 mM MgCl(2), 0.2 mM EGTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 2.5 µg/ml aprotinin, 0.01% soybean trypsin inhibitor, and 10% glycerol). Cells were kept on ice and allowed to swell for 30 min and then homogenized with a Dounce homogenizer. Nuclei and cell debris were removed by microcentrifugation at 1000 times g for 5 min at 4 °C. The supernatant was centrifuged for 45 min at 100,000 times g in a TL-100 Ultracentrifuge (Beckman Instruments). The pellets (membranes) were resuspended in 50-75 µl of homogenizing buffer minus glycerol. Total membrane protein was estimated using bicinchoninic acid (Pierce).

Western Blotting

Samples (20 µg of protein/lane) were resolved on 14% SDS-polyacrylamide gels (Novex Experimental Corp.), transblotted to Immobilon-P membrane (Millipore), and probed with primary antibody E973 raised against a peptide corresponding to alpha(q) residues 115-133. The antibody-antigen complex was detected by I-labeled goat anti-rabbit IgG (DuPont NEN). The immunoblots were exposed to Kodak XAR-5 film with Cronex lightning plus intensifying screens (DuPont) at -80 °C.

In Vitro Transcription and Translation

Wild-type and mutant cDNAs were subcloned between the KpnI/HindIII sites of the transcription vector pGEM3Zf(+) (Promega) downstream of the T(7) polymerase promoter. Supercoiled plasmid DNA from lysates of Escherichia coli JM109 cells was prepared using the RPM plasmid preparation kit (BIO 101, Inc.). The transcription and translation reactions were carried out exactly as described in the technical bulletin from Promega for the TNT T(7) coupled reticulocyte lysate system using [S]methionine (Amersham Life Science). Translation efficiency was assessed by subjecting 2-5 µl of the product to SDS-PAGE on 14% acrylamide gels. Gels were fixed, dried, and sprayed with EN^3HANCE (DuPont NEN) and exposed to Kodak XAR-5 film at -80 °C.

Trypsin Protection Assay

Trypsin protection assays were performed by a modification of the method of Lee et al.(17) . Proteins were cleaved with tosylphenylalanyl chloromethyl ketone-treated trypsin (Sigma). Translation mixtures (5 µl) were incubated at 30 °C for 30 min in the presence or absence of 20 mM MgCl(2) and 0.125 mM GTPS. Samples were digested with 25 µg/ml trypsin for 30 min at 30 °C in buffer (50 mM Tris, pH 7.6, 10 mM MgCl(2), 1 mM EDTA, 1 mM dithiothreitol). Proteolysis was terminated by the addition of equal volume of twice-concentrated sample loading buffer for SDS-PAGE. The S-labeled proteins were separated by SDS-PAGE on 14% acrylamide gels. Gels were fixed, processed, and autoradiographed as described above.


RESULTS

Analysis of Chimeric Constructs

In an attempt to define the domains in alpha(q) involved in the interaction with PLC, we constructed several recombinant cDNAs that encoded chimeric alpha-subunits. Portions of the C-terminal end of alpha(q) were progressively replaced with analogous regions of alpha(s) (Fig. 1). Chimeras 1, 2, 3, and 4 had 31, 50, 83, and 143 amino acid residues in alpha(q) replaced with residues from alpha(s). Chimera 5 had a stretch of 59 amino acids corresponding to residues Ile to Gln of alpha(s) inserted between amino acids Trp and Asp of alpha(q).


Figure 1: Chimeric constructs. Diagrammatic representation of chimeras between alpha(q) and alpha(s). The regions corresponding to alpha(q) are the open segments, whereas those corresponding to alpha(s) are the dark-striped segments. The numerical scale at the bottom is relative to the alpha(s) sequence. The numbers inside the chimeras indicate the residues at the beginning and end of segments derived from alpha(q); the numbers on the outside refer to residues of alpha(s). The mutations Q209L and R201C in alpha(q) and alpha(s) subunits, respectively, render the alpha-subunits constitutively active.



The alpha(q) used in the construction of the chimeras was mutated (alpha(q)Q209L). This mutation renders the polypeptide constitutively active by inhibiting its intrinsic GTPase(18) . Transfection of HEK-293 cells with vector containing no insert, alpha(q) wild-type and constitutively active alpha(s) (alpha(s)R201C) had very little effect on IP production (Fig. 2). Expression of alpha(q)Q209L resulted in a very large elevation of IP compared with wild-type alpha(q), as expected. Expression of chimeras 1, 2, and 3 also resulted in elevated levels of IP, comparable to that obtained with alpha(q)Q209L. Chimeras 4 and 5, however, failed to increase IP production above the basal level seen with vector alone. Fig. 3shows the accumulation of cAMP in HEK-293 cells overexpressing alpha(s)R201C, alpha(q)Q209L, and the various alpha(q)/alpha(s) chimeras. The results indicate that, besides alpha(s)R201C, only chimera 4 behaved as a functional alpha(s). Expression of this chimera produced, in fact, a higher level of cAMP than did alpha(s)R201C. These data are consistent with a previous report showing that the amino acids required for activation of adenylyl cyclase are discontinuously spaced in a domain from Gln to Arg of alpha(s)(7) . The only chimera that contained this entire domain was chimera 4 (Fig. 1). The data of Fig. 2and Fig. 3also demonstrate that chimeras 1-4 were all functional. From the results obtained with chimeras 3 and 4, it can be concluded that the sequence Ile-Lys in alpha(q) encodes a domain that is required for interaction of alpha(q) with PLC.


Figure 2: Accumulation of inositol phosphates in HEK-293 cell transfectants. Cells were transfected with 2.5 µg/ml pCMV4 or pCMV4 containing alpha(q), alpha(q)Q209L, alpha(s)R201C, or the chimeric cDNA. Cells were labeled for 24 h with myo-[^3H]inositol (2 µCi/ml), and the IP formation was measured as described under ``Materials and Methods.'' Each value represents the mean ± S.E. of 3 to 6 independent experiments.




Figure 3: Accumulation of cAMP in HEK-293 cell transfectants. Cells were transfected with 2.5 µg/ml pCMV4 or pCMV4 containing alpha(q), alpha(q)Q209L, alpha(s)R201C, or the chimeric cDNA. Cells were labeled for 24 h with [^3H]adenine (5 µCi/ml), and cAMP synthesis was measured 24 h later after incubating with 1-methyl-3-isobutylxanthine for 25 min at 37 °C, as described under ``Materials and Methods.'' Each value represented the mean ± S.E. from 3 separate experiments.



Although the data of Fig. 2and Fig. 3indicated that the full-length forms of alpha(s) and alpha(q) and chimeras 1-4 were functionally expressed in the HEK-293 cells, this was not evident for chimera 5. Fig. 4shows Western blots of membrane preparations from cells transfected with full-length and chimeric forms of alpha(q) using an alpha(q)-specific antiserum. In cells transfected with vector (pCMV4) alone, a low level of a 42-kDa protein was detected which was presumably endogenous alpha(q). Transfection of the cDNAs resulted in varying levels of expression of recombinant protein. The molecular masses of chimeras 1 and 2 were similar to that of alpha(q), as expected (Fig. 1). However, the relative mobility of chimeras 3 and 4 was slower because the alpha(s) segment contains additional amino acids in its sequence (Fig. 1). The level of 42-kDa protein corresponding to chimera 5 that was detected on the immunoblot was low, and there was a band of lower molecular mass, which probably represents a proteolytic fragment (Fig. 4).


Figure 4: Autoradiography of immunoblots showing transient expression of Galpha(q) and chimeric alpha-subunits. HEK-293 cells were transiently transfected with pCMV4 alone or pCMV carrying alpha(q)Q209L or chimeric cDNA. Membrane proteins (20 µg/lane) were resolved on SDS-14% polyacrylamide gels, transferred to Immobilon, incubated with the alpha(q)-specific antiserum E973, and developed as described under ``Materials and Methods.''



It is clear from Fig. 4that the extent of expression of the proteins was very different, but these differences were seen consistently. Since transfection of twice the amount of cDNA did not alter the pattern of IP production seen in Fig. 2(data not shown), it appears that sufficient protein was expressed with chimeras 1-4 to provide the same large stimulation of PLC or adenylyl cyclase as seen with alpha(q)Q209L or alpha(s)R201C.

The ability of an alpha-subunit to activate its effector is dependent on its capability to bind GTP and assume an active conformation. It has been shown for other alpha-subunits, e.g. alpha(s)(19) , alpha(i) and alpha(o)(20) , and alpha(t)(21) , that the conformational change induced by GTP binding results in decreased susceptibility to trypsin degradation. Although the data of Fig. 2and Fig. 3indicated that alpha(q)Q209L and chimeras 1-4 were capable of binding GTP, it was necessary to demonstrate this for chimera 5. Fig. 5shows the effect of GTPS on trypsin digestion of the in vitro translated proteins in a reticulocyte lysate. In vitro translation of alpha(q)Q209L and chimeras 1, 2, and 5 generated a major 42-kDa labeled band. Incubation with 25 µg/ml trypsin resulted in complete or almost complete degradation of the translated products. However, the extent of protection by GTPS was slight, reflecting the very low affinity of alpha(q) for GTP analogs in the absence of beta subunits and receptors(22) . Data are absent for chimeras 3 and 4 since transcription/translation of these was negligible.


Figure 5: Tryptic digestion of in vitro translated Galpha(q)Q209L and chimeric polypeptides. The in vitro translation mixture (5 µl) (see ``Materials and Methods'') was incubated at 30 °C for 30 min in the absence(-) or presence (+) of 125 µM GTPS prior to digestion with 25 µg/ml trypsin at 30 °C for 30 min. The control without trypsin is shown in the first lane of each group. The reaction was terminated by the addition of 2 times SDS-PAGE sample loading buffer. The S-labeled proteins were separated by SDS-PAGE and autoradiographed as described under ``Materials and Methods.''



Alanine Scanning Mutagenesis

Comparisons of the sequence of the putative effector interacting domain in alpha(q) (Ile-Lys) with the corresponding sequences in other alpha-subunits reveals considerable differences ( Fig. 6and see Fig. 10), although there are some highly conserved residues including some (Asn, Lys) that are involved in guanine nucleotide binding (23) . In an effort to define the residues within this domain that are required for PLC interaction, the alpha(q)Q209L and alpha(s)R201C amino acid sequences were aligned (Fig. 6). The figure shows that there are 28 identical residues within this domain, 25 of which are conserved in all alpha-subunits (see Fig. 10and (23) ). Of the 32 nonidentical residues, 28 were mutated to alanine in clusters (Fig. 6). Alanine was chosen because of the evidence that it does not alter the main chain conformation of polypeptides or impose extreme electrostatic effects. All the mutants were constructed in the alpha(q) context with the Q209L mutation and were transiently expressed in HEK-293 cells. Measurements of IP production indicated that 4 of the 14 mutants had significantly reduced ability to stimulate PLC (mutants 4, 7, 8, and 10, Fig. 7). These gave only 7 to 20% stimulation of IP production compared with alpha(q)Q209L. Western blot analysis of the mutant proteins in the cell membranes showed that mutants 7, 8, and 10 were well expressed, but that mutant 4 was poorly expressed (Fig. 8). The ability of GTPS to protect the in vitro transfected mutant proteins against trypsin digestion was also tested (Fig. 9). As noted above for the chimeras (Fig. 3), the extent of protection was not great. The nucleotide reduced the degradation of chimeras 7 and 10, but had no detectable effect on that of chimeras 4 and 8.


Figure 6: Alanine scanning mutagenesis. The sequence at the top is that of alpha(q) residues 215-276. The sequence below it is that of alpha(s), with residues identical with alpha(q) being represented by dashes. The numbered sequences below this represent the 14 separate mutant constructs in which nonidentical residues were mutated to alanine. All mutants were constructed in the alpha(q) context with the alpha(q)Q209L mutation.




Figure 10: Alignment of partial sequences of alpha(q), alpha(s), and alpha(t). Shown are the putative effector interaction sequences (underlined sequences) and the locations of the alpha-helices, beta-sheets, and switch region determined from structural studies of alpha(t)(23, 24) . This figure is modified from Fig. 1e in (24) . The residues identified in the present study and (7, 8, 9, 10) and 12 as being involved in effector activation are underlined beneath each sequence. The double-underlined sequences represent residues identified in the present study. Linker sequences between alpha-helices and beta-sheets have been omitted for clarity.




Figure 7: Effects of mutations in Galpha(q) on inositol phosphate accumulation in HEK-293 cell transfectants. Cells were transfected with 2.5 µg/ml pCMV4 alone or pCMV4 carrying mutant alpha(q) cDNA. Cells were labeled for 24 h with myo-[^3H]inositol (2 µCi/ml), and the IP accumulation was measured as described under ``Materials and Methods.'' Each value represents the mean ± S.E. of three independent experiments.




Figure 8: Autoradiography of the immunoblots showing transient expression of mutant Galpha(q) polypeptides. HEK-293 cells were transiently transfected with pCMV4 alone or pCMV4 carrying mutant alpha(q) cDNA. The other procedures were as described in the legend to Fig. 4.




Figure 9: Tryptic digestion of in vitro translated Galpha(q) mutant polypeptides. The procedures were the same as those described in the legend to Fig. 5.



The data of Fig. 8and Fig. 9indicate that the failure of mutant 4 to stimulate PLC was probably due to the effect of the mutation on expression of the protein and its capacity to bind GTP. The inability of mutant 8 to activate the enzyme may likewise be due to poor GTP binding. For these reasons, the only residues that are clearly implicated by the present study as being involved in the selective interaction of alpha(q) with PLC are Asp, Asn, Glu, Arg, and Thr.


DISCUSSION

The present studies with alpha(q)/alpha(s) chimeras indicated that a stretch of amino acids between Ile and Lys in alpha(q) was required for PLC activation. The data (Fig. 2) were very clear cut. Chimera 3, having the entire C terminus from Asp to Val replaced by the corresponding sequence from alpha(s), was fully active, whereas chimera 4, with the C-terminal sequence from Ile to Val being replaced, was completely inactive. This chimera was still capable of fully activating adenylyl cyclase (Fig. 3), indicating that it was adequately expressed and inserted into the membrane, and assumed a configuration that permitted GTP binding.

The conclusion that the sequence between Ile and Lys was required for alpha(q) activation of PLC was consistent with the data with chimera 5. However, Western blotting (Fig. 4) indicated that this chimera was poorly expressed in the membranes and underwent proteolytic degradation. The trypsin proteolysis data also indicated that the protection by GTPS was minimal (Fig. 5). Thus, the data with chimera 5 are not conclusive.

The alanine mutagenesis studies applied to the Ile-Lys sequence indicated that four mutants had a drastically impaired capacity for activating PLC (Fig. 7). However, one of these was poorly expressed (Fig. 8) and another did not bind GTPS when expressed in a reticulocyte lysate (Fig. 9). Thus, the data only unequivocally support the specific involvement of five residues (Asp, Asn, Glu, Arg, Thr). In assessing these data, it has to be emphasized that the overall purpose of the study was to identify residues specifically involved in PLC activation. Thus, the 25 conserved residues and the 3 other residues that are common to alpha(q) and alpha(s) were not mutated. A priori, these residues cannot be specific determinants, and any loss of function due to their mutation could be due to changes in the overall structure of the alpha-subunit. (^2)

It should be noted that the possibility cannot be rigorously excluded that the specific loss-of-function mutations identified in the present study prevent an activating conformational change rather than interfere with effector interaction. Although alanine substitution is reputed to produce minimal changes in the conformation of polypeptides, structural studies will be required to prove definitively that the mutations did not interfere with the conformational changes resulting from activation.

Arkinstall et al.(10) used multiple overlapping synthetic peptides to block the interaction of alpha(q) in a mixture of homogenates of yeast and Sf9 cells expressing PLCbeta1 and alpha(q) and refined their data to show that the amino acids required for PLC activation were confined to sequences Ser-Gln and Ala-Asp in alpha(q) (underlined in Fig. 10). The first sequence contains two of the residues (Arg, Thr) identified in the present study (doubly underlined in Fig. 10), but the second sequence is contained entirely within the domain of alpha(q) that was replaced by alpha(s) in chimera 3 (Fig. 1). Since this chimera was fully active in stimulating PLC (Fig. 2), there is a discrepancy between the two sets of data. Since the two studies used entirely different methodologies, there may be many technical reasons for the discrepancy. However, it should be noted that the specificity of the peptides used by Arkinstall et al.(10) was not established, i.e. they could have blocked the interactions of other alpha-subunits with their effectors. (^3)Thus, if, in fact, a PLC interaction site is present in the Ala-Asp sequence, it may not be specific.

It is interesting to compare the present data with the similar study by Berlot and Bourne (7) who extended the investigation of Osawa et al.(6) using alpha(s)/alpha(i) chimeras to define the domains in alpha(s) required for interaction with adenylyl cyclase. These studies revealed that the sequence Gln-Arg was sufficient to activate the enzyme. Homolog and alanine mutagenesis (7) further identified the following clusters of residues as specifically required for adenylyl cyclase activation: Gln-Asp, Trp-Ile, and Ser-Arg. The alpha(q) sequence (His-Asn) corresponding to alpha(s) Gln-Asp is not involved in PLC activation ( Fig. 7and Fig. 10and (10) ), and the alpha(q) sequence (Asn-Ile) corresponding to alpha(s) Ser-Arg (Fig. 10) was not identified for PLC activation in the present study and only partly overlaps the Ala-Asp sequence reported by Arkinstall et al.(10) . Thus, the individual residues specifying PLC and adenylyl cyclase activation occupy different positions in the aligned sequences of alpha(q) and alpha(s) (Fig. 10) and hence are in different locations in their three-dimensional structures (see below).

In another study of the domains in alpha(s) required for adenylyl cyclase activation, Antonelli et al.(12) employed human-Xenopus chimeras of alpha(s). This approach indicated an additional requirement for activating residues in a sequence between Gly and Lys, a conclusion reached for alpha(i) on structural grounds by Coleman et al.(11) . Antonelli et al.(12) proposed that previous chimeric studies (6, 7) failed to recognize the need for this sequence because the alpha(i) segment used for the constructs preserves the structures needed for activation of adenylyl cyclase. The possibility that the alpha(s) Gly-Lys sequence can be substituted from alpha(i) would mean that it cannot be exclusive for alpha(s). (^4)

In the study of Rarick et al.(8) who used synthetic peptides to mimic the ability of alpha(t) to activate cGMP phosphodiesterase, activation was observed with a peptide corresponding to the Glu-Glu sequence. Fig. 10shows that this sequence overlaps corresponding sequences in alpha(q) and alpha(s) identified by Arkinstall et al.(10) and Berlot and Bourne (7) as being partly involved in the activation of PLC and adenylyl cyclase, respectively. The failure of Rarick et al.(8) to identify the residue (Trp) recognized by Faurobert et al.(9) could be due to the approach used. Thus, it is possible that a combination of the Glu-Glu peptide with one containing Trp may have provided a greater stimulation than seen with Glu-Glu alone.

For full understanding of the above results, it is necessary to relate the sequences to their location in the three-dimensional structures of the various alpha-subunits. Although the crystal structures of alpha(t) and alpha(i) liganded to GDP, GTPS, or GDP-AlF(4) are known(11, 23, 24, 25) , those of alpha(q) and alpha(s) have not been reported. However, the three-dimensional structure of alpha(q) has been modeled, based on the structure of GTPSalpha(t)(10) . (^5)

The main point of the present study was to identify the residues specifically involved in the interaction of alpha(q) with PLC, and the data suggest that the selectivity is encoded by residues in alpha-helix 3 and the linker between this helix and beta-sheet 4, based on sequence alignment (Fig. 10) and computer modeling(10) .^5 This is different from alpha(t) where the activating residues are confined to alpha-helix 4 and the linker to beta-sheet 6(8, 23) . The situation with adenylyl cyclase is more complex, with the involvement of residues in alpha-helices 2, 3, and 4 and the linkers between alpha-helix 2 and beta-sheet 4 and between alpha-helix 3 and beta-sheet 5(7, 23) . As pointed out by Hamm, Sigler and associates(23, 24) , the residues in the four linkers and three alpha-helices that are involved in effector activation define a contiguous surface on the alpha-subunit.

Whereas the present study and that of Berlot and Bourne (7) were selective in that they compared alpha(q) with alpha(s) and alpha(s) with alpha(i), the other two reports (8, 10) did not test the peptides on other effector systems. Thus, it is possible that some of the domains identified in the latter studies(8, 10) , especially those in alpha-helix 4 and the adjacent linker to beta-sheet 6 may contain sequences that are required, but are not selective, for the activation of given effectors. Because of such a lack of selectivity, it is possible that the sequences could be substituted from one alpha-subunit to another in chimeric studies without apparent loss of effector activation. This explanation does not, of course, exclude the possibility that the domains could also include other sequences that designate selectivity for some effectors, e.g. residues in the linker between alpha-helix 4 and beta-sheet 6 that are specifically required for adenylyl cyclase or cGMP phosphodiesterase. In short, it is proposed that the interaction of alpha-subunits with their effectors could involve both highly selective sequences and nonselective sequences.

Finally, it is of interest to relate the domains involved in effector interaction with those whose conformation changes when the alpha-subunit is converted to the active state by GTPS binding. Lambright et al.(24) , in their crystallographic studies of alpha(t), have demonstrated that the largest conformational changes resulting from activation occur in three domains designated switches I-III. Switch I is located in a linker between the alpha-helical and GTPase components of the alpha-subunit, whereas Switch II includes part of beta-sheet 3, all of alpha-helix 2, and its linkers to beta-sheet 3 and 4 (24) . Switch III includes most of the linker between beta-sheet 4 and alpha-helix 3(24) .

Since all of the critical residues for the conformational switches in alpha(t) are present in the other alpha-subunits(24) , it is anticipated that similar switching mechanisms hold for these. Accordingly, Switches II and III would correspond to sequences Val-Thr and Asp-Arg in alpha(q) ( (24) and Fig. 10). Switch III would thus include or be adjacent to the sequences (Asp-Glu and Arg,Thr) in alpha-helix 3 and the adjacent linker that have been implicated in the activation of PLC by the present study. Thus, it is not difficult to conceive that the conformational changes resulting from activation of alpha(q) would be transmitted to the sites that specifically interact with PLC via Switch III. (^6)Understanding how this leads to activation of the enzyme will require a more precise definition of the residues in PLC that interact with alpha(q) and knowledge of the three-dimensional structure of the mammalian enzyme. (^7)


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Investigator of the Howard Hughes Medical Institute. To whom all correspondence should be addressed.

(^1)
The abbreviations used are: PLC, phosphoinositide phospholipase C; GTPS, guanosine 5`-O-(3-thiotriphosphate); DMEM, Dulbecco's modified Eagle's medium; IP, inositol phosphate; PAGE, polyacrylamide gel electrophoresis.

(^2)
The Ile-Lys sequence in alpha(q) contains one nonconserved alanine (Ala) which was not mutated. Conceivably, this could be required for PLC activation. Also, it could be argued that no definitive conclusions can be reached about Glu, Asp, and Gln, since mutant 4 was not expressed, and about Asn and Asn since, although the in vitro translated protein did not appear to bind GTPS, this may not be true for protein expressed in the cells. If the extreme view is taken that all these residues are needed, the required amino acids would still be contained in a limited sequence (Glu-Thr).

(^3)
It could also be argued that the sequence Ala-Asp is required for alpha(q) interaction with PLC, but that the corresponding sequence in alpha(s) (Ala-Ala) can substitute for it functionally. This could account for chimeras 2 and 3 being active. The alignment of these sequences in alpha(q) and alpha(s) is shown in Fig. 10. However, although some identical residues and conservative substitutions are present, it seems unlikely that the sequences are sufficiently identical to provide overlapping function.

(^4)
The possibility that an additional sequence(s) in the N-terminal domain of alpha(q) is involved in PLC activation must also be considered. This is because all of the chimeras tested in the present study (Fig. 1) contained the entire alpha(q) N terminus up to Trp. Since it is possible that such interacting sequences may not be specific to alpha(q), as argued by Antonelli et al.(12) for alpha(s), they may not be identified by a chimeric approach, but would need mutational analysis.

(^5)
E. Springman, G. Venkatakrishnan, and J. H. Exton, unpublished studies.

(^6)
Calculations by C. Berlot (Yale University) of the fractional accessibilities (26) of the side chains of the five residues identified in the present study as interacting with PLC indicate that they are highly exposed on the surface of the protein.

(^7)
The crystal structure of PLC from Bacillus cereus has recently been reported(27) , but this enzyme is Ca-independent and of much lower molecular mass than the mammalian isoforms. There is no evidence that it is regulated by G-proteins.


ACKNOWLEDGEMENTS

We thank Judy Childs for typing the manuscript and also Dr. Catherine Berlot (Yale University) for helpful comments and also for performing the ``fractional accessibility'' calculations of Footnote 6. The valuable technical assistance of Cassondra Elliott and Annette Ross is also appreciated.

Note Added in Proof-In a recent study, Skiba et al. (28) showed that the most important site for interaction of alpha(i) with the -subunit of cGMP phosphodiesterase involves a sequence encompassing alpha-helix 3 and the alpha(3)/beta(5) loop, which corresponds closely to the PLC interaction sequence identified in alpha(q) in the present study.


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