Roles of the Transducin alpha -Subunit alpha 4-Helix/alpha 4-beta 6 Loop in the Receptor and Effector Interactions*

Michael NatochinDagger , Alexey E. Granovsky, Khakim G. Muradov, and Nikolai O. Artemyev§

From the Department of Physiology and Biophysics, University of Iowa College of Medicine, Iowa City, Iowa 52242

    ABSTRACT
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ABSTRACT
INTRODUCTION
REFERENCES

The visual GTP-binding protein, transducin, couples light-activated rhodopsin (R*) with the effector enzyme, cGMP phosphodiesterase in vertebrate photoreceptor cells. The region corresponding to the alpha 4-helix and alpha 4-beta 6 loop of the transducin alpha -subunit (Gtalpha ) has been implicated in interactions with the receptor and the effector. Ala-scanning mutagenesis of the alpha 4-beta 6 region has been carried out to elucidate residues critical for the functions of transducin. The mutational analysis supports the role of the alpha 4-beta 6 loop in the R*-Gtalpha interface and suggests that the Gtalpha residues Arg310 and Asp311 are involved in the interaction with R*. These residues are likely to contribute to the specificity of the R* recognition. Contrary to the evidence previously obtained with synthetic peptides of Gtalpha , our data indicate that none of the alpha 4-beta 6 residues directly or significantly participate in the interaction with and activation of phosphodiesterase. However, Ile299, Phe303, and Leu306 form a network of interactions with the alpha 3-helix of Gtalpha , which is critical for the ability of Gtalpha to undergo an activational conformational change. Thereby, Ile299, Phe303, and Leu306 play only an indirect role in the effector function of Gtalpha .

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
REFERENCES

Upon transduction of the visual signal in vertebrate photoreceptor cells, photoexcited rhodopsin (R*)1 binds the retinal G protein, transducin (Gt), leading to Gt activation. The alpha -subunit of Gt (Gtalpha ) complexed with GTP is then released to stimulate the effector enzyme, cGMP phosphodiesterase (PDE), by reversing the inhibiton imposed by two PDE gamma  subunits (Pgamma ) on the PDE catalytic dimer (Palpha beta ). Activated PDE rapidly hydrolyzes cGMP resulting in closure of cGMP-gated channels in the photoreceptor plasma membrane (1-3).

The two central interactions of Gtalpha with R* and Pgamma during visual excitation have been extensively investigated, and the Gtalpha interaction sites have been localized. Evidence points to the C terminus of Gtalpha as the major R* contact site that is critical for Gtalpha activation (4-9). A second essential site of Gtalpha interaction with R* includes the alpha 4/beta 6 loop (residues 305-315) (6, 10, 11). A peptide, Gtalpha -311-328, competed for the Gt-R* interaction (6). The tryptic cleavage site at Arg310 of Gtalpha was protected upon Gtalpha beta gamma binding to R* (10). Several mutants with Ala substitutions of residues from the alpha 4/beta 6 loop had impaired binding to R* and reduced degrees of activation (11). Interestingly, this R* binding site overlaps with a region of Gtalpha , Gtalpha -293-314, that has been implicated in the transducin-effector interaction (12-16). A synthetic peptide, Gtalpha -293-314, corresponding to the alpha 4-beta 6 region was shown to activate PDE in vitro and to bind to Pgamma (12, 13). Sites of chemical cross-linking of the Pgamma -subunit to Gtalpha were localized to within the alpha 4-beta 6 loop (14, 15). A study using substituted peptides identified five nonconserved effector residues within this region (16). Despite the large body of evidence, the significance of the Gtalpha alpha 4-beta 6 region in the effector interaction remains unclear. An insertion of the Gtalpha -295-314 segment into Gialpha 1 only marginally improved the latter's ability to bind Pgamma (17). This finding suggests that if the alpha 4-beta 6 region is important for the interaction with PDE, then likely the conserved residues within alpha 4-beta 6 are essential for the function of the effector. Alternatively, even small differences in Gtalpha and Gialpha folding may interfere with the ability of Gtalpha -293-314 to assume the proper effector-binding conformation in the context of Gialpha . More importantly, the apparent ability of peptide Gtalpha -293-314 to potently stimulate PDE (12, 16) is inconsistent with the mutational analysis of Gtalpha (18, 19). The latter indicates the requirement of the switch II and alpha 3 regions for effector activation (18, 19).

In light of the importance of the Gtalpha alpha 4-beta 6 region for the Gt-R* interaction and substantial but conflicting evidence on its role for PDE activation, we carried out Ala-scanning mutational analysis of the alpha 4-helix (residues 293-304) and the alpha 4-beta 6 loop of Gtalpha . Our analysis of mutant Gtalpha interactions with R* and PDE has underscored the role of the alpha 4-beta 6 loop for the receptor function but revealed only indirect involvement of the alpha 4-helix in the Gtalpha effector function via requirement of the alpha 4/alpha 3 coupling for the activational conformational change.

    EXPERIMENTAL PROCEDURES

Preparation of ROS Membranes, Gtalpha GDP, Gtbeta gamma , and Pgamma BC-- Bovine ROS membranes were prepared as described previously (20). Urea-washed ROS membranes (uROS) were prepared according to protocol described by Yamanaka et al. (21). Gtbeta gamma was purified according to Kleuss et al. (22). Pgamma labeled with the fluorescent probe, 3-(bromoacetyl)-7-diethyl aminocoumarin (Pgamma BC), was obtained and purified as described previously (23).

Ala-scanning Mutagenesis of the alpha 4-beta 6 Region of Gtalpha -- Substitutions of Gtalpha residues by Ala were introduced into Gtalpha /Gialpha 1 chimeric protein, Gtalpha *, which contains only 16 residues from Gialpha . Gtalpha * was made based on another Gtalpha /Gialpha 1 chimeric protein, Chi8, which is competent to interact with R* and Gtbeta gamma (17, 19). To generate Gtalpha *, all the Gialpha residues in the alpha 3-helix and the alpha 3-beta 5 loop of Chi8, except for Met247 (corresponding to Leu243 of Gtalpha ) were replaced by Gtalpha residues. The following Gtalpha residues were introduced into Chi8: His244, Asn247, His252, Arg253, Tyr254, Ala256, and Thr257. The PCR-directed mutagenesis was carried out essentially as described in Natochin et al. (19).

Single substitutions of Gtalpha residues at positions 293, 294, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 308, 309, 310, 311, 312, 313, and 314 were introduced by PCR-directed mutagenesis. In the PCR reactions, forward mutant primers were paired with a reverse primer carrying a HindIII site and corresponding to a sequence 50 base pairs downstream of the stop codon. The pHis6-Gtalpha * plasmid was used as a template. The PCR products (~200 base pairs) were purified on agarose gel and used for a second round PCR amplification as reverse primers combined with a forward primer containing the unique Gtalpha BamHI site. The 500-base pair PCR products were digested with BamHI and HindIII and ligated into pHis6-Gtalpha * cut with the same enzymes. The sequences of all mutants were verified by automated DNA sequencing at the University of Iowa DNA Core Facility. Gtalpha * and all mutants were expressed and purified as described previously (19).

GTPgamma S Binding Assay-- Gtalpha * or mutants (0.4 µM each) were premixed with Gtbeta gamma (2 µM) in 0.5 ml of 20 mM HEPES buffer (pH 8.0) containing 100 mM NaCl and 8 mM MgSO4. The binding of GTPgamma S to Gtalpha * or mutants was initiated by addition of 5 µM [35S]GTPgamma S (0.2 µCi) and uROS membranes (100 nM rhodopsin). Aliquots (100 µl) were withdrawn at the indicated times, passed through the Whatman cellulose nitrate filters (0.45 µm), washed three times with an ice-cold binding buffer and counted. The kapp values for the binding reactions were calculated by fitting the data to the equation, GTPgamma S bound (%) = 100(1 - e-kt).

Fluorescence Assays-- Fluorescence assays of interaction between Gtalpha * and Pgamma BC were performed on a F-2000 fluorescence spectrophotometer (Hitachi) in 1 ml of 20 mM HEPES buffer (pH 7.6), 100 mM NaCl, 5 mM dithiothreitol, and 4 mM MgCl2 essentially as described in (19, 23). Where indicated, the buffer contained 30 µM AlCl3 and 10 mM NaF. Fluorescence of Pgamma BC was monitored with excitation at 445 nm and emission at 495 nm. Concentration of Pgamma BC was determined using epsilon 445 = 53,000. The AlF4--induced increases in the tryptophan fluorescence of Gtalpha *GDP and its mutants were recorded on an AB2 fluorescence spectrophotometer (Spectronic Instruments) in a stirred 1-ml cuvette with excitation at 280 nm and emission at 340 nm as described previously (19).

PDE Activation Assay-- HoloPDE was extracted from ROS membranes and purified as described earlier (24). PDE (0.2 nM) was reconstituted with 2 µM Gtalpha *GDP or the GDP-bound Gtalpha * mutants and 2 µM Gtbeta gamma in suspensions of uROS membranes containing 10 µM rhodopsin. GTPgamma S (10 µM) was added to the reaction mixture, and PDE activity was measured using [3H]cGMP similarly as described previously (19).

Miscellaneous Procedures-- Protein concentrations were determined by the method of Bradford (25) using IgG as a standard or using calculated extinction coefficients at 280 nm. Rhodopsin concentrations were measured using the difference in absorbance at 500 nm between "dark" and bleached ROS preparations. Fitting of the experimental data was performed with nonlinear least squares criteria using GraphPad Prizm (v.2) software. The results are expressed as the means ± S.E. of triplicate measurements. Examination of the crystal structure of Gtalpha was performed using RasMol (v.2.6) software.

    RESULTS

Expression and Characterization of the Receptor and Effector Competent Chimeric Gtalpha *-- We have previously found that residue Leu243 of Gtalpha is mainly responsible for the low level expression of Gtalpha /Gialpha chimeras containing the Gtalpha -237-270 (alpha 3-beta 5) segment (19). Gtalpha * was obtained based on the Gtalpha /Gialpha chimera, Chi8, which contains the alpha 3-beta 5 region of Gialpha (17). The nonconserved Gialpha residues from the alpha 3-helix and the alpha 3-beta 5 loop of Chi8 were replaced by the corresponding Gtalpha residues (except for Leu243). Two of the introduced Gtalpha residues, His244 and Asn247, are important for the Gtalpha -PDE interaction (19). The resulting chimeric Gtalpha was not only efficiently expressed in Escherichia coli (yields of soluble protein of 3-5 mg/liter culture) but was also fully competent for interaction with Gtbeta gamma and R* and capable of high affinity effector binding. The ability of Gtalpha * to interact with R* in the presence of Gtbeta gamma was evaluated using the GTPgamma S binding assay. The very slow GTPgamma S binding rate to Gtalpha * (kapp = ~0.004 s-1), which is limited by the rate of GDP dissociation (26), was significantly accelerated in the presence of R* and Gtbeta gamma (kapp = ~0.079 s-1) (Fig. 1). A fluorescence read-out assay was utilized to monitor the interaction between Gtalpha and the Pgamma subunit (23). Using this assay, Gtalpha *GDP bound fluorescently labeled Pgamma , Pgamma BC, with a Kd value of 28 nM (Fig. 2A and Table I). When Gtalpha *GDP was activated in the presence of AlF4- it bound to Pgamma BC with an almost 6-fold higher affinity (Kd of 5.1 nM) (Fig. 2B and Table I). Thus, Gtalpha *, which contains only 16 Gialpha residues, represents a well suited tool for mutational analysis to identify residues that are essential for both receptor and effector interactions of transducin.


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Fig. 1.   The time course of GTPgamma S binding to Gtalpha *. The binding of GTPgamma S to Gtalpha * (0.4 µM) was initiated by addition of 5 µM of [35S]GTPgamma S (0.2 µCi) (black-square). When the effect of R* was measured, Gtalpha * was premixed with Gtbeta gamma (2 µM), and the binding was initiated by addition of 5 µM of [35S]GTPgamma S and uROS (100 nM rhodopsin) (black-triangle). Aliquots were withdrawn at the indicated times, and GTPgamma S bound to Gtalpha * was determined using the filter binding assay. The calculated kapp values are 0.004 s-1 (black-square) and 0.079 s-1 (black-triangle).


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Fig. 2.   Binding of Gtalpha * to Pgamma BC. The relative increase in fluorescence (F/Fo) of Pgamma BC (10 nM) (excitation at 445 nm; emission at 495 nm) was determined after addition of increasing concentrations of Gtalpha *GDP in the absence (A) or in the presence (B) of AlF4-.

                              
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Table I
Interaction of Gtalpha * mutants with Pgamma BC and activation by R*

Expression of Gtalpha * Mutants with Ala Substitutions of the alpha 4-beta 6 Residues-- Residues at positions 293, 294, 297, 298, 300, 301, 302, 304, 305, 306, 308, 309, 310, 311, 312, 313, and 314 within the alpha 4-helix and the alpha 4-beta 6 loop of Gtalpha are surface exposed (27) and were substituted with Ala residues. In addition to a modestly solvent-exposed Leu306, two buried residues, Ile299 and Phe303, are involved in coupling the alpha 4-helix with the alpha 3-helix (27). This linkage might be important for stabilization of the receptor and/or effector-competent conformations of Gtalpha . Substitutions of Ile299 and Phe303 were made to test this possibility. Expression of all but three of the Gtalpha * mutants in E. coli have yielded similar amounts of soluble proteins (~3-5 mg/liter of culture). Mutants Y298A, I299A, and F303A had notably reduced expression levels (~0.5-1 mg/liter). The crystal structure of Gtalpha GTPgamma S shows that the Tyr298 side chain makes contact with Tyr286 from the alpha G-alpha 4 loop, whereas Ile299 and Phe303 interact with the alpha 3-helix (27). Perhaps, the reduction in mutant expression reflects lower rates of proper protein folding due to the lack of stabilizing contacts between alpha 4 and the alpha G-alpha 4 loop or the alpha 3-helix.

The ability of Gtalpha mutants to undergo a conformational change upon addition of AlF4- was analyzed by measuring their intrinsic tryptophan fluorescence (18). Mutants Y298A, I299A, and F303A failed to display an increase in tryptophan fluorescence upon addition of AlF4-, whereas the fluorescence change for L306A was intermediate to that for Gtalpha * (not shown).

R*-induced GTPgamma S Binding to Gtalpha * Mutants-- The ability of R* to interact with Gtalpha * mutants and cause them to release GDP was examined by measuring the rates of GTPgamma S binding to these mutants in the presence of R* and Gtbeta gamma . The release of GDP is a rate-limiting step in activation of G protein alpha  subunits, and thus it controls the rate of GTPgamma S binding (26). Three Gtalpha * mutants, Y298A, I299A, and F303A, did not appreciably bind GTPgamma S. A correlation between the low expression levels of these mutants and the lack of GTPgamma S binding indicates that defects in the overall folding might be responsible for the loss of the R*-dependent activation. However, the finding that these mutants were able to specifically interact with the effector (see below) rules out gross misfolding. Alternatively, the alpha 4-alpha 3 coupling could represent an important element in maintaining proper conformation of the R*-binding regions, or it is essential for the ability of Gtalpha to undergo a conformational change upon binding of GTPgamma S. The latter possibility is supported by the lack of the tryptophan fluorescence enhancement with addition of AlF4- to Y298A, I299A, and F303A. The GTPgamma S binding properties of the L306A mutant, in which another residue that contacts alpha 3 was substituted, were seriously compromised but not abolished. Fitting of the GTPgamma S binding data for L306A yielded a value for maximal binding at ~35% of that for Gtalpha * with an ~4-fold lower rate (kapp = ~0.019 s-1) (Fig. 3 and Table I). Gtalpha *L306A was expressed in E. coli comparably to Gtalpha * but showed diminished ability for the conformational change in the presence of AlF4-. This suggests that L306A has a similar but more mildly expressed phenotype than mutants Y298A, I299A, and F303A.


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Fig. 3.   GTPgamma S binding to Gtalpha * mutants. The binding of GTPgamma S to Gtalpha * mutants (0.4 µM mutant Gtalpha *; 2 µM Gtbeta gamma ) was initiated by addition of 5 µM [35S]GTPgamma S (0.2 µCi) and uROS membranes (100 nM rhodopsin). Protein-bound GTPgamma S was determined using the filter binding assay. The V301A mutant (black-diamond ) is representative of Gtalpha * mutants with intact kinetics of GTPgamma S binding. The L306A (black-square), R310A (black-down-triangle ) and D311A (black-triangle) mutants had impaired GTPgamma S binding characteristics.

A substantial loss of the receptor function was observed when Asp311 was replaced by Ala. The D311A mutant in comparison with Gtalpha * maximally bound only ~50% GTPgamma S with a reduced rate of 0.023 s-1 (Fig. 3 and Table I). A relatively mild alteration in R* activation was found for the R310A mutant. Gtalpha *R310A had a saturating level of GTPgamma S binding similar to that of Gtalpha *, but the rate of binding was decreased by ~2-fold (Fig. 3 and Table I). Previously, Arg309, Val312, and Lys313 were implicated in the Gtalpha -R* interaction using an assay of Gtalpha activation in microsomes of COS7 cells expressing rhodopsin and mutant Gt (11). We observed no significant changes in the kinetics of Gtalpha * activation caused by these three or other remaining mutations under our experimental conditions (Table I).

Binding of Gtalpha * Mutants to Pgamma BC-- To delineate potential effector residues within the alpha 4-beta 6 region, the Gtalpha * mutants in the GDP-bound or active AlF4--induced conformations were tested for binding to Pgamma BC. Interestingly, mutants Y298A, I299A, and F303A, which had low expression levels and lacked R*-induced GTPgamma S binding, in the GDP-bound conformations displayed affinities for Pgamma BC comparable with Gtalpha *GDP (Table I). This result indicates that in the inactive conformation their effector interface is not significantly affected. Predictably, these three Gtalpha * mutants had significant defects in binding to Pgamma in the presence of AlF4-. Addition of AlF4- produced no enhancement in the mutant interaction with Pgamma BC, evidently due to the inability of these mutants to assume an active conformation. In addition, the interaction of the L306A mutant with Pgamma BC was less sensitive than that of Gtalpha * to AlF4-. In the presence of AlF4-, L306A bound to Pgamma BC with a Kd only 2-fold lower than when AlF4- was absent (Table I). This is consistent with the limited competency of L306A to assume an active conformation. The Gtalpha * mutants, V301A and K313A, had mild defects in effector binding. These mutants retained a high affinity for Pgamma BC in the AlF4--bound conformations but revealed a somewhat reduced interaction with the effector in the absence of AlF4- (Table I). All other Gtalpha * mutants demonstrated affinities for Pgamma BC comparable with that of Gtalpha (Table I).

Activation of Rod PDE by Gtalpha * Mutants-- The ability of Gtalpha * mutants to stimulate activity of holoPDE (Palpha beta gamma 2) was tested in the reconstituted system with additions of uROS membranes and purified Gtbeta gamma in the presence of GTPgamma S. Gtalpha * as well as the majority of its mutants activated holoPDE under these conditions by ~12-18-fold. Not surprisingly, mutants Y298A, I299A, and F303A were incapable of stimulating PDE (not shown). Mutants L306A and D311A were notably less effective in the PDE activation assay (Fig. 4). This reduction in the effector function seems to correlate well with the decreased capacity of these mutants to bind GTPgamma S in the presence of R*. Therefore, residues Leu306 and Asp311 are unlikely to be directly involved in interaction with and activation of PDE.


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Fig. 4.   Activation of PDE by Gtalpha * mutants. The cGMP hydrolytic activity of rod holoPDE (0.2 nM) was measured in suspensions of uROS membranes (10 µM rhodopsin) reconstituted with 2 µM Gtbeta gamma and 10 µM GTPgamma S in the presence of 2 µM Gtalpha * mutants. The PDE activation is expressed as a percentage of that elicited by Gtalpha * (the basal and Gtalpha *-stimulated PDE activities were 14 and 210 mol cGMP/s mol PDE, respectively).


    DISCUSSION

The alpha 4-beta 6 region of Gtalpha is an essential contributor to the Gtalpha -rhodopsin interface (6, 11). The R* binding sites of Gtalpha , the alpha 4-beta 6 loop (amino acids 305-315) and Gtalpha -340-350, are positioned on the same "receptor" face of Gtalpha beta gamma as the N terminus of Gtalpha and the C terminus of Gtgamma (28). The beta 6-sheet and the alpha 5-helix project inward from the alpha 4-beta 6 loop and Gtalpha -340-350 on the Gtalpha surface to form the beta 6/alpha 5 loop. The latter contains a cluster of residues, Cys321, Ala322, and Thr323, intimately involved in binding of the guanine ring (27). Mutations of the residues within the beta 6/alpha 5 loop promote dissociation of GDP and GTP-GDP exchange on several Galpha subunits (29-31). Thus, the Gtalpha activation mechanism is likely to involve interaction of R* with the alpha 4-beta 6 loop and Gtalpha -340-350, leading to conformational changes of the beta 6/alpha 5 loop and dissociation of GDP.

The critical R*-binding region, Gtalpha -340-350, has been investigated in great detail (6-9, 32). However, the role of the Gtalpha alpha 4-beta 6 loop and its individual residues in binding to R* and Gtalpha activation is not well understood. Recently, mutants of Gtalpha with Ala substitutions of residues in the alpha 4-beta 6 loop have been translated in vitro and expressed in COS-7 cells (11). Mutational analysis revealed that substitutions of four residues in the alpha 4-beta 6 loop, Arg309, Asp311, Val312, and Lys313 impaired Gtalpha interaction with R* (11). The Ala-scanning mutagenesis of the Gtalpha alpha 4-beta 6 region in the context of Gtalpha * readily expressed in E. coli has provided us with an opportunity for in depth investigation of the roles of individual alpha 4-beta 6 residues in the receptor interaction. Reconstitution of the purified mutant Gtalpha * with Gtbeta gamma and uROS membranes has enabled the examination of the effects of mutations on the kinetics of Gtalpha * activation by R*. Our results confirm the role of Asp311 in the R*-dependent activation of Gtalpha . A substitution of this residue led to a substantial decrease in both the rate of and total R*-induced GTPgamma S binding. A moderate alteration of the kinetics of R*-induced GTPgamma S binding was caused by the substitution of Arg310. The R310A mutant bound GTPgamma S with an ~2-fold slower rate. Supporting the involvement of Gtalpha Asp311, and probably Arg310, in the interaction with R* is the fact that the trypsin cleavage site Arg310-Asp311 is protected upon binding of Gtalpha to R* (10). In addition to effective activation of Gtalpha , R* is capable of activating Gialpha (17) but has no detectable interaction with Gsalpha .2 Arg310 and Asp311 of Gtalpha align with the Lys-Asp and Ser-Gly pairs in Gialpha and Gsalpha , respectively. Therefore, Arg310 and Asp311 along with Gtalpha -340-350 may contribute to the specificity of the Gtalpha -R* interaction.

Mutations Y298A, I299A, and F303A caused the loss of Gtalpha * activation by R* or AlF4-, whereas the L306A mutation resulted in a less severe phenotype. This loss of function apparently resulted from the inability of the mutants to undergo the activational conformational change. Residues Ile299, Phe303, and Leu306 interact with Met239, Leu243, and Phe246, respectively (27). This interaction network between the alpha 4 and alpha 3 helices may secure the proper positioning of Glu241, Leu245, Ile249, and Phe255. The latter residues, upon activation of Gtalpha , engage the switch II residues Arg201, Arg204, and Trp207 to form another network of interactions, which is critical for the Gtalpha progression to the active conformation (33). Therefore, our results suggest that the coupling between helices alpha 3 and alpha 4 is critical for the transition of Gtalpha to the active state.

The key event in photoactivation of PDE is a direct interaction between the GTP-bound Gtalpha and Pgamma . Both Gtalpha GTP and Gtalpha GDP are capable of binding Pgamma . However, Gtalpha GDP binds Pgamma with ~10-30-fold lower affinity and is incapable of efficient activation of PDE (23, 34). The Gtalpha binding sites on Pgamma have been firmly established (13, 35-37). The Pgamma -binding surface on Gtalpha appears to be significantly more complex and less understood. Initially, the putative effector region of Gtalpha corresponding to the alpha 4-helix and the alpha 4-beta 6 loop was identified using synthetic Gtalpha peptides. A synthetic peptide, Gtalpha -293-314, potently (Ka of 8 µM) stimulated activity of rod holoPDE (12) via binding to Pgamma (13). Substitutions within the Gtalpha -293-314 peptide have been made, and five nonconserved residues, Asn297, Val301, Glu305, Met308, and Arg310, were found to contribute to the activational effects (16). However, evidence contradicting the role of alpha 4-beta 6 as a major effector domain of Gtalpha has emerged from analysis of chimeric Gtalpha /Gialpha proteins and mutagenesis of Gtalpha (17-19). Two other effector-interacting domains of Gtalpha , the switch II region and the alpha 3-helix-alpha 3/beta 5 loop, have been identified (17-19). These findings led to the apparent discrepancy between the ability of peptide Gtalpha -293-314 to activate PDE and the prerequisite of the switch II and alpha 3-beta 5 regions of Gtalpha for the effector stimulation. Hypothetically, the discrepancy is nonexistent if the role of switch II and alpha 3-beta 5 is only to obscure the alpha 4-beta 6 region in Gtalpha GDP. However, such a model is not supported by the crystal structures of Gtalpha (27, 33). Moreover, at least three residues, Ile208 (switch II), His244, and Asn247 (alpha 3) are likely to interact directly with Pgamma in the GTP-bound Gtalpha conformation (19).

The Ala-scanning mutational analysis performed in this study demonstrated that none of the alpha 4-beta 6 residues appear to participate directly and significantly in the Gtalpha /Pgamma binding. Even substitutions of the residues Tyr298, Ile299, Phe303, and Leu306, which disabled the activation of Gtalpha *, had no notable impact on the binding of the GDP-bound mutants to Pgamma BC. Results on activation of PDE by the Gtalpha mutants correlated well with the Pgamma binding experiments. All mutants with unimpaired capacity for R*-induced GTPgamma S binding were competent to stimulate cGMP hydrolysis by holoPDE. The studies on cross-linking of Pgamma to Gtalpha attest to a close proximity of Pgamma to the alpha 4-beta 6 region in the Gtalpha -Pgamma complex (14, 15). Although our analysis seems to rule out strong major interactions between Pgamma and Gtalpha -293-314, a relatively weak van der Waals' contact(s) at this site cannot be entirely excluded. Rather, the role of the alpha 4-beta 6 residues, Ile299, Phe303, and Leu306, is that they are critical for the activational conformational change via the interaction with the alpha 3-helix and thus indirectly are important for the effector function of Gtalpha .

The most surprising finding in this work is that none of the mutations of five Gtalpha residues identified using synthetic peptides (16) meaningfully affected the Gtalpha *-PDE interaction. A greater sensitivity of the peptide structure than that of Gtalpha * to mutations may explain the different results. Although the NMR analysis of substituted peptides ruled out gross misfolding, inactivation of mutant peptides due to a conformational change remains a possibility (16). However, a more plausible explanation is that the peptide Gtalpha -293-314 and Gtalpha activate PDE via different mechanisms. This raises a general concern regarding potential problems with interpretation of effects that might be observed using synthetic peptides as probes of protein-protein interactions. The conclusion that Gtalpha -293-314 likely represents a major effector-activating domain of Gtalpha was reached based on the ability of the peptide to "mimic" Gtalpha in PDE activation (12, 16) and provided the best explanation of the data in the absence of an alternative approach. Yet, the puzzling mimicking effect of the Gtalpha peptide does not appear to reflect the role of the corresponding region in Gtalpha .

    FOOTNOTES

* This work was supported by National Institutes of Health Grant EY-10843 and American Heart Association Grant-in-Aid 9750334N. National Institutes of Health Grant DK-25295 supported the services provided by the Diabetes and Endocrinology Research Center of the University of Iowa.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 Recipient of the American Heart Association Iowa Affiliate Postdoctoral Fellowship.

§ To whom correspondence should be addressed. Tel.: 319-335-7864; Fax: 319-335-7330; E-mail: nikolai-artemyev{at}uiowa.edu.

2 M. Natochin and N. O. Artemyev, unpublished observations.

    ABBREVIATIONS

The abbreviations used are: R*, light-activated (bleached) rhodopsin; Gtalpha , rod G-protein (transducin) alpha -subunit; PDE, rod outer segment cGMP phosphodiesterase; Palpha beta and Pgamma , alpha , beta , and gamma  subunits of PDE; ROS, rod outer segment(s); uROS, urea-stripped ROS membranes; Pgamma BC, Pgamma labeled with 3-(bromoacetyl)-7-diethyl aminocoumarin; GTPgamma S, guanosine 5'-O-(3-thiotriphosphate); PCR, polymerase chain reaction.

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ABSTRACT
INTRODUCTION
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