Roles of the Transducin α-Subunit α4-Helix/α4-β6 Loop in the Receptor and Effector Interactions*

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 α4-helix and α4-β6 loop of the transducin α-subunit (Gtα) has been implicated in interactions with the receptor and the effector. Ala-scanning mutagenesis of the α4-β6 region has been carried out to elucidate residues critical for the functions of transducin. The mutational analysis supports the role of the α4-β6 loop in the R*-Gtα interface and suggests that the Gtα 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 Gtα, our data indicate that none of the α4-β6 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 α3-helix of Gtα, which is critical for the ability of Gtα to undergo an activational conformational change. Thereby, Ile299, Phe303, and Leu306play only an indirect role in the effector function of Gtα.

Upon transduction of the visual signal in vertebrate photoreceptor cells, photoexcited rhodopsin (R*) 1 binds the retinal G protein, transducin (G t ), leading to G t activation. The ␣-subunit of G t (G t ␣) complexed with GTP is then released to stimulate the effector enzyme, cGMP phosphodiesterase (PDE), by reversing the inhibiton imposed by two PDE ␥ subunits (P␥) on the PDE catalytic dimer (P␣␤). Activated PDE rapidly hydrolyzes cGMP resulting in closure of cGMP-gated channels in the photoreceptor plasma membrane (1)(2)(3).
The two central interactions of G t ␣ with R* and P␥ during visual excitation have been extensively investigated, and the G t ␣ interaction sites have been localized. Evidence points to the C terminus of G t ␣ as the major R* contact site that is critical for G t ␣ activation (4 -9). A second essential site of G t ␣ interaction with R* includes the ␣4/␤6 loop (residues 305-315) (6,10,11). A peptide, G t ␣-311-328, competed for the G t -R* interaction (6). The tryptic cleavage site at Arg 310 of G t ␣ was protected upon G t ␣␤␥ binding to R* (10). Several mutants with Ala substitutions of residues from the ␣4/␤6 loop had impaired binding to R* and reduced degrees of activation (11). Interestingly, this R* binding site overlaps with a region of G t ␣, G t ␣-293-314, that has been implicated in the transducin-effector interaction (12)(13)(14)(15)(16). A synthetic peptide, G t ␣-293-314, corresponding to the ␣4-␤6 region was shown to activate PDE in vitro and to bind to P␥ (12,13). Sites of chemical cross-linking of the P␥-subunit to G t ␣ were localized to within the ␣4-␤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 G t ␣ ␣4-␤6 region in the effector interaction remains unclear. An insertion of the G t ␣-295-314 segment into G i ␣ 1 only marginally improved the latter's ability to bind P␥ (17). This finding suggests that if the ␣4-␤6 region is important for the interaction with PDE, then likely the conserved residues within ␣4-␤6 are essential for the function of the effector. Alternatively, even small differences in G t ␣ and G i ␣ folding may interfere with the ability of G t ␣-293-314 to assume the proper effector-binding conformation in the context of G i ␣. More importantly, the apparent ability of peptide G t ␣-293-314 to potently stimulate PDE (12,16) is inconsistent with the mutational analysis of G t ␣ (18,19). The latter indicates the requirement of the switch II and ␣3 regions for effector activation (18,19).
In light of the importance of the G t ␣ ␣4-␤6 region for the G t -R* interaction and substantial but conflicting evidence on its role for PDE activation, we carried out Ala-scanning mutational analysis of the ␣4-helix (residues 293-304) and the ␣4-␤6 loop of G t ␣. Our analysis of mutant G t ␣ interactions with R* and PDE has underscored the role of the ␣4-␤6 loop for the receptor function but revealed only indirect involvement of the ␣4-helix in the G t ␣ effector function via requirement of the ␣4/␣3 coupling for the activational conformational change.
Ala-scanning Mutagenesis of the ␣4-␤6 Region of G t ␣-Substitutions of G t ␣ residues by Ala were introduced into G t ␣/G i ␣ 1 chimeric protein, G t ␣*, which contains only 16 residues from G i ␣. G t ␣* was made based on another G t ␣/G i ␣ 1 chimeric protein, Chi8, which is competent to interact with R* and G t ␤␥ (17,19). To generate G t ␣*, all the G i ␣ residues in the ␣3-helix and the ␣3-␤5 loop of Chi8, except for Met 247 (corresponding to Leu 243 of G t ␣) were replaced by G t ␣ residues. The following G t ␣ residues were introduced into Chi8: His 244 , Asn 247 , His 252 , Arg 253 , Tyr 254 , Ala 256 , and Thr 257 . The PCR-directed mutagenesis was carried out essentially as described in Natochin et al. (19).
Single substitutions of G t ␣ 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-G t ␣* plasmid was used as a template. The PCR products (ϳ200 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 G t ␣ BamHI site. The 500-base pair PCR products were digested with BamHI and Hin-dIII and ligated into pHis6-G t ␣* cut with the same enzymes. The sequences of all mutants were verified by automated DNA sequencing at the University of Iowa DNA Core Facility. G t ␣* and all mutants were expressed and purified as described previously (19). Fluorescence Assays-Fluorescence assays of interaction between G t ␣* and P␥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 MgCl 2 essentially as described in (19,23). Where indicated, the buffer contained 30 M AlCl 3 and 10 mM NaF. Fluorescence of P␥BC was monitored with excitation at 445 nm and emission at 495 nm. Concentration of P␥BC was determined using ⑀ 445 ϭ 53,000. The AlF 4 Ϫ -induced increases in the tryptophan fluorescence of G t ␣*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 M G t ␣*GDP or the GDP-bound G t ␣* mutants and 2 M G t ␤␥ in suspensions of uROS membranes containing 10 M rhodopsin. GTP␥S (10 M) was added to the reaction mixture, and PDE activity was measured using [ 3 H]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 Graph-Pad Prizm (v.2) software. The results are expressed as the means Ϯ S.E. of triplicate measurements. Examination of the crystal structure of G t ␣ was performed using RasMol (v.2.6) software.

Expression and Characterization of the Receptor and Effector
Competent Chimeric G t ␣*-We have previously found that residue Leu 243 of G t ␣ is mainly responsible for the low level expression of G t ␣/G i ␣ chimeras containing the G t ␣-237-270 (␣3-␤5) segment (19). G t ␣* was obtained based on the G t ␣/G i ␣ chimera, Chi8, which contains the ␣3-␤5 region of G i ␣ (17). The nonconserved G i ␣ residues from the ␣3-helix and the ␣3-␤5 loop of Chi8 were replaced by the corresponding G t ␣ residues (except for Leu 243 ). Two of the introduced G t ␣ residues, His 244 and Asn 247 , are important for the G t ␣-PDE interaction (19). The resulting chimeric G t ␣ 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 G t ␤␥ and R* and capable of high affinity effector binding. The ability of G t ␣* to interact with R* in the presence of G t ␤␥ was evaluated using the GTP␥S binding assay. The very slow GTP␥S binding rate to G t ␣* (k app ϭ ϳ0.004 s Ϫ1 ), which is limited by the rate of GDP dissociation (26), was significantly accelerated in the presence of R* and G t ␤␥ (k app ϭ ϳ0.079 s Ϫ1 ) (Fig. 1). A fluorescence read-out assay was utilized to monitor the inter-action between G t ␣ and the P␥ subunit (23). Using this assay, G t ␣*GDP bound fluorescently labeled P␥, P␥BC, with a K d value of 28 nM ( Fig. 2A and Table I). When G t ␣*GDP was activated in the presence of AlF 4 Ϫ it bound to P␥BC with an almost 6-fold higher affinity (K d of 5.1 nM) ( Fig. 2B and Table I). Thus, G t ␣*, which contains only 16 G i ␣ residues, represents a well suited tool for mutational analysis to identify residues that are essential for both receptor and effector interactions of transducin.
Expression of G t ␣* Mutants with Ala Substitutions of the ␣4-␤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 ␣4-helix and the ␣4-␤6 loop of G t ␣ are surface exposed (27) and were substituted with Ala residues. In addition to a modestly solvent-exposed Leu 306 , two buried residues, Ile 299 and Phe 303 , are involved in coupling the ␣4-helix with the ␣3-helix (27). This linkage might be important for stabilization of the receptor and/or effector-competent conformations of G t ␣. Substitutions of Ile 299 and Phe 303 were made to test this possibility. Expression of all but three of the G t ␣* mutants in E. coli have yielded similar amounts of soluble proteins (ϳ3-5 mg/liter of culture). Mutants Y298A, I299A, and F303A had notably reduced expression levels (ϳ0.5-1 mg/liter). The crystal structure of G t ␣GTP␥S shows that the Tyr 298 side chain makes contact with Tyr 286 from the ␣G-␣4 loop, whereas Ile 299 and Phe 303 interact with the ␣3-helix (27). Perhaps, the reduction in mutant expression reflects lower rates of proper protein folding due to the lack of stabilizing contacts between ␣4 and the ␣G-␣4 loop or the ␣3-helix.
The ability of G t ␣ mutants to undergo a conformational change upon addition of AlF 4 Ϫ 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 AlF 4 Ϫ , whereas the fluorescence change for L306A was intermediate to that for G t ␣* (not shown).
R*-induced GTP␥S Binding to G t ␣* Mutants-The ability of R* to interact with G t ␣* mutants and cause them to release GDP was examined by measuring the rates of GTP␥S binding to these mutants in the presence of R* and G t ␤␥. The release of GDP is a rate-limiting step in activation of G protein ␣ subunits, and thus it controls the rate of GTP␥S binding (26). Three G t ␣* mutants, Y298A, I299A, and F303A, did not appreciably bind GTP␥S. A correlation between the low expression levels of these mutants and the lack of GTP␥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 ␣4-␣3 coupling could represent an important element in maintaining proper conformation of the R*-binding regions, or it is essential for the ability of G t ␣ to undergo a conformational change upon binding of GTP␥S. The latter possibility is supported by the lack of the tryptophan fluorescence enhancement with addition of AlF 4 Ϫ to Y298A, I299A, and F303A. The GTP␥S binding properties of the L306A mutant, in which another residue that contacts ␣3 was substituted, were seriously compromised but not abolished. Fitting of the GTP␥S binding data for L306A yielded a value for maximal binding at ϳ35% of that for G t ␣* with an ϳ4-fold lower rate (k app ϭ ϳ0.019 s Ϫ1 ) (Fig. 3 and Table I). G t ␣*L306A was expressed in E. coli comparably to G t ␣* but showed diminished ability for the conformational change in the presence of AlF 4 Ϫ . This suggests that L306A has a similar but more mildly expressed phenotype than mutants Y298A, I299A, and F303A.
A substantial loss of the receptor function was observed when Asp 311 was replaced by Ala. The D311A mutant in comparison with G t ␣* maximally bound only ϳ50% GTP␥S with a reduced rate of 0.023 s Ϫ1 (Fig. 3 and Table I). A relatively mild alteration in R* activation was found for the R310A mutant. G t ␣*R310A had a saturating level of GTP␥S binding similar to that of G t ␣*, but the rate of binding was decreased by ϳ2-fold ( Fig. 3 and Table I). Previously, Arg 309 , Val 312 , and Lys 313 were implicated in the G t ␣-R* interaction using an assay of G t ␣ activation in microsomes of COS7 cells expressing rhodopsin and mutant G t (11). We observed no significant changes in the kinetics of G t ␣* activation caused by these three or other remaining mutations under our experimental conditions ( Table I).
Binding of G t ␣* Mutants to P␥BC-To delineate potential effector residues within the ␣4-␤6 region, the G t ␣* mutants in the GDP-bound or active AlF 4 Ϫ -induced conformations were tested for binding to P␥BC. Interestingly, mutants Y298A, I299A, and F303A, which had low expression levels and lacked R*-induced GTP␥S binding, in the GDP-bound conformations displayed affinities for P␥BC comparable with G t ␣*GDP (Table  I). This result indicates that in the inactive conformation their effector interface is not significantly affected. Predictably, these three G t ␣* mutants had significant defects in binding to P␥ in the presence of AlF 4 Ϫ . Addition of AlF 4 Ϫ produced no   enhancement in the mutant interaction with P␥BC, evidently due to the inability of these mutants to assume an active conformation. In addition, the interaction of the L306A mutant with P␥BC was less sensitive than that of G t ␣* to AlF 4 Ϫ . In the presence of AlF 4 Ϫ , L306A bound to P␥BC with a K d only 2-fold lower than when AlF 4 Ϫ was absent (Table I). This is consistent with the limited competency of L306A to assume an active conformation. The G t ␣* mutants, V301A and K313A, had mild defects in effector binding. These mutants retained a high affinity for P␥BC in the AlF 4 Ϫ -bound conformations but revealed a somewhat reduced interaction with the effector in the absence of AlF 4 Ϫ (Table I). All other G t ␣* mutants demonstrated affinities for P␥BC comparable with that of G t ␣ ( Table I).
Activation of Rod PDE by G t ␣* Mutants-The ability of G t ␣* mutants to stimulate activity of holoPDE (P␣␤␥ 2 ) was tested in the reconstituted system with additions of uROS membranes and purified G t ␤␥ in the presence of GTP␥S. G t ␣* as well as the majority of its mutants activated holoPDE under these conditions by ϳ12-18-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 GTP␥S in the presence of R*. Therefore, residues Leu 306 and Asp 311 are unlikely to be directly involved in interaction with and activation of PDE.

DISCUSSION
The ␣4-␤6 region of G t ␣ is an essential contributor to the G t ␣-rhodopsin interface (6,11). The R* binding sites of G t ␣, the ␣4-␤6 loop (amino acids 305-315) and G t ␣-340 -350, are positioned on the same "receptor" face of G t ␣␤␥ as the N terminus of G t ␣ and the C terminus of G t ␥ (28). The ␤6-sheet and the ␣5-helix project inward from the ␣4-␤6 loop and G t ␣-340 -350 on the G t ␣ surface to form the ␤6/␣5 loop. The latter contains a cluster of residues, Cys 321 , Ala 322 , and Thr 323 , intimately involved in binding of the guanine ring (27). Mutations of the residues within the ␤6/␣5 loop promote dissociation of GDP and GTP-GDP exchange on several G␣ subunits (29 -31). Thus, the G t ␣ activation mechanism is likely to involve interaction of R* with the ␣4-␤6 loop and G t ␣-340 -350, leading to conformational changes of the ␤6/␣5 loop and dissociation of GDP.
The critical R*-binding region, G t ␣-340 -350, has been inves-tigated in great detail (6 -9, 32). However, the role of the G t ␣ ␣4-␤6 loop and its individual residues in binding to R* and G t ␣ activation is not well understood. Recently, mutants of G t ␣ with Ala substitutions of residues in the ␣4-␤6 loop have been translated in vitro and expressed in COS-7 cells (11). Mutational analysis revealed that substitutions of four residues in the ␣4-␤6 loop, Arg 309 , Asp 311 , Val 312 , and Lys 313 impaired G t ␣ interaction with R* (11). The Ala-scanning mutagenesis of the G t ␣ ␣4-␤6 region in the context of G t ␣* readily expressed in E. coli has provided us with an opportunity for in depth investigation of the roles of individual ␣4-␤6 residues in the receptor interaction. Reconstitution of the purified mutant G t ␣* with G t ␤␥ and uROS membranes has enabled the examination of the effects of mutations on the kinetics of G t ␣* activation by R*.
Our results confirm the role of Asp 311 in the R*-dependent activation of G t ␣. A substitution of this residue led to a substantial decrease in both the rate of and total R*-induced GTP␥S binding. A moderate alteration of the kinetics of R*induced GTP␥S binding was caused by the substitution of Arg 310 . The R310A mutant bound GTP␥S with an ϳ2-fold slower rate. Supporting the involvement of G t ␣ Asp 311 , and probably Arg 310 , in the interaction with R* is the fact that the trypsin cleavage site Arg 310 -Asp 311 is protected upon binding of G t ␣ to R* (10). In addition to effective activation of G t ␣, R* is capable of activating G i ␣ (17) but has no detectable interaction with G s ␣. 2 Arg 310 and Asp 311 of G t ␣ align with the Lys-Asp and Ser-Gly pairs in G i ␣ and G s ␣, respectively. Therefore, Arg 310 and Asp 311 along with G t ␣-340 -350 may contribute to the specificity of the G t ␣-R* interaction. Mutations Y298A, I299A, and F303A caused the loss of G t ␣* activation by R* or AlF 4 Ϫ , 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 Ile 299 , Phe 303 , and Leu 306 interact with Met 239 , Leu 243 , and Phe 246 , respectively (27). This interaction network between the ␣4 and ␣3 helices may secure the proper positioning of Glu 241 , Leu 245 , Ile 249 , and Phe 255 . The latter residues, upon activation of G t ␣, engage the switch II residues Arg 201 , Arg 204 , and Trp 207 to form another network of interactions, which is critical for the G t ␣ progression to the active conformation (33). Therefore, our results suggest that the coupling between helices ␣3 and ␣4 is critical for the transition of G t ␣ to the active state.
The key event in photoactivation of PDE is a direct interaction between the GTP-bound G t ␣ and P␥. Both G t ␣GTP and G t ␣GDP are capable of binding P␥. However, G t ␣GDP binds P␥ with ϳ10 -30-fold lower affinity and is incapable of efficient activation of PDE (23,34). The G t ␣ binding sites on P␥ have been firmly established (13,(35)(36)(37). The P␥-binding surface on G t ␣ appears to be significantly more complex and less understood. Initially, the putative effector region of G t ␣ corresponding to the ␣4-helix and the ␣4-␤6 loop was identified using synthetic G t ␣ peptides. A synthetic peptide, G t ␣-293-314, potently (K a of 8 M) stimulated activity of rod holoPDE (12) via binding to P␥ (13). Substitutions within the G t ␣-293-314 peptide have been made, and five nonconserved residues, Asn 297 , Val 301 , Glu 305 , Met 308 , and Arg 310 , were found to contribute to the activational effects (16). However, evidence contradicting the role of ␣4-␤6 as a major effector domain of G t ␣ has emerged from analysis of chimeric G t ␣/G i ␣ proteins and mutagenesis of G t ␣ (17)(18)(19). Two other effector-interacting domains of G t ␣, the switch II region and the ␣3-helix-␣3/␤5 loop, have been identified (17)(18)(19). These findings led to the apparent discrepancy between the ability of peptide G t ␣-293-314 to activate PDE and the prerequisite of the switch II and ␣3-␤5 regions of G t ␣ for the effector stimulation. Hypothetically, the discrepancy is nonexistent if the role of switch II and ␣3-␤5 is only to obscure the ␣4-␤6 region in G t ␣GDP. However, such a model is not supported by the crystal structures of G t ␣ (27,33). Moreover, at least three residues, Ile 208 (switch II), His 244 , and Asn 247 (␣3) are likely to interact directly with P␥ in the GTP-bound G t ␣ conformation (19).
The Ala-scanning mutational analysis performed in this study demonstrated that none of the ␣4-␤6 residues appear to participate directly and significantly in the G t ␣/P␥ binding. Even substitutions of the residues Tyr 298 , Ile 299 , Phe 303 , and Leu 306 , which disabled the activation of G t ␣*, had no notable impact on the binding of the GDP-bound mutants to P␥BC. Results on activation of PDE by the G t ␣ mutants correlated well with the P␥ binding experiments. All mutants with unimpaired capacity for R*-induced GTP␥S binding were competent to stimulate cGMP hydrolysis by holoPDE. The studies on cross-linking of P␥ to G t ␣ attest to a close proximity of P␥ to the ␣4-␤6 region in the G t ␣-P␥ complex (14,15). Although our analysis seems to rule out strong major interactions between P␥ and G t ␣-293-314, a relatively weak van der Waals' contact(s) at this site cannot be entirely excluded. Rather, the role of the ␣4-␤6 residues, Ile 299 , Phe 303 , and Leu 306 , is that they are critical for the activational conformational change via the interaction with the ␣3-helix and thus indirectly are important for the effector function of G t ␣.
The most surprising finding in this work is that none of the mutations of five G t ␣ residues identified using synthetic peptides (16) meaningfully affected the G t ␣*-PDE interaction. A greater sensitivity of the peptide structure than that of G t ␣* 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 G t ␣-293-314 and G t ␣ 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 G t ␣-293-314 likely represents a major effector-activating domain of G t ␣ was reached based on the ability of the peptide to "mimic" G t ␣ 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 G t ␣ peptide does not appear to reflect the role of the corresponding region in G t ␣.