©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Characterization of Rhodopsin Mutants That Bind Transducin but Fail to Induce GTP Nucleotide Uptake
CLASSIFICATION OF MUTANT PIGMENTS BY FLUORESCENCE, NUCLEOTIDE RELEASE, AND FLASH-INDUCED LIGHT-SCATTERING ASSAYS (*)

Oliver P. Ernst (1) (2)(§), Klaus Peter Hofmann (2)(§), Thomas P. Sakmar (1)(¶)

From the (1) Howard Hughes Medical Institute, Laboratory of Molecular Biology and Biochemistry, Rockefeller University, New York, New York 10021 and the (2) Institut für Biophysik und Strahlenbiologie der Universität Freiburg, Albertstrasse 23, D-79104 Freiburg, Federal Republic of Germany

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The photoreceptor rhodopsin is a seven-transmembrane helix receptor that activates the G protein transducin in response to light. Several site-directed rhodopsin mutants have been reported to be defective in transducin activation. Two of these mutants bound transducin in response to light, but failed to release the bound transducin in the presence of GTP (Franke, R. R., König, B., Sakmar, T. P., Khorana, H. G., and Hofmann, K. P. (1990) Science 250, 123-125). The present study was carried out to determine the nucleotide-binding state of transducin as it interacts with rhodopsin mutants. Five mutant bovine opsin genes were prepared by site-specific mutagenesis. Three mutant genes had deletions from one cytoplasmic loop each: AB 70-71; CD 143-150; and EF 237-249. Two additional loop CD mutant genes were prepared: E134R/R135E had a reversal of a conserved charge pair, and CD r140-152 had a 13-amino acid sequence replaced by a sequence derived from the amino-terminal tail. Three types of assays were carried out: 1) a fluorescence assay of photoactivated rhodopsin (R*)-dependent guanosine 5`- O-(3-thiotriphosphate) uptake by transducin, 2) an assay of R*-dependent release of labeled GDP from the -subunit of transducin holoenzyme (G)GDP, and 3) a light-scattering assay of R*G complex formation and dissociation. We show that the mutant pigments, which are able to bind transducin in a light-dependent manner but lack the ability to activate transducin, most likely form R*GGDP complexes that are impaired in GDP release.


INTRODUCTION

Rhodopsin, the visual photoreceptor of the rod cell, is a member of the family of seven-transmembrane helix receptors that activate guanine nucleotide-binding regulatory proteins (G proteins).() The opsin apoprotein and its chromophore 11 -cis-retinal are covalently linked via a protonated Schiff base at Lys. Photoisomerization of the chromophore to all- trans-retinal is followed by several conformational changes and results in the formation of metarhodopsin II (MII), the active photoproduct which catalyzes nucleotide exchange in the rod cell G protein, transducin. The photoactivated form of rhodopsin can be referred to as R*.

The postulated secondary structure of rhodopsin and its topology with respect to the membrane bilayer (1) has been supported by a projection structure of rhodopsin obtained by cryoelectron microscopy (2) and by comparison with other G protein-coupled receptors (3) . Three cytoplasmic loops, AB, CD, and EF link successive transmembrane helices Fig. 1 ). A fourth putative loop is formed by a region of the carboxyl-terminal tail between the seventh helix and two palmitoylated cysteines (Cys and Cys). The cytoplasmic surface of the receptor interacts with transducin, which is peripherally bound to the membrane surface in the GDP-bound state, and with other cytoplasmic proteins such as rhodopsin kinase and arrestin (4) .


Figure 1: Schematic representation of rhodopsin mutants studied. The seven putative transmembrane helices are depicted as cylinders ( A-G). According to secondary structure predictions (1) and a projection structure (2), the helices are successively connected by loops (3). Interactions with other proteins of the signaling cascade occur at the cytoplasmic surface of the receptor toward the top of the figure. The cytoplasmic loops are designated AB, CD, and EF. A fourth cytoplasmic loop is formed by a portion of the carboxyl-terminal tail between helix G and a pair of palmitoylated cysteines. Five mutant rhodopsins are shown schematically. Three mutants contained partial deletions of one of the loops ( AB 70-71; CD 143-150; EF 237-249). Deleted residues are boxed. A fourth mutant ( E134R/R135E) contained a reversal of two amino acids of the CD loop. In the fifth mutant ( CD r140-152) the original sequence of residues 140-152 was replaced by the amino acid sequence GTEGPNFYVPFTS (see Table I). The amino termini of the recombinant rhodopsins may be acetylated as is the case in bovine rhodopsin.



The assignment of the transducin-interacting domains of rhodopsin has been largely based upon analysis of site-directed mutants (5, 6, 7, 8) . In addition, biochemical (9) , peptide competition (10) , and antibody competition (11) approaches have been employed. Cytoplasmic loops CD, EF, and the fourth loop were proposed to be the interacting sites between rhodopsin and transducin (5, 6, 7, 8, 10) . Cytoplasmic loop AB also may be involved in rhodopsin-transducin interaction (11, 12) . In general, all of the assays used to define rhodopsin-transducin interaction in recombinant pigments have relied upon a loss of the ability of R* to catalyze guanine-nucleotide exchange in transducin. However, according to the transducin activation scheme shown in Fig. 2, it is clear that an inability to form GGTPS could result from a defect at any one of four steps: 1) R* binding to GGDP, 2) GDP dissociation from the R*GGDP complex, 3) GTPS uptake by the R*G(empty) complex, or 4) dissociation of GGTPS and G from R*.


Figure 2: Experimental reaction scheme for light-induced transducin activation by rhodopsin. Photoactivated rhodopsin ( R*) binds to transducin (G) and induces GDP release followed by GTPS uptake by the nucleotide-depleted R*G(empty) complex leading to dissociation of the R*, GGTPS, and G. R*-dependent GDP release ( step 3) is studied by a nucleotide release assay. Nucleotide uptake of the nonhydrolyzable GTP analogue GTPS ( step 4) is studied by the fluorescence assay.



Two rhodopsin mutants have been reported that bound transducin in response to light but failed to release the bound transducin in the presence of GTP (6) . These rhodopsin mutants formed spectrally normal R* species, which bound transducin. However, it was not determined whether the mutant R*G complex contained GDP, GTP, or no nucleotide. It is important for a detailed understanding of the mechanism of receptor-mediated G-protein activation to be able to develop methods to study receptor mutants that are inactive in standard GTPase or filter-binding assays.

In this report, five rhodopsin mutants with amino acid replacements or deletions in either the cytoplasmic loop AB, CD, or EF were characterized. Three types of assays were carried out on the mutant pigments: 1) assay of R*-dependent GTPS uptake by transducin, 2) assay of R*-dependent GDP release from GGDP, and 3) assay of R*G complex formation and dissociation. The mutants, which are able to bind transducin in a light-dependent manner but lack the ability to activate transducin, are shown to form R*GGDP complexes that are impaired in GDP release.


EXPERIMENTAL PROCEDURES

Materials

Sources of most materials have been previously reported (7, 8, 13) . Detergents were purchased from Anatrace, Inc. (Cleveland, OH). Radionuclides were from DuPont NEN and BA85 nitrocellulose filters were from Schleicher & Schuell. Nucleotides were purchased from Boehringer Mannheim.

Preparation of Rhodopsin Mutants

The five mutants studied are shown schematically in Fig. 1. The mutant opsin genes were prepared by cassette mutagenesis of a synthetic gene for bovine rhodopsin (8, 14) that had been cloned into the expression vector as described previously (5) . Individual mutant genes were prepared as described previously: AB 70-71 (12) ; CD 143-150 (7) ; EF 237-249 (6) ; E134R/R135E (8) ; CD r140-152 (6) . Opsin genes were expressed in COS-1 cells following transient transfection by a DEAE-dextran procedure as described (15) . COS cells expressing the mutant apoproteins were harvested and then incubated in the presence of 11- cis-retinal under dim red light to reconstitute pigments as described elsewhere (7, 8) . The purification procedure employed was based on the immunoaffinity procedure of Oprian et al.(15) , which was modified as previously described (7, 8, 16) . Pigments were generally prepared in 10 mM BTP, pH 7.0, 130 mM NaCl, 1 mM MgCl, and 0.02% (w/v) DM or 1.5% (w/v) OG detergent. For some experiments, pigments AB 70-71 and CD r140-152 in DM detergent buffer were concentrated slightly using Microcon-30 filters (Amicon). All pigments prepared in OG detergent buffer were concentrated 2-3-fold using Centricon-30 filters. Each pigment displayed a visible value of 500 nm as previously reported (6, 7, 8, 12) . Each pigment concentration was determined based on its absorbance at 500 nm ( = 42,700 M cm).

Fluorescence Assay of GGTPS Formation

Bovine transducin was purified from rod outer segment extracts by hexyl-agarose chromatography (17) . A stock solution of transducin (approximately 25 µM) was stored at -20 °C in 10 mM MOPS, pH 7.5, 2 mM MgCl, 1 mM DTT, 5 µM GDP, 50% glycerol. Active transducin concentration was determined precisely by fluorometric titration (18) . Fluorescence assay of GGTPS formation rate catalyzed by rhodopsin and mutant pigments in light was carried out as described previously (18) except as noted below. Briefly, 1.5 ml of a mixture of 2 nM pigment and 200 nM transducin (10 mM BTP, pH 7.0, 130 mM NaCl, 1 mM MgCl, 1 mM DTT, 0.01% (w/v) DM) was continuously stirred at 10 °C and continuously illuminated in the cuvette through a fiber optic guide with 543.5-nm light from a HeNe laser (Melles-Griot). The sample was excited at 300 nm and fluorescence emission at 345 nm was recorded. After 7 min, 50 µl of GTPS was injected into the cuvette to give a final concentration of 5 µM, and fluorescence increase over time was recorded.

Nucleotide Release Assay

A nucleotide-release assay was employed to measure the ability of rhodopsin and of mutant pigments to catalyze GDP release by GGDP. Transducin used for this assay was purified as described previously (19) and was stored at -80 °C at concentrations of 6-8 µM in storage buffer (10 mM BTP, pH 7.0, 130 mM NaCl, 1 mM MgCl, and 1 mM DTT). Transducin concentration was precisely determined by fluorometric titration (18) . The intrinsic GTPase activity of transducin was used to prepare G[-P]GDP from transducin pre-loaded with [-P]GTP. Transducin samples were thawed and mixed with a catalytic amount of rhodopsin and [-P]GTP to give a final solution of 1 µM transducin, 1.5 nM rhodopsin, and approximately 60 or 150 pM [-P]GTP (3200 or 800 Ci/mmol, respectively) in storage buffer. To ensure uptake of the labeled GTP and its hydrolysis to GDP, the reaction mixture was incubated for 1 h at room temperature under continuous illumination (495 nm long-pass filter). During this incubation, the R* present decayed to opsin and free all- trans-retinal. The resulting [-P]GDP-loaded transducin was kept on ice, and the assay was carried out immediately as described. An assay mixture, containing 200 nM [-P]GDP-loaded transducin and 400 nM rhodopsin or mutant pigment in a total volume of 100 µl, was illuminated for 5 min. The light source was a 150-watt projector lamp fitted with a 495-nm long-pass filter (Oriel, Inc.). The assay was carried out in parallel with a reference sample in dim-red light (dark sample). The dark sample measures the total amount of GDP-loaded transducin present under the conditions of the assay. After 5 min of illumination, aliquots (20 µl 4) were removed from each of the two (light and dark) samples and vacuum filtered through a nitrocellulose membrane. The filters were rapidly washed (6 ml) and then air dried. The amount of bound [-P]GDP was quantitated using the PhosphorImager system (Molecular Dynamics). The average value of four measurements from the reference (dark) experiment was normalized to 100%. The standard error was in the range of ±5%. The mean of the four values obtained from the illuminated sample was then expressed as percentage of the reference (dark) sample. The photolyzed rhodopsin sample catalyzed the release of 96.6 ± 0.7% of the labeled GDP from transducin during the course of the assay. This corresponds to 3.4% G[-P]GDP bound.

Measurement of Mutant Pigment Schiff Base Stability

An acid denaturation method was used to determine the extent of Schiff base hydrolysis (8) . Under acidic conditions, rhodopsin denatures, but the chromophore-opsin protonated Schiff base linkage is stable, resulting in a broad peak with a value of 440 nm. Free all- trans-retinal gives a peak with a of about 380 nm. After an initial dark spectrum was recorded on a 90-µl aliquot, the remaining sample was illuminated for 20 s. After 5 min, 10 µl of 2 M hydrochloric acid was added to denature the pigment, and a spectrum was recorded.

Reconstitution of Rhodopsin and Mutant Pigments into Egg-Lecithin Vesicles

Purified rhodopsin or mutant pigment solubilized in OG detergent buffer was reconstituted into phospholipid vesicles in a 1:350 (pigment:phospholipid) molar ratio. All manipulations were carried out under dim red light. A suspension of 5 mg/ml egg-yolk phosphatidylcholine (Fluka) in deionized water was added to pigment (1.5-1.7 µM) in OG solution. The sample was dialyzed in a microdialysis unit (Pierce) against 2 liters of buffer (10 mM BTP, pH 7.0, 130 mM NaCl, and 1 mM MgCl) for 20 h at 4 °C. Vesicles were subjected to UV-visible spectroscopy to determine the concentration of reconstituted pigment. Freshly prepared vesicles were used for light-scattering measurements.

Light-Scattering Assay

Light-scattering measurements were performed using an apparatus previously described (20) that recently has been used to study rhodopsin kinase/rhodopsin (21) and transducin/PDE (19) interaction. The samples contained 700 nM recombinant pigment in reconstituted vesicles and 4 µM transducin in 10 mM BTP, pH 7.0, 130 mM NaCl, 1 mM MgCl, and 0.3 mM DTT in a final volume of 260 µl. Transducin used in this assay was prepared as described previously (19) and stored in 10 mM BTP, pH 7.0, 130 mM NaCl, 1 mM MgCl, 1 mM DTT at -80 °C. Measurements were carried out in a 10-mm path length cuvette. Light-scattering changes were monitored in an angular range of 16 ± 2 degrees by a continuous 840-nm incident light beam. To achieve a stable baseline, samples were incubated for up to 20 min. For measurements in the presence of GTP, 100 µM hydroxylamine was present during the 20-min incubation period (22) . When required, GTP was added immediately prior to measurement. Binding signals were recorded after photolysis of samples with a 500 ± 20-nm flash. The light-scattering signals are interpreted as binding signals and dissociation signals of transducin from the photoactivated pigment (23) . The physical correlate of the light-scattering signals is the light-induced gain or loss of protein mass by the pigment-phospholipid vesicle (20) .


RESULTS

Preparation of Recombinant Pigments

Five mutant bovine opsin genes were prepared by site-specific mutagenesis as shown schematically in Fig. 1. Three mutant genes had deletions from one cytoplasmic loop: AB 70-71; CD 143-150, and EF 237-249 (numbers indicate deleted amino acid residues). Two additional loop CD mutant genes were prepared: E134R/R135E had a reversal of a conserved charge pair and CD r140-152 had a 13-amino acid sequence replaced by a sequence derived from the amino-terminal tail region (). The genes were expressed by transient transfection of COS-1 cells and the expressed opsins were regenerated with 11- cis-retinal chromophore. The resulting pigments were purified in detergent solution and showed absorption maxima identical to rod outer segment rhodopsin ( = 500 nm).

Illumination of each pigment yielded a species with a value of 380 nm characteristic of MII. The decay of the Schiff base linkage in the MII-like species for each mutant was identical to that measured for native MII. In DM detergent solution at room temperature, less than 5% of the Schiff base decayed to opsin plus all- trans-retinal after 5 min. As reported previously, the expression level and regeneration efficiency for mutants AB 70-71 (12) and CD r140-152 (6) were significantly lower than for rhodopsin (not shown). Three types of assays were carried out on recombinant rhodopsin and on the mutant pigments as described below.

Assay of R*-dependent GTPS Uptake by Transducin

A fluorescence assay was employed to measure the ability of each pigment to catalyze GTPS uptake by transducin. Each of the mutants prepared was reported previously to be significantly impaired in light-dependent activation of transducin. The mutants had been studied in a GTPase assay (5, 6, 8) or in a GTPS uptake assay using nitrocellulose filters (12) . The ability of each mutant to catalyze the uptake of GTPS by transducin in response to light was evaluated here using a more sensitive fluorescence assay (18, 24) . In this assay, the accumulation of GGTPS as depicted in the scheme in Fig. 2is monitored as an increase in intrinsic fluorescence. As shown in Fig. 3 A, the addition of GTPS to a mixture of transducin and R* results in an increase of relative fluorescence as a function of time. The signal reaches a maximum when GTPS uptake, in a 1:1 stoichiometry with G, is complete.


Figure 3: Measurement of R*-catalyzed GGTPS formation and R*-dependent GDP release from GGDP. A, a fluorescence assay was employed to measure the rate of R*-catalyzed GGTPS formation. The increase in relative fluorescence is recorded as a function of time and is directly related to the increase in the concentration of GGTPS. GTPS was injected ( arrow) into a cuvette containing known concentrations of R* and G to start the reaction. The assay was carried out as described under ``Experimental Procedures.'' Assay conditions are: 2 nM pigment, 200 nM transducin, 10 mM BTP, pH 7.0, 130 mM NaCl, 1 mM MgCl, 1 mM DTT, 0.01% (w/v) DM, GTPS 5 µM at 10 °C. Under these conditions, R* catalyzes the uptake of 48 pmol GTPS/min (1.5-ml reaction volume). B, a nucleotide-release assay was employed to measure the R*-catalyzed GDP release by GGDP. The intrinsic GTPase activity of transducin was used to prepare G[-P]GDP from transducin pre-loaded with [-P]GTP. The assay was carried out as described under ``Experimental Procedures.'' Briefly, [-P]GDP-loaded transducin was incubated with rhodopsin for 5 min in the dark or in the light. The mixture was filtered through a nitrocellulose membrane, and the filter-bound radioactivity was quantitated. The amount of bound G[-P]GDP for the dark sample was defined as 100%. In the light, R* binds transducin and catalyzes GDP release from GGDP. The decrease in G[-P]GDP bound to the filter is directly proportional to the R*-dependent release of [-P]GDP from its nucleotide-binding pocket. The values plotted represent the means of five individual experiments. An error bar (S.E., n = 5) is shown for the light experiments. Assay conditions are: 400 nM pigment, 200 nM transducin, 10 mM BTP, pH 7.0, 130 mM NaCl, 1 mM MgCl, 0.2 mM DTT, 0.016% (w/v) DM, at 20 °C.



The activation rate for recombinant rhodopsin was determined by linear regression analysis of a 35-50 s time interval starting 6 s after GTPS addition (Fig. 4). Under the conditions of the assay, 2 nM recombinant rhodopsin (3 pmol of R*) catalyzed the uptake of 48 pmol of GTPS/min. Each of the mutant pigments was tested for its ability to activate transducin in the fluorescence assay (Fig. 4). The activation rates were measured as follows: transducin without pigment, 0.4%; AB 70-71, 6.7%; E134R/R135E, 0.6%; CD 143-150, 0.3%; CD r140-152, 0.8%; EF 237-249, 1.1%. Only mutant AB 70-71 showed a significant transducin activation rate (6.7% of the rate for rhodopsin). Rates of GGTPS formation slower than about 1% of that of native rhodopsin are within the noise of the assay and the corresponding mutants are considered to be unable to induce significant GGTPS formation under conditions of the assay.


Figure 4: Mutant rhodopsins are deficient in catalyzing GTPS uptake by transducin. A fluorescence assay was employed to measure the rate of pigment-catalyzed GGTPS formation. The increase in relative fluorescence is recorded as a function of time and is directly related to the increase in the concentration of GGTPS. GTPS was injected ( arrow) into a cuvette containing known concentrations of light activated pigment and G to start the reaction. Purified recombinant rhodopsin was compared with five mutant pigments as described in Fig. 1. In the control measurement no pigment was present. For rhodopsin the slope of the curve in the linear phase starting 6 s after GTPS addition was calculated from a linear regression analysis over a time interval of 35-50 s. For each of the mutant pigments the slope of the curve starting 60 s after GTPS addition was calculated from a linear regression analysis over a time interval of 500 s. The rates of fluorescence increase expressed as a percentage of the rhodopsin rate for the experiments shown were: transducin without pigment, 0.4%; AB 70-71, 6.7%; E134R/R135E, 0.6%; CD 143-150, 0.3%; CD r140-152, 0.8%; EF 237-249, 1.1%. The rates determined from independent preparations were reproducible to within ±1%.



Assay of R*G Complex Formation and Dissociation

A light-scattering assay was developed to measure directly the ability of each pigment to bind transducin in response to light and to release bound transducin in the presence of GTP. Binding and dissociation signals for recombinant rhodopsin reconstituted into phosphatidylcholine vesicles are shown in Fig. 5. A flash-induced scattering increase over time is noted in the presence (binding signal), but not in the absence of transducin. This signal reflects binding of the soluble fraction of the protein to the vesicles after photolysis as observed with reconstituted systems of disk membranes and transducin (19, 20) or rhodopsin kinase (21) . In presence of transducin and GTP, a flash produces a decrease of scattering intensity. This is due to the R*-catalyzed activation of the transducin fraction bound to the vesicle at the time of the flash. GTP binds to the activated R*G(empty) complex and then GGTP and G dissociate from the R* in the vesicle, resulting in a loss of scattering mass and a decrease in scattering intensity (dissociation signal). The mutant EF 237-249 is able to bind transducin as shown by the positive scattering change. However, the dissociation signal, which is normally observed in the presence of GTP, is absent with this mutant. Photolysis of mutants E134R/R135E and CD 143-150 does not cause scattering changes in presence of transducin alone, nor in the presence of transducin and GTP.


Figure 5: Binding and dissociation signals for rhodopsin and mutant pigments. Flash-induced near-infrared light-scattering changes were measured on recombinant pigments reconstituted into phosphatidylcholine vesicles. Samples containing 700 nM pigment and 4 µM transducin were bleached with a green flash as indicated by arrows. The mole fraction of flash-excited rhodopsin (R*/R) was 55% for signals in the a and b traces, or 0.2% for signals in the c traces. Light-scattering changes as a function of time in the presence ( a) or absence ( b) of transducin are shown. The dissociation signal caused by the presence of 1 mM GTP in addition to transducin is shown ( c). After GTP uptake, vesicle-bound GGTP and G dissociate from R and become soluble, resulting in a decrease in light-scattering intensity. The very small scattering decrease in the control without transducin ( b) is due to the N-signal (21, 41), a rhodopsin-related signal, which is superimposed on all signals caused by transducin. The N-signal in vesicles is much smaller than in disk membranes. As shown, rhodopsin binds transducin in response to light and releases bound transducin in the presence of GTP. The photoactivated mutant EF 237-249 binds transducin somewhat less well than rhodopsin, but does not release bound transducin in the presence of GTP. Mutants E134R/R135E and CD 143-150 fail to bind transducin. I/I is the normalized scattering change where I represents the flash induced scattering change and I represents the scattering intensity of the sample before flash minus the scattering intensity of the buffer.



Assay of R*-dependent GDP Release from GGDP

A nucleotide-release assay was developed to measure the ability of each pigment to catalyzed the release of GDP from GGDP (Fig. 3 B). Photoactivated rhodopsin induces almost complete release of GDP from the nucleotide-binding pocket of transducin after 5 min of continuous illumination. Only 3.4% of the dark reference sample was not released. This small residual GDP binding can be attributed to incomplete hydrolysis of [-P]GTP in the preloading step (see ``Experimental Procedures'') or to transducin which is not able to bind to R*. The results of this assay for the recombinant pigments are shown in Fig. 6and . Mutants E134R/R135E, CD 143-150, and CD r140-152 did not show a significant release of GDP. The difference in the mean amount of GGDP complex between dark and light experiments was less than 10%. Mutant pigments AB 70-71 and EF 237-249 catalyzed significant GDP-nucleotide releases of about 84 and 34%, respectively.


Figure 6: Light-dependent GDP release from transducin in the presence of recombinant pigments. The assay was carried out to determine the effect of various photoactivated rhodopsin mutants on the nucleotide occupancy of transducin. The fluorescence assay in Fig. 4 shows that mutants CD r140-152 and EF 237-249 do not induce GTPS uptake by transducin. The GDP-release assay addresses the question of whether the transducin that binds to these mutants contains GDP or an empty nucleotide-binding site. The results are consistent with the failure of transducin to release GDP upon binding to mutant pigments CD r140-152 and EF 237-249. Mutant pigments E134R/R135E and CD 143-150 were shown not to bind transducin (Fig. 5) (6). GDP release is not detected from transducin exposed to these light-activated mutant pigments. Mutant AB 70-71 shows GDP release consistent with its small transducin-activating ability (Fig. 4). The assay was performed as described for rhodopsin in Fig. 3. The values plotted represent the means of three to five individual experiments. The means of the dark values are normalized to 100%. An error bar (S.E., n = 3-5) is shown for the light experiments. Numerical values are presented in Table II.




DISCUSSION

The aim of this work was to further characterize rhodopsin mutants with alterations of their cytoplasmic loops. These mutants were chosen because of their particular properties with respect to transducin interaction. Mutant AB 70-71 showed a very low but measurable ability to activate transducin in a filter-binding assay (12) . Mutant CD 143-150 was unable to activate transducin in a GTPase assay (6) . Mutant E134R/R135E was also unable to activate transducin in a GTPase assay (8) , possibly because of alteration of the conserved Glu residue, which has been shown to regulate light-dependent transducin binding and activation (18, 25) . Mutants CD r140-152 and EF 237-249 were also shown to be inactive in a GTPase assay, but had the additional very interesting property that they bound transducin in response to light but failed to release the bound transducin in the presence of GTP (6) .

Mutants CD r140-152, EF 237-249, and E134R/R135E were previously studied by an assay which measured the ability of transducin to shift the equilibrium between MI- and MII-like forms of the pigment photoproduct toward MII (extra-MII assay) (6, 26) . Mutant E134R/R135E did not bind transducin in the extra-MII assay (6) . Mutants CD r140-152 and EF 237-249 formed extra-MII upon photolysis in the presence of transducin in both the presence and absence of GTP. It was postulated that photoactivated mutants CD r140-152 and EF 237-249 formed stable complexes with transducin, and that the bound transducin had an empty nucleotide-binding pocket that was unable to bind GTP (6) . However, the combination of GTPase and extra-MII assays could not distinguish among three possibilities for defects in mutants CD r140-152 and EF 237-249.

According to the transducin activation pathway (Fig. 2), an inability of these mutants to catalyze GGTP formation could result from a defect at any one of three steps: 1) GDP dissociation from the R*GGDP complex, 2) GTP uptake by the R*G(empty) complex, or 3) dissociation of GGTP and G from R*.

In the present study, a set of five rhodopsin mutants was studied to elucidate the particular step impaired in the activation pathway of transducin. A fluorometric assay was used to measure the accumulation of GGTPS over time. As shown in Fig. 3 A, the addition of GTPS to a mixture of R* and G causes an increase in fluorescence resulting from the formation of GGTPS (27) . Each CD-loop mutant (CD r140-152, CD 143-150, and E134R/R135E) and mutant EF 237-249 was unable to catalyze a significant GTPS uptake under conditions of the assay. This result supports the GTPase and filter-binding assay results of earlier studies (6, 7, 8) . Mutant AB 70-71 displayed about 7% of the activity of rhodopsin. This activation rate is somewhat higher than that reported from a filter-binding assay (12) . The different rates reported are most likely due to a higher pH (0.5 unit) in the filter-binding assay. This pH increase is known to cause a lower activation rate for rhodopsin (18) .

Direct binding of transducin to photoactivated pigments reconstituted into phosphatidylcholine vesicles was measured in a light-scattering assay (Fig. 5). Both transducin-dependent binding and dissociation signals can be obtained using the assay. Compared with the extra MII assay this has the advantage that stable binding need not be inferred from an effect on the MI/MII equilibrium between pigment photoproducts. The light-scattering assay is therefore not dependent on the effects of a particular mutation on the MI-like/MII-like equilibrium for a particular mutant pigment. Mutant pigments have been described with markedly altered photoactivation pathways. For example, a mutant rhodopsin photoproduct with a protonated Schiff base and a value of 474 nm has been shown to activate transducin (28, 29, 30) . Such mutants pigments can be studied by the light-scattering assay but not by the extra-MII assay.

Recombinant rhodopsin and mutants EF 237-249, E134R/R135E, and CD 143-150 were studied by the light-scattering assay (Fig. 5). Mutant EF 237-249 bound but failed to activate transducin in response to light. The magnitude of binding of transducin by the mutant pigment was about 40% of that of rhodopsin. Mutants E134R/R135E and CD 143-150 did not bind transducin. Mutants EF 237-249 and E134R/R135E were studied previously by the extra-MII assay (6) . The results of the light-scattering assay for these mutants are consistent with the results from the earlier extra-MII assay study (6) .

A nucleotide-release assay (Fig. 3 B) measured the ability of each pigment to catalyze the release of GDP from GGDP (Fig. 6). Photoactivated rhodopsin induced almost complete release of GDP from the nucleotide-binding pocket of transducin. The nucleotide release assay is a rigorous test as to whether a rhodopsin mutant is competent to induce the release of GDP from bound transducin. Even very low catalytic activity would be detected in this assay since it extends over minutes and measures the accumulated GDP release over the entire period of the assay. For rhodopsin, as calculated from the GGTPS formation rate, total GDP release is reached after only a few seconds. This is consistent with the time of formation of the R*GGDP complex, for which the light scattering 1/e time provides an upper limit (20) . Therefore, defects detected by this assay must be severe. For example, in mutant EF 237-249, only 34% of the GDP in the transducin pool was released after 5 min, while at least 40% of the mutant pigment preserved the ability to form a complex with transducin based on the magnitude of the light-scattering binding signal. It can be concluded that the rate of catalyzed GDP release from transducin was specifically slowed. A more direct comparison between the results of the nucleotide-release assay and the light-scattering assay is not possible and an intrinsic relationship does not necessarily exist between the values for GDP release and complex formation.

The photoproducts of mutants E134R/R135E and CD 143-150 do not bind transducin as shown by the light-scattering assay (Fig. 5). Therefore, they are not expected to induce GDP release ( Fig. 6and ). Mutant AB 70-71 had a small signal in the fluorescence GTPS-uptake assay (Fig. 4), and the significant GDP release in the nucleotide release assay of about 84% ( Fig. 6and ) is consistent with that activity.

Based upon the results discussed above, the mutant pigments can be grouped according to defects in discrete transducin activation steps depicted in Fig. 2 . Mutants E134R/R135E and CD 143-150 are profoundly defective in binding to GGDP. Mutant AB 70-71 may have an incomplete defect in binding GGDP or in release of GDP from the R*GGDP complex. Consequently, GDP release and GTPS uptake are diminished but detectable. These three mutants are defective to varying degrees in each of the three assays carried out. Mutants EF 237-249 and CD r140-152 are able to bind GGDP, but appear defective in catalyzing GDP dissociation from the mutant R*GGDP complex. A mutant defective in dissociation from GGTPS and G was not found among the five tested. It is likely that such a mutant phenotype will be rare based upon current understanding of the nucleotide-dependent switch regions of G from the x-ray crystal structure (31, 32, 33) , and from energetic considerations where the binding of GTP to the R*G(empty) complex has been shown to occur in a thermally activated state (34) .

The relationship between the properties of mutants CD 143-150 and CD r140-152 is particularly interesting. The 8-amino acid deletion from loop CD in mutant CD 143-150 causes an inability of the pigment to interact with transducin in all assays. No transducin activation occurs and no binding is detected in the light-scattering assay. However, replacement of the deleted amino acids by a sequence derived from the intradiscal domain in mutant CD r140-152 restores binding activity as previously reported (6) . It has been noted as shown in that the amino acid replacement retained the same amino acid residues at positions Asn and Phe(6) . Thus, the structure of loop CD appears to be important, although specific interactions between the central portion of loop CD, including positions 145 and 146, and transducin require further study. Only one point mutation in this loop has been reported, R147Q, which had normal ability to activate transducin in a GTPase assay (7) . Interestingly, a hydrophobic amino acid residue in the CD loop in the muscarinic cholinergic receptor was identified by alanine scanning mutagenesis to play a key role in G protein coupling (35) .

Detailed information is now available about the role of the conserved residue Glu at the cytoplasmic border of helix C (6, 8, 18, 25, 36) . Glu in the protonated form regulates a transducin binding and activation domain that must be made up of additional titratable surface groups (18, 25) . The present data provide direct support for the G protein activation model depicted in Fig. 2. The specific impairment of catalyzed GDP release in certain mutants confirms the existence of this discrete step in the activation pathway. This conclusion is supported by previous indirect evidence: 1) that binding of R* and nucleotide to transducin are antagonistic (37) , 2) that GDP dissociates the R*G(empty) complex (38, 39) , and 3) that R*-bound transducin cannot be solubilized in the absence of nucleotide (40) . The present findings provide a framework for further study to determine the extent of involvement of particular regions of the cytoplasmic surface of rhodopsin in mediating discrete steps in the transducin activation pathway.

  
Table: Amino acid sequence replacement in rhodopsin mutant CD r140-152


  
Table: Results of assay of light-dependent GDP release from G[GDP]



FOOTNOTES

*
This work was supported in part by the Deutsche Forschungsgesellschaft. 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.

§
Present address: Universitätsklinikum Charité, Medizinische Fakultät der Humboldt-Universität zu Berlin, Institut für Medizinische Physik und Biophysik, Ziegelstrasse 5-9, 10098 Berlin, FRG.

To whom correspondence should be addressed: Howard Hughes Medical Institute, Rockefeller University, Box 284, 1230 York Ave., New York, NY 10021. Tel.: 212-327-8288; Fax: 212-327-8370; E-mail: sakmar@rockvax.rockefeller.edu.

The abbreviations used are: G protein, guanine nucleotide-binding regulatory protein; BTP, 1,3-bis[tris-(hydroxymethyl)-methylamino]propane; DM, n-dodecyl--D-maltoside; DTT, dithiothreitol; GTPS, guanosine 5`- O-(3-thiotriphosphate); OG, n-octyl--D-glucoside; MI, metarhodopsin I; MII, metarhodopsin II; MOPS, 3-( N-morpholino)propanesulfonic acid; R*, photoactivated rhodopsin; G or G, transducin holoenzyme; G, -subunit of transducin; G(empty), transducin with empty nucleotide-binding pocket.


ACKNOWLEDGEMENTS

-We thank T. Zvyaga, M. Heck, K. Fahmy, C. Min, and R. R. Franke for assistance.


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