©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Transducin Activation by the Bovine Opsin Apoprotein (*)

(Received for publication, July 28, 1994; and in revised form, December 7, 1994)

Arjun Surya (1) (2) Kenneth W. Foster (2) Barry E. Knox (1)(§)

From the  (1)Department of Biochemistry and Molecular Biology, State University of New York Health Science Center, Syracuse, New York 13210 and the (2)Department of Physics, Syracuse University, Syracuse, New York 13244

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The interaction of the bovine opsin apoprotein with transducin in rod outer segment membranes was investigated using a guanyl nucleotide exchange assay. In exhaustive binding experiments, opsin activates transducin, with half-maximal exchange activity occurring at 0.8 mol of opsin/mol of transducin. The opsin activity was light-insensitive, hydroxylamine-resistant, unaffected by stoichiometric concentrations of retinaloxime, and more heat-labile than rhodopsin. The t of transducin activation in the presence of excess opsin was 8.5 min, compared with 0.7 min for metarhodopsin(II). The second-order rate constants were determined to be 0.012 pmol of guanosine 5`-(-thio)triphosphate (GTPS) bound per min/nM opsin and 0.35 pmol of GTPS bound per min/nM metarhodopsin(II). Opsin was able to activate more than one transducin, although there appeared to be a turnover-dependent inactivation of the apoprotein. Opsin showed a broad pH range (5.8-7.4) for optimal activity, with no activity in buffers of pH >9, whereas metarhodopsin(II) exhibited activity at pH >9. Regulation of opsin activity by stoichiometric amounts of retinal was observed, with inhibition by 11-cis-retinal and stimulation by all-trans-retinal. A model for opsin activity is proposed.


INTRODUCTION

Visual transduction in the rod cell is mediated by the photoreceptor, rhodopsin (for a recent review, see Hargrave and McDowell(1992)). The rhodopsin holoprotein consists of an 11-cis-retinylidene chromophore covalently attached to the apoprotein, opsin, in a protonated Schiff base with the -amino group of Lys. Upon absorbing a photon, the chromophore isomerizes from an 11-cis to an all-trans form, and rhodopsin undergoes a series of conformational changes, characterized by several photostationary intermediates, the most stable of which is metarhodopsin(II) ((max) = 380 nm). Accompanying the conformational changes are the deprotonation of the Schiff base, followed by the hydrolysis and release of the chromophore, yielding the apoprotein and all-trans-retinal (for a recent review, see Birge(1990)). Under bright illumination of the retina, the apoprotein is a major form of the photoreceptor. When the lighting conditions change from light to dark, the holoprotein is regenerated from the apoprotein by the binding of 11-cis-retinal (Dowling, 1960). Thus, there is a continual cycling of rhodopsin among the holoprotein, the metastable photointermediates, and the apoprotein.

Rhodopsin is a member of the family of G-protein-coupled transmembrane receptors and interacts with the retinal G-protein, transducin, in a light-dependent manner (reviewed in Hargrave and McDowell(1992)). Although there are a number of conformational states of light-activated rhodopsin, several lines of evidence suggest that metarhodopsin(II) is a key catalyst of transducin activation in the rod outer segment. First, the kinetics of formation of the metarhodopsin(II) coincide with the kinetics for interaction of transducin with metarhodopsin(II) containing membranes (Vuong et al., 1984). Second, the amount of metarhodopsin(II) relative to metarhodopsin(I), an earlier formed and less stable intermediate, is increased in the presence of transducin and decreased when transducin release is stimulated by addition of GTP (Hofmann et al., 1983). Third, conditions which stabilize or promote the formation of metarhodopsin(II) stimulate the light-dependent activation of transducin (Bennett et al., 1982; Emeis et al., 1982; Bornancin et al., 1989; Longstaff et al., 1986; Kibelbek et al., 1991). Thus, the main active conformation of rhodopsin, as defined by its ability to activate transducin and initiate the photoreceptor response to light, appears to be metarhodopsin(II).

It is not known whether other conformations of rhodopsin can activate transducin or regulate the cell's responsiveness. Physiological studies of bleaching adaptation have suggested that a non-metarhodopsin(II) conformation, possibly opsin, plays a role in controlling the sensitivity of the cell to light (Brin and Ripps, 1977; Pepperberg et al., 1978; Catt et al., 1982; Clack and Pepperberg, 1982; Jones et al., 1989; Corson et al., 1990; Cornwall et al., 1990; Jin et al., 1993). Biochemical studies of transducin activation by non-metarhodopsin(II) conformations have produced conflicting reports. Several groups have concluded that only metarhodopsin(II) is able to activate transducin (Hofmann et al., 1983; Longstaff et al., 1986; Kibelbek et al., 1991). These workers studied rhodopsin-transducin interactions after treatment of metarhodopsin(II) with hydroxylamine, a compound which causes the release of retinal from the apoprotein (Wald and Brown, 1953). Complete inhibition by hydroxylamine was found in experiments using either light-scattering, centrifugation, or transducin assays (Hofmann et al., 1983; Bornancin et al., 1989; Calhoon and Rando, 1985; Pepperberg et al., 1987; Kibelbek et al., 1991). On the other hand, Okada et al.(1989) have reported a hydroxylamine-resistant form, which they termed ``post-metarhodopsin(II),'' which stimulated GTP hydrolysis by transducin to levels similar to metarhodopsin(II). In more recent experiments, unregenerated opsin produced in transfected COS cells exhibited some ability to activate transducin at acidic pH (Cohen et al., 1993). Since different properties of the rhodopsin-transducin interaction are measured in the various assays, and the reaction conditions vary significantly, it is not apparent how to reconcile these findings.

In order to better define the conformations able to activate transducin, we have prepared the bovine opsin apoprotein in rod outer segment membranes and studied its interaction with transducin, using a guanyl nucleotide binding assay. We have found that the apoprotein is capable of stimulating the exchange of guanyl nucleotides by transducin in a light-independent manner. Furthermore, we have characterized and compared the opsin activity with the activity of metarhodopsin(II) and found significant differences in the rate, extent, sensitivity to hydroxylamine, and pH. We have also found that the opsin activity can be modulated by 11-cis- and all-trans-retinal and propose a model for the opsin structure to account for the observed activity.


EXPERIMENTAL PROCEDURES

Protein and Lipid Preparations

Rod outer segments were prepared in dim red light from frozen bovine retinae (J. A. Lawson) (Wilden and Kuhn, 1982), and peripheral membrane proteins were removed by treatment with urea (Shichi and Somers, 1978). The urea-stripped rod outer segment membranes are designated ``rhodopsin,'' and when exposed to light are termed ``metarhodopsin(II).'' The apoprotein, opsin, was prepared as follows (Bhattacharya et al., 1992). In the dark, frozen aliquots (5-10 mg) of rhodopsin were thawed and vortexed in 10 mM sodium phosphate, pH 7.0, containing 50 mM hydroxylamine. The concentration of the rhodopsin was adjusted to 0.1 mg/ml, and the suspension was then bleached for 10 min on ice, using a 300-watt projector (Kodak) fitted with a > 515-nm cut-off filter. All subsequent procedures were carried out at 4 °C under fluorescent light. The suspension was centrifuged at 100,000 times g, and the pellet was resuspended in 10 mM sodium phosphate, pH 6.5, containing 2% bovine serum albumin. Retinaloxime was separated from the opsin-containing membranes by five washes with this buffer. Bovine serum albumin was then removed by washing the pellet three times in 10 mM sodium phosphate, pH 6.5. Aliquots of opsin-containing membranes, hereafter designated ``opsin,'' were frozen and stored at -70 °C. UV-visible absorption spectra were recorded on aliquots of the membranes after solubilization in 1% dodecyl maltoside (Anatrace). Although residual retinal was usually absent (Fig. 1), there were preparations in which residual (<5%) retinaloxime was present. A range of 8-25 in the ratio of A/A was observed for the preparations used in the experiments reported here. However, no other difference in properties or activities were noted. The opsin concentration was estimated using an extinction coefficient of 81,200 M cm at 280 nm, which was derived from the ratio of A/A of 1.9-2.1 for urea-stripped membranes (Fig. 1), and an extinction coefficient at 500 nm of 40,600 M cm (Wald and Brown, 1953).


Figure 1: UV-visible absorption spectra of opsin. Bovine opsin apoprotein containing membranes were prepared from bleached rod outer segments. Spectra of opsin (solid line) and unbleached rhodopsin (broken line) were determined after the membranes were solubilized with 1% dodecyl maltoside. Removal of retinal chromophore is indicated by the absence of absorbance in the visible wavelengths.



Pigments were regenerated by incubating 5 µM opsin with 5-fold molar excess of either 11-cis-retinal, 11-cis-locked retinal (Bhattacharya et al., 1992), or all-trans-retinal, at 22-24 °C for 1 h in 10 mM sodium phosphate, pH 7.0. The chromophores were added from ethanol stock solutions, and the final ethanol concentration during regeneration was 2%. A UV-visible spectrum was taken after solubilization in 1% dodecyl maltoside. In the case of 11-cis-retinal, dark-light difference spectra were obtained after bleaching the sample for 1 min with projector light ( > 515 nm) to determine the extent of regeneration.

Transducin was purified from frozen bovine retinae (Bubis and Khorana, 1991). The purified transducin was dialyzed against 5 mM Tris-HCl, 100 mM NaCl, 5 mM MgCl(2), 50% glycerol (w/v), 10 mM 2-mercaptoethanol, and stored at -20 °C. Protein concentrations were determined by a modified Lowry assay using bovine serum albumin as standard (Peterson, 1977). The purified transducin bound between 0.9-1.0 mol of GTPS(^1)/mol of protein, as determined by exhaustive reaction with [S]GTPS in the presence of excess light-activated rhodopsin (see below).

Phospholipids were extracted from 50 bovine retinae according to the procedure of Weigand and Anderson(1982), except that retinal was first removed from the rod outer segment membranes by bleaching in the presence of 50 mM hydroxylamine followed by a washing step before extraction. Vesicles were made by solubilizing the phospholipids on ice in 2% CHAPS followed by overnight dialysis against 10 mM Tris-HCl, pH 7.0.

Assays

Stimulation of the binding of [S]GTPS to transducin by various membrane preparations was measured using a nitrocellulose filter binding assay (Wessling-Resnick and Johnson, 1987a). Reaction volumes were 250 µl, and assays were carried out at room temperature (22-24 °C). The reaction mixtures contained varying amounts of opsin or metarhodopsin(II) and 50-200 pmol of transducin in 10 mM Tris acetate, pH 7.4, 100 mM NaCl, 5 mM MgCl(2), 5 mM 2-mercaptoethanol, and 2 µM [S]GTPS (DuPont NEN). The specific activity of the [S]GTPS ranged from 600 to 3000 cpm/pmol and was estimated for each experiment by determining the total amount of radionucleotide that could bind to excess transducin. Typically, between 70 and 95% of the total radioactivity bound to transducin, depending on the age of the [S]GTPS.

Exhaustive Binding

In experiments to determine the total number of transducin molecules activated by opsin and metarhodopsin(II), reactions were allowed to proceed at room temperature for 120 min. For light-exposed samples, continuous illumination was provided with light from a projector ( > 515 nm). To remove unbound nucleotide, 100-µl aliquots of the reaction mix were diluted into 5 ml of ice-cold wash buffer (5 mM Tris-HCl, pH 7.4, 100 mM NaCl, 5 mM MgCl(2), 5 mM 2-mercaptoethanol) and immediately filtered through nitrocellulose filters (Millipore, HAWP 02500). The filters were removed, dried, and dissolved in scintillation fluid for liquid scintillation counting. All the data was corrected for [S]GTPS binding to transducin in the absence of opsin or metarhodopsin(II). This background was typically 5-8% of the binding obtained in the presence of excess metarhodopsin(II). Controls were also performed with rhodopsin in the dark. Each point was run in independent duplicates and each duplicate was spotted on duplicate filters. When assays were performed at a different pH, the buffers used were: pH 4-5.2, sodium acetate; pH 5-6.5, Na-MES; pH 6.5-8.5, Na-HEPES; pH 7.4-9.7 Na-TAPS; pH 7.0-9.0, Tris acetate; and pH 8.5-9.7, Na-CHES. For these buffers, the concentration was 20 mM, and pH was adjusted at room temperature. No buffer effects in the assays were detected. Three to five independent experiments were carried out at each pH. The mean value of the activity was determined for all experiments. The standard error of the mean varied from 3 to 20%, with a mean value of 12%. The background activity due to transducin was subtracted from all the data shown. This activity was negligible above pH 6.5, but became significant below pH 6.0 (Cohen et al., 1992). Due to the precipitation of magnesium salts from solution, experiments could not be performed at pH >9.7.

Initial Rate Measurements

Transducin (150 pmol) was added in the dark to 750 µl containing 10 mM Tris acetate, pH 7.4, 100 mM NaCl, 5 mM MgCl(2), 5 mM 2-mercaptoethanol, 2 µM [S]GTPS and either opsin or rhodopsin. After 1 min, the rhodopsin samples were exposed to light. At the times indicated in the figures, 50-µl aliquots were removed from the reaction, diluted into 10 ml of ice-cold wash buffer, and washed as described above. Duplicate samples were processed for each time point. Experiments with limiting opsin or metarhodopsin(II) used 0.2 µM or 0.024 µM, respectively, whereas those with excess opsin or metarhodopsin(II) used 1.2 µM or 0.2 µM, respectively.

Curve Fitting and Data Analysis

Curve fitting software (SigmaPlot, Jandel) based on the least squares method was used to fit the data for the various experiments. Data from the exhaustive binding experiments (Fig. 2) was fit to the Hill equation, which yielded estimates for the concentration of opsin or metarhodopsin(II) required to activate 50% of the transducin (K).


Figure 2: Titration of transducin activation by opsin. Opsin containing membranes, having either no detectable chromophore ((A/A = 25, closed triangles) or residual retinaloxime (A/A = 8, open triangles), were incubated with 60 pmol of transducin and 500 pmol of [S]GTPS (1100 cpm/pmol) in 250 µl for 2 h. Also, rhodopsin was assayed both in the light (open circles) and in the dark (closed circles). Transducin activation was determined by measuring the bound [S]GTPS using a nitrocellulose filtration assay. The solid lines are sigmoidal fits to the data using the Hill equation. Concentrations of opsin and metarhodopsin(II) required to activate 50% of the transducin were 41 ± 7 and 6.6 ± 0.7 pmol, respectively. The Hill coefficient was 1.9 for opsin and 1.2 for light-activated rhodopsin. The background activity from transducin alone, which was 6% of the maximal activity, was subtracted from all data shown.



The pH profile for opsin was generated by fitting the data to the equation below which represents the sum of two titratable groups with pK(a) values of k(1) and k(2), respectively,

where B(t) is the maximum value of the activity, and n(1) and n(2) are parameters related to the steepness of the titration curve. The pH profile for metarhodopsin(II) was fitted by adding a third ionizable group to the above equation to yield the following equation.

The initial rate data for both opsin and metarhodopsin(II) were fitted to single parameter exponential decay equations. These equations yielded values for the t for the reaction. The second-order rate constant was determined from the slope of the linear portion of the fit for the opsin or metarhodopsin(II) concentration in the linear range for transducin activation.


RESULTS

Opsin Activates Transducin

Opsin, essentially free of chromophore, was prepared by bleaching rhodopsin in the presence of excess hydroxylamine according to Bhattacharya et al. (1992). A UV-visible absorption spectrum of opsin solubilized in 1% dodecyl maltoside showed minimal residual retinal or retinaloxime, as judged by the absence of absorbance in the 365-380 nm regions (Fig. 1). Occasionally, traces of residual chromophore (<5% of the starting material) remained in the membranes. To show that the opsin-induced transducin activation was independent of these contaminants, a number of opsin preparations were used in the assays described below ( Fig. 2and Fig. 3).


Figure 3: Kinetics of transducin activation by opsin. Left panel, excess opsin (900 pmol, triangles) or metarhodopsin(II) (150 pmol, open circles) was added to 150 pmol of transducin and 750 pmol of [S]GTPS in 750 µl. At the indicated times reactions were terminated by dilution and aliquots were assayed for bound nucleotide. The inset shows early kinetic points. The time required to activate half the transducin was 8.6 ± 0.1 min for opsin and 0.67 ± 0.1 min for rhodopsin, as determined from a single parameter exponential fit (solid lines). Right panel, initial rate of transducin activation by limiting concentrations of opsin (0.2 µM). The time required to activate half the transducin was 0.012 pmol of transducin/min/nM opsin, as determined from a single parameter fit (solid line). The closed triangles represent opsin with A/A = 25, and open triangles represent opsin with A/A = 8.



Stimulation of guanyl nucleotide exchange on purified transducin by opsin, measured by the binding of [S]GTPS to transducin, was used to assay the interaction between opsin and transducin (Wessling-Resnick and Johnson, 1987a). The reaction of opsin with transducin was compared in two complementary sets of experiments. In the first set, an exhaustive binding reaction was performed using a fixed concentration of transducin, in order to measure the total number of interactions that occur. The opsin apoprotein caused a concentration-dependent stimulation of nucleotide exchange on transducin (Fig. 2). The concentration dependence was sigmoidal, with half-maximal activity occurring at 41 ± 7 pmol, which is 0.80 ± 0.1 mol of opsin/mol of transducin. Opsin preparations with residual retinaloxime (A/A=8, Fig. 2, open triangles) gave results that were identical to preparations having undetectable retinaloxime (closed triangles). Metarhodopsin(II) also stimulated transducin (Fig. 2); however, the half-maximal activity occurred at 6.6 ± 0.7 pmol which was 0.13 ± 0.01 mol of rhodopsin/mol of transducin. This specific activity is similar to the activities found earlier using different reaction conditions: limited bleaching of rhodopsin in reconstituted phospholipid membranes (Fung et al., 1981), limited GTPS (Wessling-Resnick and Johnson, 1987a), and in detergent-lipid micelles (Knox and Khorana, 1991). At the highest concentrations of opsin used, 84% of the transducin was activated, whereas metarhodopsin(II) was able to activate all of the transducin. The least active species tested was rhodopsin in the dark, for which significant transducin activation was observed only at the highest amounts (>200 pmol). The activity observed in the dark for rhodopsin may reflect the intrinsic activity of dark rhodopsin or may be due to artifacts arising from the presence of either unregenerated opsin or a minor fraction of rhodopsin that was exposed to light during isolation.

Kinetics of Opsin Activity

In the second set of experiments, initial rate measurements were made using excess opsin as determined from the saturating region of the exhaustive binding curve (Fig. 2). This experiment determined the maximal rate of opsin-induced guanyl nucleotide exchange. The apoprotein activated transducin in a time-dependent manner that was fit by a single-parameter exponential having a t value of 8.6 ± 0.1 min (Fig. 3, left panel). The reaction was essentially complete in 60 min. In contrast, metarhodopsin(II) activated transducin with a t value of 0.7 ± 0.1 min, and the reaction was complete by 5 min (Fig. 3, left panel). In order to determine the second-order rate constants, a second experiment was performed using limiting concentrations of opsin and metarhodopsin(II). The rate constants were found to be 0.35 pmol of transducin/min/nM metarhodopsin(II) (data not shown), and 0.012 pmol of transducin/min/nM opsin (Fig. 3, right panel). Again, a comparison of opsin preparations containing residual retinaloxime (open triangles) with chromophore-free preparations (closed triangles) showed no difference in transducin activation.

Even in reaction mixtures containing excess transducin, the opsin-induced nucleotide exchange was finished by 1 h at room temperature. The cessation of nucleotide exchange was not due to inactivation of transducin or depletion of nucleotide during the assay (data not shown). Unlike metarhodopsin(II), which decays at room temperature (Hofmann et al., 1983) and thus exhibits a decay in the activation of transducin, opsin did not lose the ability, when incubated at room temperature, to activate transducin (data not shown). Furthermore, preincubation of opsin at room temperature in the presence of transducin before addition of nucleotide, only caused a 10% decrease in subsequent nucleotide exchange assays (data not shown). Thus, the cessation of opsin-stimulated nucleotide exchange appears to require interaction and activation of transducin.

Effect of Light and Hydroxylamine

As shown above, the concentrations of opsin necessary to activate transducin were about six to eight times those for metarhodopsin(II). Experiments were performed to determine whether a small quantity of unbleached rhodopsin or residual metarhodopsin(II) contributed to the observed activity. Opsin activity was measured in the light and dark and no difference was detected (Table 1: Experiment A). Thus, the opsin activity is light-independent and has no contribution from unbleached rhodopsin. Hydroxylamine reacts rapidly with bleached rhodopsin to form retinaloxime and opsin (Hubbard and Kropf, 1958). Therefore, trace amounts of metarhodopsin(II) in the opsin preparation would be sensitive to treatment with hydroxylamine. Three experiments were performed using hydroxylamine. In the first, opsin membranes were extensively rewashed with hydroxylamine. No effect on transducin activation was observed (Table 1: Experiment B). In the second, opsin activity was assayed in the presence of different concentrations of hydroxylamine. Although transducin activation by metarhodopsin(II) was dramatically inhibited (Fig. 4; Hofmann et al., 1983), opsin was only slightly affected. In the third, opsin was incubated with hydroxylamine and all-trans-retinal to control for any possible stimulation of transducin activation by retinaloxime (Table 1: Experiment C). No effect on the activity was observed, confirming the observation that residual retinaloxime did not contribute to the opsin-induced nucleotide exchange. Thus, neither metarhodopsin(II) nor retinaloxime-opsin complexes contribute to the observed activity and indicate that the opsin activity is independent of retinoids. As another control, experiments were performed to determine whether rod outer segment phospholipids stimulate nucleotide exchange by transducin. No significant stimulation of nucleotide exchange was observed using phospholipid concentrations that were up to 10 times higher than used in the opsin experiments (Table 1: Experiment D). Thus nonspecific adsorption to the membranes cannot account for the observed activity. Taken together, the above experiments demonstrate that the opsin apoprotein is able to interact with transducin and accelerate nucleotide exchange.




Figure 4: Transducin activation by opsin is insensitive to hydroxylamine. Opsin (50 pmol, closed triangles) and rhodopsin (5 pmol, open circles), were incubated for 1 min with hydroxylamine at the indicated concentrations at pH 7.4 in the light ( > 515 nm). Transducin (85 pmol) and [S]GTPS (500 pmol) were then added to a final volume of 250 µl, and the reaction was carried out for 2 h. Light-dependent transducin activation by rhodopsin is severely inhibited by hydroxylamine, with 50% inhibition occurring at 0.4 mM, whereas opsin activity was inhibited less than 10% at 5 mM hydroxylamine. The line for the rhodopsin data is an exponential fit, whereas the line for the opsin data is a linear regression.



In order to determine whether the activity induced by the apoprotein depended on a specific three-dimensional conformation, the heat stability of opsin was investigated. Earlier studies had found that opsin and rhodopsin thermally denature at different temperatures, 56 and 72 °C, respectively (Miljanich et al., 1985; Khan et al., 1991). Therefore, opsin and rhodopsin were preincubated in the dark at 0, 70, or 100 °C and then used to activate transducin in an exhaustive binding assay at 25 °C (Table 2). No transducin activation was observed in heated opsin samples, whereas rhodopsin displayed 100% light-dependent activity when preincubated at 70 °C. Thus, the opsin conformation responsible for interaction with transducin is disrupted by heat. This experiment shows that residual rhodopsin does not contribute to the observed activity and also indicates that the opsin activity is not merely due to the activation of transducin by the cytoplasmic loop residues in a manner similar to that observed by the addition of synthetic peptides (Okamoto et al. 1991; Hamm et al., 1988). Rather, it suggests that a specific arrangement of the loops, necessary for interaction with transducin, occurs in opsin.



Activation of Multiple Transducins

To determine whether opsin is able to interact with and activate more than one transducin, a fixed amount of opsin (0.2 µM) was assayed with different amounts of transducin (0.04-0.68 µM). A linear dependence of guanyl nucleotide exchange was observed (Fig. 5). At transducin levels above 0.30 µM, multiple turnovers were observed, with 2.4 transducins activated per opsin at the highest amount tested. For comparison, metarhodopsin(II) was able to stimulate about 50-100 turnovers (Fung et al., 1981; also see Fig. 2). Opsin was less effective than the same amount of metarhodopsin(II) at each transducin concentration as can be seen by the difference in slope, which was 1.0 for metarhodopsin(II) and 0.7 for opsin. Thus, only about 70% of a given amount of transducin could be induced by opsin to bind GTPS. This was typically the case, although at extremely high opsin concentrations, all of the transducin could be activated (e.g.Fig. 3). The direct binding of opsin and transducin was investigated using a centrifugation assay (Kuhn, 1980). Through many attempts, no opsin-specific binding was observed, although metarhodopsin(II) binding was clear (data not shown; also see Pepperberg et al.(1987)). From these experiments and the high concentrations needed to see complete activation, it appears that the binding of opsin and transducin is of low affinity.


Figure 5: Turnover of transducin by opsin. 50 pmol of opsin (closed triangles) or metarhodopsin(II) (open circles) and 1000 pmol of [S]GTPS was assayed with varying amounts of transducin for 2 h in 250 µl. At the highest transducin concentrations tested, 2.4 mol of transducin were activated per mol of opsin. Linear regression (solid lines) gave slopes of 0.7 for opsin and 1.0 for metarhodopsin(II) induced activity.



pH Dependence of Opsin Activity

Previous work has shown that the equilibrium of photostationary states, and thus the active conformation of rhodopsin, is sensitive to pH (Mathews et al., 1963; Wong and Ostroy, 1973; Sakmar et al., 1989; Weitz and Nathans, 1992). These experiments suggest that the conformation(s) of rhodopsin, and potentially opsin, are regulated by the ionization state of certain amino acid side chains. Moreover, Cohen et al.(1993), report opsin activity only under acidic conditions, unlike conditions used above. Therefore, the effect of pH on the opsin induced transducin activation was assayed over the pH range 4.0-9.7. The activity showed a broad maximum in the range of 5.9-7.4 and dropped steeply outside this range (Fig. 6). The profile could be fit by assuming two titratable groups, with pK(a) values of 5.3 and 7.9. The loss of opsin activity at alkaline pH was reversible, since a 2-h incubation at pH 9.7, followed by an assay at pH 6.5, gave the usual activity (data not shown).


Figure 6: pH profile of transducin activation by opsin. Opsin (30 pmol, closed triangles) and metarhodopsin(II) (3.8 pmol, open circles) were assayed in six different buffers, pH 4-10, in 250 µl containing 500 pmol of [S]GTPS and 100 pmol of transducin. The pH activity profiles of opsin shows a maximum around pH 6.5 with a steep decline above pH 8.0. The activity of transducin alone, which has been subtracted from the data, rises below pH 6.0, and at pH 5.0 about 20% of the transducin has bound [S]GTPS in 2 h.



In contrast, the profile for metarhodopsin(II) was complex, with activity observed at pH >8.5 (Fig. 6). The reduced extent of nucleotide exchange at high pH was caused by a slower rate of transducin activation at pH 9.7 compared with pH 6.5 (the ratio of the rates was found to be 0.64 (data not shown)). The distinct drop in activity around pH 8.0 followed by a recovery at higher pH was observed in three buffers. The metarhodopsin(II) profile in Fig. 6was fitted assuming that three titratable groups underwent changes in their ionization state. The data in the region below pH 8.0 were fitted by two groups, with pK(a) values of 5.3 and 7.7, similar to the values found for opsin. For data in the region of alkaline pH, the fit with a pK(a) of 8.2 was not as good. In summary, pH profiles present another property in which the activities of opsin and metarhodopsin(II) differ.

Modulation of Opsin Activity by Retinal

The data presented thus far show an active apoprotein, whereas the least active conformation was rhodopsin in the dark. This suggests that 11-cis-retinal may modulate the activity of the apoprotein. To investigate this point, opsin was incubated with either 11-cis-retinal or 11-cis-locked retinal. Approximately 90% of the opsin was regenerated as determined from light-dark difference spectra of the 11-cis retinal incubated opsin samples. Transducin activation was assayed in the dark using the regenerated samples. As a control, assays were also performed in the light. In the dark, 11-cis- and 11-cis-locked retinal reduce transducin activation by 85% of that due to the apoprotein alone (Fig. 7A). In the light, the 11-cis-retinal regenerated control showed activity equivalent to metarhodopsin(II), whereas the locked analog showed a minor stimulation above the dark sample confirming an earlier report (Bhattacharya et al., 1992; data not shown). Thus the interaction of 11-cis-retinal with opsin and the reformation of rhodopsin reduces the activity of the apoprotein. The residual activity is not due to isomerization, either thermally or light-induced, since both 11-cis- and 11-cis-locked retinal exhibit the same activity. Rather it is most likely due to either unregenerated opsin or some intrinsic activity of rhodopsin in the dark.


Figure 7: Retinal modulates the opsin activity. Opsin was incubated in the dark with 5-fold excess of 11-cis-retinal, 11-cis-locked retinal or all-trans-retinal. A, 20 pmol of regenerated pigment was assayed with 60 pmol of transducin and 500 pmol of [S]GTPS in 250 µl for 2 h in the dark. Both 11-cis- and 11-cis-locked retinal inhibit transducin activation by opsin in the dark. B, 4 pmol of regenerated material or metarhodopsin(II) was assayed as in A. All-trans-retinal caused a dramatic enhancement of the opsin activity in the dark, to levels comparable with metarhodopsin(II).



In contrast, regeneration of opsin with all-trans-retinal results in a dramatic stimulation of nucleotide exchange (Fig. 7B). The activity was comparable with metarhodopsin(II) and could be measured immediately after the addition of stoichiometric amounts of all-trans-retinal. Previously, stimulation of opsin activity by all-trans-retinal has been reported (Yoshizawa and Fukada, 1983; Cohen et al., 1992) although at significantly lower levels than found here. Thus, it appears that a metarhodopsin(II)-like conformation is formed upon the addition of all-trans-retinal to the apoprotein. In summary, 11-cis-retinal inhibits the activity of the apoprotein, whereas all-trans-retinal enhances it.


DISCUSSION

Using opsin in rod outer segment membranes and purified transducin, we have found that the bovine opsin apoprotein activates nucleotide exchange on transducin in a light- and hydroxylamine-insensitive manner. From these experiments, as well as the unique characteristics of the activity, we have shown that the activity is not due to contaminating chromophore containing photoproducts. It is possible to reconcile various other reports and our main findings. First, Okada et al.(1989) have reported a hydroxylamine-resistant stimulation of GTP hydrolysis on transducin by a post-metarhodopsin(II) species. The rates of transducin activation were comparable with that of metarhodopsin(II), in contrast to those here, in which opsin had a 10-fold slower rate. The discrepancy may be due to saturating concentrations of post-metarhodopsin(II) which were used, their more indirect and slower assay of GTP hydrolysis (Vuong and Chabre, 1990), or residual all-trans-retinal in the preparations. Second, although opsin activates transducin, the binding interaction of these two proteins appears substantially weaker than that between transducin and metarhodopsin(II), since transducin remains in the supernatant following centrifugation with opsin membranes (data not shown; Pepperberg et al., 1987). This may also explain why light-scattering experiments, which require tight association, failed to detect apoprotein interaction with transducin (Hofmann et al., 1983; Bruckert et al., 1988). Third, activation of transducin by post-metarhodopsin(II) products was observed by Kibelbek et al.(1991) who attributed this activity to the reversibility of the metarhodopsin(II)-metarhodopsin(III) transition. However in our experiments, these photoproducts were not present, since we have removed the chromophore. Furthermore, the activity is insensitive to hydroxylamine. In addition, several reports contain data in which apparent opsin activity was attributed to background nucleotide exchange (Wessling-Resnick and Johnson, 1987b) or to the retinaloxime in the preparations (Yoshizawa and Fukada, 1983; Fukada et al., 1982). The experiments reported here clearly eliminate these explanations. Finally, the lipid may affect the opsin activity, since Rando and colleagues (Calhoon and Rando, 1985; Longstaff et al., 1986) failed to observe apoprotein activity with opsin reconstituted into egg phosphatidylcholine. The ability of metarhodopsin(II) to activate transducin is very sensitive to the hydrophobic environment (^2)(Knox and Khorana, 1991), and it additionally appears that the apoprotein may exhibit similar sensitivity. (^3)Therefore the lipid environment may be crucial to apoprotein structure and function. This may help to explain why a loss in activity was observed when retinaloxime-containing preparations were extracted with organic solvents, which would alter the lipid composition of the membrane (Yoshizawa and Fukada, 1983; Fukada et al., 1982). This point may have relevance to the observations of Cohen et al. (1993) in which no apoprotein activity was observed at pH 6.7, and some activity was observed at pH 6.1. In those experiments, COS cell membranes containing transfected opsin were used for assay, which might have altered properties when compared with opsin in rod outer segment membranes. Therefore, we conclude that opsin is able to activate transducin under physiological conditions, but with a significantly lower activity than metarhodopsin(II).

One of the striking differences between transducin activation by opsin and metarhodopsin(II) is the dependence of transducin activation on pH. Opsin shows a profile with peak activity occurring in the range 6.0-7.4 and a decline in activity above this range. By pH 9, no activity can be detected. On the other hand, metarhodopsin(II) exhibits a more complex behavior, having a profile similar to opsin below pH 7.5, but retaining up to 70% of the maximal activity at higher pH. Rhodopsin expressed in COS cell membranes appears to be less active at high pH than rhodopsin in rod outer segment membranes (Cohen et al., 1992; Fahmy and Sakmar, 1993). For both opsin and metarhodopsin(II), the effects of high pH are fully reversible. Thus, the active conformation of both opsin and metarhodopsin(II) may contain one or more titratable groups that control the transition from active to less active. However, the sensitivity to pH is dramatically different, and the nature of the amino acid residue(s) that respond differently to pH changes in opsin versus metarhodopsin(II) are not known. The fact that opsin is inactive at higher pH, and metarhodopsin(II) is not, appears to rule out artifacts due to surface membrane effects. A number of workers have pointed to the importance of charges located at or near Lys, the site of retinal attachment, for transducin activation (Longstaff et al., 1986; Arnis and Hofmann, 1993; Cohen et al., 1993; Fahmy and Sakmar, 1993). Although the major difference between opsin and metarhodopsin(II) involves the removal of the chromophore, it is not clear whether any significant changes in protonation state, accounting for the activity difference, occur at Lys as the pH is varied from pH 7 to 9. There have been several reports concerning pH-sensitive conformations of rhodopsin. Koutalos(1992) has shown that there is a spectral blue shift in the dark in rhodopsin at high pH, leading to a proposal that chromophore-protein interactions are sensitive to pH. Our experiments suggest that there may be similar conformational changes associated with high pH occurring in metarhodopsin(II). In fact, Arnis and Hofmann(1993) have proposed two conformations of metarhodopsin(II) that are related by the protonation of an unidentified residue and may have different abilities to interact with transducin.

It appears that the binding of transducin to opsin is the rate-limiting step in activation, rather than other steps that occur after opsin and transducin interact. In control experiments, preincubation of opsin and transducin, in the absence of guanyl nucleotide, for 2 h at room temperature, had little effect on the subsequent activation of transducin (data not shown). Furthermore, we were unable to isolate an opsin-transducin complex by centrifugation (data not shown; Pepperberg et al., 1987).

The nature of the binding site for transducin on metarhodopsin(II) has been studied by site-directed mutagenesis (Khorana, 1992) and by peptide competition (Konig et al., 1989). From these studies, a structural model has emerged in which the transducin binding site on rhodopsin is created by three cytoplasmic loops. This interaction leads to high affinity binding, and subsequent activation of transducin. In order to explain the activation of transducin by opsin, we propose that one of the three loops involved in the interaction is in a different relative position in opsin compared with metarhodopsin(II). According to this hypothesis, there will be a lower interaction energy of opsin with transducin and hence a lower apparent affinity leading to fewer transducin molecules activated. This model can also explain the regulation of opsin activity by retinal. The experiments presented here show that when 11-cis-retinal binds to the apoprotein, reforming rhodopsin, a less active conformation is formed. In the model, the binding of 11-cis-retinal to opsin disturbs the relative position of the two loops, contributing to the transducin binding site, and further reduces the binding energy, effectively preventing transducin activation in the dark. A different effect was observed with all-trans-retinal, which enhanced opsin activity to the level observed for metarhodopsin(II). Binding of all-trans-retinal to opsin could move the loop to the position occupied in metarhodopsin(II), reforming the high affinity binding site. Although the model explains the basic observations, certain properties of opsin regenerated with all-trans-retinal are different from metarhodopsin(II) (Cohen et al., 1992; Hofmann et al. 1992), (^4)suggesting that the conformations are distinct. Future work will be directed to determining the cytoplasmic loops involved in the opsin activation of transducin.

Although the activation of transducin by opsin has structural implications, as discussed in the model, the physiological relevance of an active apoprotein remains uncertain. Numerous groups (Barlow, 1964; Brin and Ripps, 1977; Pepperberg et al., 1978; Catt et al., 1982; Clack and Pepperberg, 1982; Jones et al., 1989; Corson et al., 1990; Cornwall et al., 1990; Jin et al., 1993) have implicated the apoprotein in bleaching adaptation (Rushton, 1961). The results presented in our work provide several possible explanations for this process. In one scheme, opsin could desensitize the photoreceptor by triggering the same adaptation mechanisms as metarhodopsin(II), through transducin. This would explain both the excess adaptation beyond the loss of quantum catch and recovery of the photoreceptor when opsin recombines with retinal or its analogs. In another scheme, opsin could affect photoreceptor sensitivity by competing for the available transducin molecules, thus limiting metarhodopsin(II) interaction with transducin. In both schemes, the control of apoprotein activity by retinal would provide a means for cycling through the various sensitivities observed in bleaching adaptation.


FOOTNOTES

*
Partial support of this work was obtained from National Institutes of Health Grant EY09409 (to B. E. K.) and a Basil O'Connor March of Dimes Award. 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.

§
To whom correspondence should be addressed. Tel.: 315-464-8719; Fax: 315-464-8750; knoxb{at}VAX.cs.hscsyr.edu.

(^1)
The abbreviations used are: GTPS, guanosine 5`-(-thio)triphosphate; 11-cis-locked retinal, cyclohexatrienylidene; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; MES, 4-morpholineethanesulfonic acid; TAPS, 3-{[2-hydroxy-1,1-bis (hydroxymethyl)ethyl]amino}-1-propanesulfonic acid; CHES, 2-(cyclohexylamino)ethanesulfonic acid.

(^2)
B. E. Knox, S. Bhattacharya, K. Ridge, and H. G. Khorana, manuscript in preparation.

(^3)
A. Surya and B. E. Knox, unpublished observations.

(^4)
A. Surya and B. E. Knox, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank Drs. Kevin Ridge, Dorine Starace, Tomoko Nakayama, and Harvey Penefsky for helpful discussions throughout this work and Drs. David Turner, Enrico Nasi, Robert Barlow, Wes Corson, and Ruth Yokoyama for critical reading of the manuscript.


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