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J. Biol. Chem., Vol. 275, Issue 26, 19713-19718, June 30, 2000

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Signaling States of Rhodopsin

RETINAL PROVIDES A SCAFFOLD FOR ACTIVATING PROTON TRANSFER SWITCHES^*

Christoph K. Meyer, Monika Böhme, Andreas Ockenfels^§¶, Wolfgang Gärtner^§, Klaus Peter Hofmann, and Oliver P. Ernst

From the  Institut für Medizinische Physik und Biophysik, Humboldt Universität zu Berlin, Universtitätsklinikum Charité, Schumannstrasse 20-21, 10098 Berlin and the ^§ Max-Planck-Institut für Strahlenchemie, Stiftstrasse 34-36, 45470 Mülheim an der Ruhr, Germany

Received for publication, January 27, 2000, and in revised form, March 24, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The G-protein-coupled receptor rhodopsin is activated by photoconversion of its covalently bound ligand 11-cis-retinal to the agonist all-trans-retinal. After light-induced isomerization and early photointermediates, the receptor reaches a G-protein-dependent equilibrium between active and inactive conformations distinguished by the protonation of key opsin residues. In this report, we study the role of the 9-methyl group of retinal, one of the crucial steric determinants of light activation. We find that when this group is removed, the protonation equilibrium is strongly shifted to the inactive conformation. The residually formed active species is very similar to the active form of normal rhodopsin, metarhodopsin II. It has a deprotonated Schiff base, binds to the retinal G-protein transducin, and is favored at acidic pH. Our data show that the normal proton transfer reactions are inhibited in 9-demethyl rhodopsin but are still mandatory for receptor activation. We propose that retinal and its 9-methyl group act as a scaffold for opsin to adjust key proton donor and acceptor side chains for the proton transfer reactions that stabilize the active conformation. The mechanism may also be applicable to related receptors and may thus explain the partial agonism of certain ligands.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The retinal photoreceptor rhodopsin is one of the archetypes of the G-protein-coupled receptor superfamily. It is composed of the apoprotein opsin comprising seven transmembrane helices (TMs)¹ and the chromophore 11-cis-retinal, which is covalently bound to Lys²⁹⁶ in TM7 via a protonated Schiff base (SB), keeps the receptor in the inactive conformation, and acts as a highly effective inverse agonist. Following the absorption of a photon, the retinal isomerizes to a strained all-trans-conformation (1), which induces a series of conformational rearrangements of the opsin moiety. The final product of this reaction sequence is the active conformation (R*) that contains all-trans-retinal as a covalently bound agonist and is capable of catalyzing the activation of the retinal G-protein transducin (G_t) (for reviews see Refs. 2-4).

These events can be summarized as shown in Fig. 1A). After cis-trans-isomerization and initial, short lived intermediates, illuminated rhodopsin progresses from the lumirhodopsin form to the first long lived state, metarhodopsin I (meta I). Up to and including meta I, the SB of the pigment remains protonated. Deprotonation of the SB and protonation of its counterion Glu¹¹³ mark the transition from the meta I (_max = 478 nm) to the metarhodopsin II (meta II) conformation (5), which is characterized by a strongly blue-shifted absorbance maximum (_max = 380 nm). SB deprotonation is known to be necessary for interaction with G_t (6-8) and is accompanied by the uptake of a proton from the aqueous solution (9-11). pH rate profiles of G_t activation and proton uptake measurements have shown the opsin residue Glu¹³⁴ to be an important mediator of the uptake or even the acceptor of this proton (12, 13). Based on computer simulations and mutagenesis studies, similar results have been obtained for the _1B-adrenergic receptor (14, 15).

View larger version (44K): [in this window] [in a new window]   Fig. 1.   Photoactivation schemes of rhodopsin and 9-demethyl rhodopsin. A, rhodopsin (see text); B or C, 9-demethyl rhodopsin. In B, altered chromophore-protein interactions prevent rhodopsin from reaching the normal active form, instead yielding a photoproduct with reduced activity. In C, the equilibrium between active and inactive forms is shifted in favor of the inactive form, because of the only partial agonism of all-trans-9-dm retinal, also leading to reduced Gt activation. According to our data, scheme C is correct.

The current understanding of this sequence is that although the initial phase of the photoactivation cascade is dominated by steric mechanisms (16), the later stages are characterized by proton transfer and proton uptake processes (10, 17, 18). The "steric trigger" (16, 19) and "salt bridge breaking" (20) concepts have been formulated to describe this in graphic terms.

The 9-methyl group of the retinal, which has been shown to interact directly with the Gly¹²¹ residue of opsin in the middle of TM3 (21, 22), is known to be one of the crucial steric determinants of photoactivation. Removal of this group (see Fig. 2) causes steric defects that reduce the ability of the pigment to progress to the meta II state and impair its catalytic efficiency (23). Fig. 1, B and C, summarize the two conceivable reaction pathways that may be adopted by the defective pigment formed with this chromophore. Either a breakdown of the steric mechanisms prevents light-activated 9-demethyl (9-dm) rhodopsin from reaching the protonation equilibrium and instead causes the formation of a weakly active product (b) or the lack of retinal's 9-methyl group allows the photoreaction to proceed to the protonation equilibrium, but shifts this equilibrium in favor of the inactive conformation analogous to meta I (c). Experimental results until now have not been able to decide between the two possibilities.

View larger version (12K): [in this window] [in a new window]   Fig. 2.   Structural formula of 9-demethyl-11-cis-retinal.

In the present study, we provide proof that c is the correct scheme. We show that illuminated 9-dm rhodopsin is in an equilibrium between SB-protonated and SB-deprotonated forms that is strongly shifted toward the protonated form when compared with the normal pigment. Further, we show that this equilibrium is affected by transducin and pH much like the meta I  meta II equilibrium of the wild type pigment. We also find that many of the defects of this pigment can be rescued by the replacement mutation E134Q,² which has previously been shown to mimic the effects of proton uptake (12, 13, 24, 25).

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- Bovine eyes were purchased from the local slaughter house. 11-cis-retinal was a gift from Rosalie Crouch (University of South Carolina and the National Eye Institute, National Institutes of Health). Monoclonal antibody, rho 1D4, specific for the carboxyl terminus of rhodopsin (26), was kindly provided by Mark Hirschel (National Center for Research Resources and the National Culture Center, Minneapolis, MN) and was coupled to CNBr-activated Sepharose 4 Fast Flow (Amersham Pharmacia Biotech) largely following the manufacturer's instructions. n-Dodecyl--D-maltoside (dodecyl maltoside, DM) was purchased from Biomol (Hamburg, Germany). Reagents for tissue culture were from Life Technologies, Inc. or Sigma. COS-1 cells (ATCC No. CRL-1650) were from American Type Culture Collection (Manassas, VA). Mammalian expression vectors for wild type opsin and the rhodopsin mutant E134Q (27) were kindly provided by Dr. T. P. Sakmar (Rockefeller University, New York). Complete^TM protease inhibitor mixture was from Roche Molecular Biochemicals and was used at a concentration of 1 tablet/75 ml of buffer.

Expression and Preparation of Recombinant Rhodopsins-- Expression of opsin genes was performed basically as described (28) with the following modifications: COS-1 cells were grown in monolayer in plastic cell culture roller bottles (surface area 850 cm²) under a humidified atmosphere with 5% CO₂ at 37 °C. Growth medium was Dulbecco's modified Eagle's medium containing 4.5 g/liter D-glucose and 0.11 g/liter sodium pyruvate, supplemented with 100 µg/ml streptomycin, 100 units/ml penicillin, 2 mM L-glutamine, and 10% (v/v) heat-inactivated fetal bovine serum. At 80-95% confluence, transient transfection of the cells was initiated using a transfection mixture composed of 150 µg of plasmid DNA, 6 ml of 1 M Tris buffer (pH 7.4), 48 ml of serum-free growth medium, and 6 ml of DEAE-dextran stock solution (2.5 mg/ml in Dulbecco's modified Eagle's medium). After 5.5 h, the transfection mixture was replaced with 75 ml of 0.1 mM chloroquine in growth medium for 90 min. The cells were washed with Dulbecco's modified Eagle's medium and further incubated in growth medium until harvest 64-68 h later. By incubation with 1 mM Na₂EDTA in phosphate-buffered saline (137 mM NaCl, 2.7 mM KCl, 8.1 mM Na₂HPO₄, 1.5 mM KH₂PO₄, pH 7.0) for 10 min, cells expressing the apoproteins were detached from the plastic surface. The cell suspension was supplemented with protease inhibitors and incubated with 30 µM (final concentration) 11-cis-retinal or 9-demethyl-11-cis-retinal, respectively, for 2-4 h at 4 °C to reconstitute the pigment. The rhodopsins were purified by immunoaffinity adsorption based on a procedure described by Oprian et al. (29). Briefly, cells were solubilized by the addition of dodecyl maltoside (1% (w/v) final concentration), and nuclei were removed by centrifugation. Pigments from two roller bottles were incubated with 1D4-Sepharose and washed twice with 0.03% (w/v) DM in phosphate-buffered saline and once with elution buffer (0.015% (w/v) DM, 10 mM BTP, pH 6.0; 50 ml/wash). Elution of the pigments from the 1D4-Sepharose was carried out in elution buffer supplemented with 100 µM peptide corresponding to the carboxyl-terminal 18 amino acids of rhodopsin.

Preparation of Transducin (G_t)-- G_t was purified from bovine retinae essentially as described (30) and stored in 20 mM BTP, pH 7.1, 130 mM NaCl, 1 mM MgCl₂, 1 mM dithiothreitol.

Preparation of 9-Demethyl Retinal-- The first three steps of the synthesis of 9-demethyl-retinal were performed according to the following procedure of Van den Tempel and Huisman (31): oxidation of -ionone with sodium hypochlorite, reduction of the resulting acid with LiAlH₄, followed by reoxidation with MnO₂ resulted in 3-(2,6,6-trimethyl-cyclohexene-1-yl)-2-propenal. Elongation of the chain was achieved by Wittig-Horner coupling of the aldehyde with 2-(diethylphosphono)-acetonitrile and subsequent reduction with diisopropylaluminiumhydride, followed by a second Wittig-Horner coupling with 4-(diethylphosphono)-3-methyl-2-butene-nitrile. Subsequent reduction of the nitrile with diisopropylaluminiumhydride formed 9-demethyl-retinal. The all-trans isomer was separated by silicagel column chromatography (n-hexane/15% diethylether). Irradiation of a solution of the all-trans isomer of 9-demethylretinal in acetonitrile (1 mg/ml, GE EHJ 250 W with a Schott KG5-Filter, 120 min) resulted in a photostationary mixture of six geometric isomers (20% 11-cis isomer), which were separated by isocratic preparative HPLC (Kromasil Si-100-5 µ, n-hexane, 7% diethylether) and characterized by ¹H NMR spectroscopy. The purity of the isolated 11-cis isomer was verified with analytic HPLC and was greater than 95%.

UV-visible Spectroscopy-- The buffers used were 130 mM NaCl, 1 mM MgCl₂, 0.01% (w/v) DM, and 20 mM MES (for pH 5.5, 6.0, and 6.5) or BTP (for pH 6.5, 7.0, 7.5, and8.0) adjusted to pH. The spectra at pH 6.5 were measured with both buffers. Acid denaturation of the pigment was performed by adding 10% of the sample volume of 1 M HCl. Samples were illuminated for 10 s using a fiber optic light source equipped with a >480-nm longpass and a heat protection filter. The concentration of rhodopsin was determined spectrophotometrically using  = 42,700 mol¹ cm¹ for all pigments.

Assay of G_t Activation-- Experiments were performed essentially as described (12, 32). The instrument settings were _exc = 300 nm and _em = 345 nm, and the integration time was 2 s at 20 °C. Pigment concentration in samples was 2 nM with 0.01% (w/v) DM and 250 nM G_t in 20 mM BTP, pH 7.5, 130 mM NaCl, and 1 mM MgCl₂. Illumination of the sample using the same equipment as for UV-visible spectroscopy was maintained throughout the experiment. Activation of G_t was started by adding GTPS into the cuvette. Traces were corrected for the dilution because of the addition of GTPS and normalized relative to the fluorescence intensity before the addition. Initial slopes of the traces were determined by linear regression to the first 15% of the amplitude gain.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Illuminated 9-Demethyl Rhodopsin Shows a Strongly Reduced but pH-dependent Schiff Base Deprotonation-- Opsin was expressed in COS cells, reconstituted with 11-cis-9-demethyl-retinal or 11-cis-retinal, and purified in dodecyl maltoside yielding up to 100 µg of purified rhodopsin/cell culture roller bottle with spectral ratios of 1.9 (A₂₈₀/A₄₆₄ for 9-dm rhodopsin) to 1.7 (A₂₈₀/A₅₀₀ for rhodopsin). UV-visible spectra of 9-dm wild type (wt) rhodopsin at pH 5.5, 6.5, and 7.5 are shown in the main panel of Fig. 3. The dotted line is the spectrum taken of the sample at pH 6.5 before illumination. It displays the visible _max at 464 nm, in accordance with data reported in the literature (22). The dark spectra of the samples at pH 5.5 and 7.5 are nearly identical (data not shown). The solid lines are the spectra taken after illumination. At pH 7.5, the spectrum shows the formation of a photoproduct with the visible _max at 466 nm. This species is slightly red-shifted from the dark form and has been described before (22). The spectrum also shows an increase of absorption around 380 nm. The spectra taken after illumination at pH 6.5 and 5.5 clearly show this to be a second species with a _max of 380 nm. This species is in a pH-dependent equilibrium with the 466-nm form and is favored at acidic pH.

View larger version (24K): [in this window] [in a new window]   Fig. 3.   Spectra of recombinant 9-dm wild type rhodopsin before and after illumination, at pH 5.5, 6.5, and 7.5. Dark spectra at different pH values were nearly identical, only the dark spectrum taken at pH 6.5 is shown (dotted line). The solid lines are spectra taken after illumination, at the pH indicated. The dashed line is the spectrum of the illuminated pH 5.5 sample after acid denaturation performed by adding 10% (v/v) 1 M HCl. The inset shows a similar experiment performed on wt rhodopsin bearing normal retinal (dotted line, spectrum before illumination; solid lines, spectra after illumination). Samples contained 250 and 350 nM pigment, respectively.

The dashed line is a spectrum taken after the illuminated pH 5.5 sample was denatured with 1 M HCl (10% of sample volume added). It shows a _max of 432 nm, characteristic of an intact, protonated SB. Spectra taken of dark samples denatured with acid display the same _max (data not shown). The shift in _max between the 432 nm observed after acidification and the 440 nm found with normal retinal roughly corresponds to the shift between the absorption maxima of the free retinals in solution (22).

An absorption maximum around 380 nm is indicative of a deprotonated Schiff base. From its UV-visible absorption spectrum, the 380-nm absorbing species of 9-dm rhodopsin is similar to meta II, the Schiff base-deprotonated intermediate of light-activated rhodopsin, which also shows a _max of 380 nm. Also, the pH dependence of this species resembles that found for bovine rhodopsin in disc membrane preparations (9, 11), where meta II formation is increased at acidic pH.

However, there is an important difference; when native rhodopsin or recombinant wild type opsin regenerated with normal retinal are purified in dodecyl maltoside, the formation of meta II following illumination is increased such that the meta I  meta II equilibrium becomes almost independent of pH and temperature (Fig. 3, inset, and Ref. 10). Hence, when compared with the normal pigment, SB deprotonation by 9-dm rhodopsin is clearly deficient.

Purified 9-dm rhodopsin prepared from native, i.e. not expressed, opsin also shows pH-dependent SB deprotonation after illumination (data not shown). Interestingly though, SB deprotonation is less than with the expressed pigment.

Transducin Stabilizes the Schiff Base Deprotonated Form of Illuminated 9-Demethyl Rhodopsin in a GTPS-sensitive Way-- Fig. 4 shows a sequence of spectra taken before and after illumination of 9-dm rhodopsin in the presence of 500 nM G_t and after addition of 50 µM GTPS. The spectra labeled "dark" and "illuminated" in Fig. 4A show that the presence of G_t stimulates the formation of the 380-nm absorbing species to a level higher than that observed in the absence of Gt, which is seen in Fig. 4B. This effect is similar to a static measurement of "extra meta II," an assay commonly applied to rhodopsin in disc membranes (6, 32); G_t stabilizes meta II at the expense of its tautomeric SB-protonated form meta I. The result shows that 9-dm rhodopsin can form a 380-nm absorbing species that is capable of forming a stable complex with G_t.

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Spectra of recombinant 9-dm wild type rhodopsin with and without Gt. A, sample containing 9-dm rhodopsin and Gt. Spectra were taken before (dark) and after illumination, as indicated. The spectrum labeled +GTPS was taken after this nonhydrolyzable GTP analog was added to the illuminated sample. Note the high absorption around 280 nm because of the presence of Gt. B, sample containing only 9-dm rhodopsin, without Gt. Spectra were taken before and after illumination, as indicated. pH 7.5 in both samples.

In the case of native rhodopsin, this complex catalyzes GTP uptake into G_t, which results in complex dissociation. The spectrum labeled "+GTPS" in Fig. 4A was taken after the addition of the nonhydrolyzable GTP analog GTPS to the complex of illuminated 9-dm rhodopsin and G_t. It shows a decrease of the absorption at 380 nm and a concurrent increase around 464 nm, presumably because GTPS uptake into G_t has caused the dissociation of the complex. By showing that the G_t-induced accumulation of the SB-deprotonated over the SB-protonated species is reversible, this experiment demonstrates that light-activated 9-dm rhodopsin exists in a true, G_t-sensitive equilibrium between these forms, in analogy to the meta I  meta II equilibrium in native rhodopsin. It also proves that the SB is still intact in the 380-nm absorbing species (additionally verified by acid denaturation, data not shown).

The Neutralization of Residue Glu¹³⁴ in the E134Q Replacement Mutant Enhances Spontaneous Schiff Base Deprotonation in 9-Demethyl Pigments-- We investigated the effects of the E134Q replacement mutation, which has been shown to mimic exogenous protonation (12, 13, 25), in combination with 9-dm retinal. Fig. 5 shows a series of spectra of the pigment 9-dm E134Q, with the pH varying from 5.5 to 7.5. Most importantly, the extent of SB deprotonation greatly exceeds that shown by 9-dm wild type pigment, demonstrating a significant functional rescue of this 9-dm-induced deficiency. Especially at basic pH, the extent of SB deprotonation in illuminated 9-dm E134Q is comparable to that displayed by wild type pigments bearing the normal retinal and yields complete deprotonation.

View larger version (18K): [in this window] [in a new window]   Fig. 5.   Spectra of the mutant 9-dm E134Q pigment, at pH 5.5, 6.5, and 7.5. Dark spectra at different pH values were nearly identical, the dotted line is the dark spectrum taken at pH 6.5. The solid lines are spectra taken after illumination, at the pH indicated. The dashed line is the spectrum of the illuminated pH 5.5 sample after acid denaturation, performed by adding 10% (v/v) 1 M HCl.

However, a striking result is that increasing acidity leads to an increase of absorption around 466 nm and a concurrent decrease in a 380-nm absorption. This behavior is the opposite of that shown by the 9-dm wild type pigment or bovine rhodopsin but similar to what is known from the E113Q counterion mutant (33). In the case of E113Q in the dark, it was shown that the SB was protonated directly with solution protons. As an explanation for the present observation, we suggest that the pK_a of the Schiff base in 9-dm meta II may be less acidic than that of normal meta II. This could force exogenous reprotonation of the SB at acidic pH and would lead to two counteracting processes; one is the low pH-induced transition from the meta I-analog to 9-dm meta II and the other is the exogenous reprotonation of the SB in a presumably meta II-like conformation. In the 9-dm E134Q mutant, the first process would always proceed to completion, leaving only the second process to be observed. This hypothesis implies that the lack of retinal's 9-methyl group has a direct influence on the pK_a of the Schiff base, probably because of a different geometric arrangement between the SB and the counterion. It will be interesting to investigate whether the SB counterion Glu¹¹³ changes its state of protonation when the SB is reprotonated in this pigment.

Quantification of SB Deprotonation-- For 9-dm wt rhodopsin and 9-dm E134Q, we have recorded the light-induced absorbance increase at 380 and absorbance decrease at 464 nm, at increments of 0.5 between pH 5.5 and 8.0 (Fig. 6A, open symbols, at 464 nm; closed symbols, at 380 nm). Note that plotting the decrease inverts the sign of the absorbance change, which is actually negative. Because both absorbance increase at 380 nm and absorbance decrease at 464 nm indicate SB deprotonation, plotting the data in this way gives higher ordinate values for more extensive SB deprotonation. At acidic pH, the data show an increase of SB deprotonation by 9-dm wt (circles) but a decrease of deprotonation by 9-dm E134Q (triangles). One possible explanation for the behavior shown by 9-dm E134Q is reprotonation of the SB from solution. If this is true, it might be expected to also occur with 9-dm wt pigment (for details, see "Discussion"). To eliminate this counteracting process from the titration curve of 9-dm wt, we have expressed SB deprotonation by 9-dm wt as a percentage of deprotonation by 9-dm E134Q, at every pH (Fig. 6B). A formal derivation shows that this yields data that only includes the first step, i.e. pH-induced SB deprotonation. These data were numerically fitted to yield an estimated pK_a for this transition of 5.7. The basic end of the fitted curve was left open to account for pH-independent absorbance changes.

View larger version (11K): [in this window] [in a new window]   Fig. 6.   A, SB deprotonation by 9-dm wt and 9-dm E134Q, at varying pH. Solid symbols are absorbance increases at 380 ± 2 nm generated by the illumination of 9-dm wt (circles) and 9-dm E134Q (triangles) pigments plotted versus pH. Absorbance increases in this region indicate SB deprotonation. Open symbols are simultaneously measured absorbance decreases at 464 ± 2 nm. Absorbance decreases in this region also indicate SB deprotonation. Note that plotting the decrease at 464 nm has made the negative absorbance change positive for the purpose of the graph. B, titration curve of SB deprotonation by 9-dm rhodopsin. The plot shows SB deprotonation by 9-dm wt as a percentage of SB deprotonation by 9-dm E134Q (for details see "Results"). At every pH, the 9-dm wt data from a were averaged and expressed as percentage of the averaged 9-dm E134Q data at that pH (plotted points). The solid line is a numerical fit of a Henderson-Hasselbalch type curve. The basic end of the curve was left open for the fit. The resulting pKa was 5.7.

The E134Q Replacement Mutation Rescues the Deficient G_t Activation Capacity of 9-dm Pigments-- Fig. 7 shows recordings of the change of intrinsic fluorescence over time caused by rhodopsin-catalyzed GTPS uptake into G_t. Table I shows the results of a quantification of these experiments performed using a linear regression to determine the initial rate of activation (i.e. initial slope of the trace). Fig. 7A shows traces recorded in the presence of 2 nM wild type or 9-dm wild type pigments. At the indicated time, GTPS was added to reach final concentrations of 0.2, 1.0, or 5 µM GTPS as indicated. Between 1 and 5 µM GTPS, the rate of activation is virtually independent of the concentration of GTPS. Under these circumstances, the rate-limiting step in catalysis is the formation of the complex between R* and G_t (an exact derivation yields k₊ [R*] [G_t] for the rate, with k₊ being the rate constant for complex formation). The results show that this step is slowed by a factor of 4 in 9-dm rhodopsin. This is to be expected if the decrease in the catalytic activity is mainly because of a reduced formation of the active species and hence a reduced efficiency of collisional coupling.

View larger version (21K): [in this window] [in a new window]   Fig. 7.   A, Gt activation by wild type and 9-dm wild type pigments. The traces show a percentage change of fluorescence versus time as readout of pigment-catalyzed Gt activation. All samples contained identical amounts of wild type (top traces) and 9-dm wild type (lower traces) pigment and Gt. At the time indicated, GTPS was added at the final concentration given in the figure. B, Gt activation by wild type, 9-dm wild type, E134Q, and 9-dm E134Q pigments. A similar experiment as a is shown but with 5 µM GTPS final concentration in all samples. Traces generated using the different pigments are marked in the figure. The scale bars indicate a 10% fluorescence increase and 200 s, respectively.

Fig. 7B shows traces recorded in the presence of 2 nM wild type, 9-dm wild type, E134Q, and 9-dm E134Q rhodopsins. At the indicated time, GTPS was added to reach a final concentration of 5 µM. In this assay also, the E134Q replacement mutation shows a virtually complete functional rescue of defects caused by the 9-dm retinal, as rates of activation by 9-dm E134Q, wt, and E134Q pigments are within 5% percent of each other. This parallels the increased formation of meta II exhibited by this pigment.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

It is a general property of G-protein-coupled receptors that their interaction with G-proteins depends on the stereochemistry of the respective ligands, defining their agonistic or antagonistic properties. In the case of the photoreceptor rhodopsin, the crucial effect of retinal stereochemistry on the early photochemically defined stages of activation is a well established fact. In the present study, we have addressed the question if and how retinal stereochemistry also co-determines the late proton transfer reactions leading to breaking of the salt bridge between the protonated Schiff base and the counterion. These are the intermediates that display the most obvious analogies to receptors activated by diffusible ligands, e.g. as seen in the extended ternary complex model (34).

We have investigated the origin of the low G_t-activating capacity of the photoreceptor 9-dm rhdopsin, which is formed by reconstituting the apoprotein, opsin, with a retinal chromophore lacking the 9-methyl group. The data published in the literature have shown the importance of this group. When it was removed, yielding 9-dm rhodopsin, the deprotonation of the SB, which is a prerequisite for the transition to the active state (7, 8, 12, 35), was not observed, and the catalytic activity of this pigment was several times lower than that of opsin bearing the normal chromophore (22, 23).

We have presented data that show that, although the transition to the active form may be greatly impeded in light-activated 9-dm rhodopsin, it is not impossible and is rather the manifestation of a very unfavorable equilibrium between inactive, SB-protonated and active, SB-deprotonated forms (Fig. 1C). The G_t stabilizes the deprotonated form, showing that this form represents the interacting, active conformation of the receptor. This is further supported by the fact that the interaction can be terminated by the addition of GTPS. Additionally, we have found that neutralization of the opsin residue Glu¹³⁴ by replacing it with Gln recovers much of the functionality lost due to removal of retinal's 9-methyl group. Published data suggest that the E134Q mutation anticipates the uptake of a proton from solution, which in wild type rhodopsin accompanies the light-induced formation of the active state (12, 13, 24, 25). This effect seems to be strong enough to force the transition of 9-dm rhodopsin to the active state.

We arrived at the following conclusions. (i) The 9-methyl group of retinal is the strongest factor among several that influence transition to metarhodopsin II. When native rhodopsin is illuminated, it finally enters an equilibrium between the inactive, SB-protonated meta I and the active form meta II with a deprotonated SB. A number of factors are known to influence this equilibrium, low pH and high temperature both shift the equilibrium toward meta II. The influence of the receptor's hydrophobic environment, e.g. when rhodopsin is embedded in detergent micelles (dodecyl maltoside in this study) after purification, can override both these factors and cause a complete shift to meta II in a wide range of pH and temperatures (10). It is interesting to note that a similar detergent-induced shift from the inactive toward the active receptor conformation has been observed for the ₂-adrenergic receptor (34). The data presented in this study show that the action of the 9-methyl group of retinal is a further, perhaps even the decisive factor in the meta I  meta II equilibrium. When this group is removed, even the normally dominating influence of the detergent environment does not suffice to shift this equilibrium toward meta II at basic pH. Instead, the equilibrium becomes pH-dependent, with SB deprotonation favored at acidic pH as known from bovine rhodopsin in retinal disc membranes.

(ii) Interaction with G_t requires deprotonation of the Schiff base. Potentially, 9-dm rhodopsin may be able to form several SB-deprotonated species with the concomitant 380-nm absorption maximum. As witnessed by the increased formation of these species when transducin is present after illumination, at least one of these is able to interact with G_t, demonstrating that the form capable of interaction is stabilized at the expense of those SB-protonated forms that are not. It also shows that, even under conditions that are very unfavorable for Schiff base deprotonation, the proton transfer event is required for the binding of G_t.

(iii) Reduced G_t activation by 9-dm rhodopsin is explained by a strongly shifted equilibrium between an inactive and an active conformation, 9-dm meta II. Besides the similar pH dependence, the ability to bind and activate G_t is a further common feature of meta II and the 380-nm form of 9-dm rhodopsin, which we term 9-dm meta II. The data, especially the reversal of the shift after the addition of GTPS, show that the interactive species must be linked to the SB-protonated forms via a true equilibrium. Hence, the all-trans-form of this chromophore, resulting from the photoactivation of 9-demethyl-11-cis-rhodopsin, shows itself to behave as a partial agonist, i.e. removal of the 9-methyl group reduces the capability of the bound ligand to shift the receptor from its inactive towards its active conformation.

(iv) Retinal provides the scaffold for adjustment of proton donor and acceptor groups. We have recently proposed that meta I may be the state in which proton donor and acceptor groups in light-activated rhodopsin are arranged in such a way with respect to angle, distance, and electric field as to prepare for the proton translocations that mark the transition to the meta II state (18, 36). In their infrared spectroscopic study, Ganter et al. (23) found that the photoactivation reaction of 9-dm rhodopsin shows its clearest defect in the progression from lumirhodopsin. We have shown that the defects of the dysfunctional meta I conformer become visible in the proton translocations involving the SB and Glu¹¹³. Taken together, this could mean that retinal acts as a scaffold to adjust the apoprotein moiety, which allows the proton transfer reactions that stabilize the active conformation to occur. To reach the signaling state of rhodopsin, TM3 and TM6 move relative to another (37, 38), which has been suggested to cause the observed exposure of the cytoplasmic end of TM7 (39). Assuming such movement of TM3, which carries the key residues Gly¹²¹, Glu¹¹³, and Glu¹³⁴, relative to its surroundings, the 9-methyl group possibly makes use of the helix structure to prime meta I for the transition to meta II. In the case of 9-demethyl retinal, insufficient priming occurs, because either TM3 is left free to fluctuate or it is adjusted in a position unsuitable for the transition to meta II (Fig. 8). It will be interesting to see whether other stereochemical elements of retinal that are known to have an influence on the rhodopsin activation pathway, namely the -ionone ring (40) and methyl modification of C-10 (41), fit into this scheme.

View larger version (26K): [in this window] [in a new window]   Fig. 8.   Proposed scaffolding function of retinal in the arrangement of the apoprotein. In native rhodopsin, steric interaction of the 9-methyl group of retinal with Gly121 holds TM3 of opsin in a position where Glu113 and Glu134 on this helix are properly adjusted for the protonation reactions linked to the transition to the signaling state. In 9-demethyl rhodopsin, 9-demethyl retinal cannot provide a functional scaffold for the activating protonation switches because TM3 is misaligned.

	ACKNOWLEDGEMENTS

We thank Dr. Tom Sakmar (Rockefeller University, NY) for providing the expression vectors and helpful advice about rhodopsin expression. We also thank C. Koch, R. Kukina, J. Engelmann, and I. Semjonow for technical assistance.

	FOOTNOTES

^* This work was supported by Grants Sfb 449 (to O. P. E. and K. P. H.) and Sfb 498 (to K. P. H.) from the Deutsche Forschungsgemeinschaft.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ Recipient of a grant from the Friedrich-Ebert-Stiftung, Bonn, Germany.

To whom correspondence may be addressed. Tel.: 49-30-2802-6141; Fax: 49-30-2802-6377; E-mail: kph@charite.de or oliver.ernst{at}charite.de.

Published, JBC Papers in Press, April 17, 2000, DOI 10.1074/jbc.M000603200

² Pigments consisting of the opsin with the Glu  Gln replacement at position 134 are designated by E134Q.

	ABBREVIATIONS

The abbreviations used are: TM, transmembrane helix; SB, Schiff base; R and R*, inactive and active receptor conformations, respectively; G_t, retinal G-protein transducin; meta I, metarhodopsin I; meta II, metarhodopsin II; dm, demethyl; DM, n-dodecyl--D-maltoside; HPLC, high pressure liquid chromatography; MES, 2(N-morpholino)ethanesulfonic acid; BTP, 1,3-bis[tris(hydroxymethyl)methylamino]propane; GTPS, guanosine 5'-3-O-(thio)triphosphate; G-protein, guanine nucleotide-binding regulatory protein; wt, wild type.

	REFERENCES

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