Signaling states of rhodopsin: Retinal provides a scaffold ... · Meyer et al. 24.03.00 MS...

Meyer et al. 24.03.00

MS 240300.doc

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*, Oliver P. Ernst*

*please address correspondence to either author

Address:

Institut für Medizinische Physik und Biophysik, Humboldt Universität zu Berlin,

Universtitätsklinikum Charité – CCM, Schumannstr. 20-21, 10098 Berlin

Phone: ++49 – 30 – 2802 – 6141

Fax: ++49 – 30 – 2802 – 6377

Email: [email protected] or [email protected]

§Max-Planck-Institut für Strahlenchemie, Stiftstr. 34-36, 45470 Mülheim an der Ruhr

Running Title:

Retinal provides a scaffold for proton transfer switches

Keywords:

rhodopsin, G-Protein, GPCR, agonist, 9-demethyl-retinal, receptor activation,

steric trigger, salt-bridge breaking

Copyright 2000 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on April 17, 2000 as Manuscript M000603200 by guest on February 17, 2020

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SUMMARY

The G-protein coupled receptor rhodopsin is activated by photoconver-

sion 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 con-

formations distinguished by the protonation of key opsin residues. In this re-

port, we study the role of retinal´s 9-methyl group, one of the crucial steric de-

terminants of light activation. We find that when this group is removed, the pro-

tonation 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 ex-

plain the partial agonism of certain ligands.

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INTRODUCTION

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 (TM)[see footnote], and the chromophore 11-cis-

retinal, which is covalently bound to Lys296 in TM7 via a protonated Schiff base (SB)

and keeps the receptor in the inactive conformation, acting as a highly effective in-

verse agonist. Following the absorption of a photon, the retinal isomerizes to a

strained all-trans-conformation (1), which induces a series of conformational rear-

rangements 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 (Gt) (for

reviews see (2-4)).

These events can be summarized as shown in Fig. 1.a.): 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

(λmax = 478 nm) to the meta II conformation (5), which is characterized by a strongly

blue-shifted absorbance maximum (λmax = 380 nm). SB deprotonation is known to be

necessary for interaction with Gt (6-8), and is accompanied by the uptake of a proton

from the aqueous solution (9-11). pH-rate profiles of Gt-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 simula-

tions and mutagenesis studies, similar results have been obtained for the α1B-

adrenergic receptor (14, 15).


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The current understanding of this sequence is that while 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 formu-

lated to describe this in graphic terms.

The 9-methyl group of the retinal, which has been shown to interact directly

with opsin’s Gly121 residue 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 pigment’s ability to progress to the meta II state

and impairs its catalytic efficiency (23). Fig. 1 panels b) and c) summarize the two

conceivable reaction pathways which may be adopted by the defective pigment

formed with this chromophore: Either a breakdown of the steric mechanisms pre-

vents light-activated 9-demethyl rhodopsin (9-dm rhodopsin) from reaching the pro-

tonation equilibrium and instead causes the formation of a weakly active product

(panel 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 con-

formation analogous to meta I (panel c.). Experimental results until now have not

been able to decide between the two possibilities.

In the present study, we provide proof that panel c.) is the correct scheme: We

show that illuminated 9-dm-rhodopsin is in an equilibrium between SB-protonated

and SB-deprotonated forms which is strongly shifted towards the protonated form

when compared to the normal pigment. Further, we show that this equilibrium is af-

fected 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


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by the replacement mutation E134Q, which has previously been shown to mimic the

effects of proton uptake (12, 13, 24, 25).


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EXPERIMENTAL PROCEDURES

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 Na-

tional Eye Institute, National Institute of Health, USA). 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, USA) and was coupled to CNBr-activated Sepharose 4 Fast Flow (Am-

ersham Pharmacia Biotech) largely following the manufacturers instructions.

n-dodecyl-β-D-maltoside (dodecyl maltoside, DM) was purchased from Biomol (Ham-

burg, Germany). Reagents for tissue culture were from Life Technologies/ GIBCO

BRL or Sigma. COS-1 cells (ATCC-No. CRL-1650) were from American Type Culture

Collection (Manassas, VA, USA). 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 protease inhibitor cocktail was from

Roche Molecular Biochemicals and was used at a concentration of 1 tablet per 75 ml

buffer.

Expression and Preparation of Recombinant Rhodopsins. Expression of

opsin genes was performed basically as described (28) with the following modifica-

tions: COS-1 cells were grown in monolayer in plastic cell culture roller bottles (sur-

face area 850 cm2) under a humidified atmosphere with 5% CO2 at 37° C. Growth

medium was Dulbecco’s modified Eagle's medium (DMEM) containing 4.5 g/l D-

glucose and 0.11g/l Na-pyruvate, supplemented with 100 mg/ml streptomycin, 100

units/ml penicillin, 2 mM L-glutamine, and 10% (v/v) heat-inactivated FBS. At 80-95%

confluence, transient transfection of the cells was initiated using a transfection cock-

tail composed of 150 µg plasmid DNA, 6 ml 1 M Tris buffer (pH 7.4), 48 ml serum


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free growth medium and 6 ml DEAE-dextran stock solution (2.5 mg/ml in DMEM).

After 5.5 hr, the transfection cocktail was replaced with 75 ml of 0.1 mM chloroquine

in growth medium for 90 minutes. The cells were washed with DMEM and further in-

cubated in growth medium until harvest 64-68 hr later. By incubation with 1 mM

Na2EDTA in PBS (137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4,

pH 7.0) for 10 minutes, 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,

resp., for 2-4 hr 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 addition of dodecyl maltoside (1% (w/v) final con-

centration) and nuclei were removed by centrifugation. Pigments from two roller bot-

tles were incubated with 1D4-Sepharose, washed twice with 0.03% (w/v) DM in PBS

and once with elution buffer (0.015% (w/v) DM, 10 mM BTP, pH 6.0; 50 ml per

wash). Elution of the pigments from the 1D4-Sepharose was carried out in elution

buffer supplemented with 100 µM peptide corresponding to the carboxy-terminal 18

amino acids of rhodopsin.

Preparation of Transducin (Gt). Gt was purified from bovine retinae essen-

tially as described (30) and stored in 20 mM BTP pH 7.1, 130 mM NaCl, 1 mM

MgCl2, 1 mM DTT.

Preparation of 9-demethyl-retinal. The first three steps of the synthesis of 9-

demethyl-retinal were performed according to the procedure of v. d. Tempel and

Huisman (31): Oxidation of β-Ionone with sodium hypochlorite, reduction of the re-

sulting acid with LiAlH4 followed by reoxidation with MnO2 resulted in 3-(2,6,6-

trimethyl-cyclohexen-1-yl)-2-propenal. Elongation of the chain was achieved by Wit-


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tig-Horner coupling of the aldehyde with 2-(diethylphosphono)-acetonitrile and sub-

sequent reduction with DIBAH, followed by a second Wittig-Horner coupling with 4-

(diethylphosphono)-3-methyl-2-butene-nitrile. Subsequent reduction of the nitrile with

DIBAH formed 9-demethylretinal. 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 6 geometric iso-

mers (20 % 11-cis isomer) which were separated by isocratic preparative HPLC

(Kromasil Si-100-5µ, 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/Vis-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, 6.5) or BTP (for pH 6.5, 7.0, 7.5,

8.0) 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 ε = 42700 mol-1cm-1 for all pigments.

Assay of Gt-activation. Experiments were performed essentially as described

(12, 32). The instrument settings were λexc = 300 nm, λem = 345 nm, integration time

2 s, T = 20°C. Pigment concentration in samples was 2 nM, with 0.01% (w/v) DM and

250 nM Gt in 20 mM BTP pH 7.5, 130 mM NaCl, 1 mM MgCl2. Illumination of the

sample using the same equipment as for UV/ Vis spectroscopy was maintained

throughout the experiment. Activation of Gt was started by adding GTPγS into the

cuvette. Traces were corrected for the dilution due to addition of GTPγS and normal-


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ized 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.


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RESULTS

Illuminated 9-demethyl-rhodopsin shows a strongly reduced but pH-

dependent Schiff base deprotonation. Opsin was expressed in COS cells, recon-

stituted with 11-cis-9-demethyl retinal or 11-cis-retinal and purified in dodecyl malto-

side yielding up to 100 µg of purified rhodopsin per cell culture roller bottle with spec-

tral ratios of 1.9 (A280/A464 for 9-dm rhodopsin) to 1.7 (A280/A500 for rhodopsin).

UV/Vis-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 λmax 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 illu-

mination. At pH 7.5, the spectrum shows the formation of a photoproduct with the

visible λmax 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 7.5 clearly show

this to be a second species with a λmax of 380 nm. This species is in a pH-dependent

equilibrium with the 466 nm form and is favored at acidic pH.

The dashed line is a spectrum taken after the illuminated pH 5.5 sample was

denatured with 1M HCl (10% of sample volume added). It shows a λmax of 432 nm,

characteristic of an intact, protonated Schiff base (SB). Spectra taken of dark sam-

ples denatured with acid display the same λmax (data not shown). The shift in λmax

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).


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An absorption maximum around 380 nm is indicative of a deprotonated Schiff

base. From its UV/Vis-absorption spectrum, 9-dm-rhodopsin’s 380 nm absorbing

species is similar to metarhodopsin II (meta II), the Schiff base deprotonated inter-

mediate of light activated rhodopsin, which also shows a λmax of 380 nm. Also, the

pH-dependence of this species resembles that found for bovine rhodopsin in disk

membrane preparations (9, 11), where meta II formation is increased at acidic pH.

There is an important difference however: When native rhodopsin or recombi-

nant 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

(inset of Fig. 3 and (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). Interest-

ingly though, SB-deprotonation is less than with the expressed pigment.

Transducin stabilizes the Schiff base deprotonated form of illuminated 9-

demethyl-rhodopsin in a GTPγγS sensitive way— Fig. 4 shows a sequence of

spectra taken before and after illumination of 9-dm rhodopsin in the presence of

500 nM of the retinal G-protein transducin (Gt), and after addition of 50 µM GTPγS.

The spectra labeled “dark” and “illuminated” in Fig. 4a show that the presence of Gt

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

disk membranes (6, 32): Gt stabilizes meta II at the expense of its tautomeric SB-


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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 Gt.

In the case of native rhodopsin, this complex catalyzes GTP uptake into Gt,

which results in complex dissociation. The spectrum labeled “+GTPγS” in Fig. 4a was

taken after the addition of the non-hydrolyzable GTP analog GTPγS to the complex of

illuminated 9-dm-rhodopsin and Gt. It shows a decrease of the absorption at 380 nm

and a concurrent increase around 464 nm, presumably because GTPγS uptake into

Gt has caused the dissociation of the complex. By showing that the Gt-induced ac-

cumulation of the SB-deprotonated over the SB-protonated species is reversible, this

experiment demonstrates that light-activated 9-dm rhodopsin exists in a true, Gt-

sensitive equilibrium between these forms, in analogy to the meta I meta II equi-

librium in native rhodopsin. It also proves that the SB is still intact in the 380 nm ab-

sorbing 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-wildtype 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.

However, a striking result is that increasing acidity leads to an increase of absorption

around 466 nm and a concurrent decrease in 380 nm absorption. This behavior is the


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opposite of that shown by the 9-dm-wild type pigment or bovine rhodopsin, but simi-

lar 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 pKa of the Schiff base

in 9-dm meta II may be less acidic than that of normal meta II. This could force ex-

ogenous 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, 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 hy-

pothesis implies that the lack of retinal’s 9-methyl group has a direct influence on the

pKa of the Schiff base, probably because of a different geometric arrangement be-

tween 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 absorb-

ance decrease at 464 nm, at increments of 0.5 between pH 5.5 and 8.0 (Fig. 6.a;

open symbols: at 464 nm; closed symbols: at 380 nm). Note that plotting the de-

crease inverts the sign of the absorbance change, which is actually negative. Since

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


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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 the Discussion). To eliminate this counteract-

ing process from the titration curve of 9-dm wt, we have expressed SB-deprotonation

by 9-dm wt as percentage of deprotonation by 9-dm E134Q, at every pH (Fig. 6b). A

formal derivation shows that this yields data which only includes the first step, i.e. pH-

induced SB-deprotonation. This data was numerically fitted to yield an estimated pKa

for this transition of 5.7. The basic end of the fitted curve was left open to account for

pH-independent absorbance changes.

The E134Q replacement mutation rescues the deficient Gt-activation ca-

pacity of 9-dm-pigments. Fig. 7 shows recordings of the change of intrinsic fluo-

rescence over time caused by rhodopsin catalyzed GTPγS uptake into Gt. Table 1

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 of wild type or 9-dm-wild

type pigments. At the indicated time, GTPγS was added to reach final concentrations

of 0.2, 1.0 or 5 µM GTPγS as indicated. Between 1 and 5 µM GTPγS, the rate of acti-

vation is virtually independent of the concentration of GTPγS. Under these circum-

stances, the rate limiting step in catalysis is the formation of the complex between R*

and Gt. (An exact derivation yields [ ] [ ]tGRk ⋅⋅+ * for the rate, with +k the rate con-

stant 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 due to a reduced formation of the active species and hence a reduced effi-

ciency of collisional coupling.

Fig. 7b.) shows traces recorded in the presence of 2 nM of wild type, 9-dm-

wild type, E134Q and 9-dm-E134Q rhodopsins. At the indicated time, GTPγS was


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added to reach a final concentration of 5 µM. In this assay also, the E134Q replace-

ment 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 ex-

hibited by this pigment.


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DISCUSSION

It is a general property of GPCRs that their interaction with G-proteins de-

pends 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 com-

plex model (34).

We have investigated the origin of the low Gt-activating capacity of the photo-

receptor 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-demethyl-

rhodopsin (9-dm-rhodopsin), the deprotonation of the Schiff base (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 bear-

ing the normal chromophore (22, 23).

We have presented data that show that, while 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. 1.c). The retinal G-protein trans-

ducin (Gt) stabilizes the deprotonated form, showing that this form represents the

interacting, active conformation of the receptor. This is further supported by the fact


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that the interaction can be terminated by the addition of GTPγS. 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 solu-

tion, which in wild type rhodopsin accompanies the light-induced formation of the ac-

tive state (12, 13, 24, 25). This effect seems to be strong enough to force 9-dm rho-

dopsin’s transition to the active state.

We arrive at the following conclusions:

i.) Retinal’s 9-methyl group is the strongest factor among several that influence

transition to metarhodopsin II. When native rhodopsin is illuminated, it finally en-

ters 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 equi-

librium: low pH and high temperature both shift the equilibrium towards meta II. The

influence of the receptor’s hydrophobic environment, e.g. when rhodopsin is embed-

ded in detergent micelles (dodecyl maltoside in this study) after purification, can over-

ride both these factors and cause a complete shift to meta II in a wide range of pH

and temperature (10). It is interesting to note that a similar detergent-induced shift

from the inactive towards the active receptor conformation has been observed for the

β2-adrenergic receptor (34). The data presented in this study show that the action of

retinal’s 9-methyl group is a further, perhaps even the decisive factor in the

meta I meta II equilibrium. When this group is removed, even the detergent envi-

ronment’s normally dominating influence does not suffice to shift this equilibrium to-

wards 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 disk

membranes.


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ii.) Interaction with Gt requires deprotonation of the Schiff base. Potentially, 9-

dm rhodopsin may be able to form several SB-deprotonated species with the con-

comitant 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 the retinal G-protein transducin (Gt), 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 binding of Gt.

iii.) Reduced Gt-activation by 9-dm rhodopsin is explained by a strongly shifted

equilibrium between an inactive and an active conformation, 9-dm meta II. Be-

sides the similar pH-dependence, the ability to bind and activate Gt is a further com-

mon 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 GTPγS,

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


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19

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

rhodosin’s signaling state, 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 β-ionone ring (40)

and methyl modification of C-10 (41), fit into this scheme.


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ACKNOWLEDGEMENTS

We thank Dr. Tom Sakmar (Rockefeller University, New York) for providing the

expression vectors and helpful advice about rhodopsin expression. We also thank

Ch. Koch, R. Kukina, J. Engelmann and I. Semjonow for technical assistance. This

work was supported by grants from the Deutsche Forschungsgemeinschaft Sfb 449

(to O.P.E and K.P.H.) and Sfb 498 (to K.P.H.). A. O. is the recipient of grant from the

Friedrich-Ebert-Stiftung, Bonn, Germany.


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REFERENCES

1. Ganter, U. M., Gärtner, W., and Siebert, F. (1988) Biochemistry 27, 7480-7488

2. Stryer, L. (1986) Annu. Rev. Neurosci. 9, 87-119

3. Sakmar, T. P. (1998) Prog. Nucleic. Acid Res. Mol. Biol. 59, 1-34

4. Helmreich, E. J. and Hofmann, K. P. (1996) Biochim. Biophys. Acta 1286, 285-

322

5. Jäger, F., Fahmy, K., Sakmar, T. P., and Siebert, F. (1994) Biochemistry 33,

10878-10882

6. Emeis, D., Kuhn, H., Reichert, J., and Hofmann, K. P. (1982) FEBS Lett. 143,

29-34

7. Longstaff, C., Calhoon, R. D., and Rando, R. R. (1986) Proc. Natl. Acad. Sci.

USA 83, 4209-4213

8. Kibelbek, J., Mitchell, D. C., Beach, J. M., and Litman, B. J. (1991) Biochemistry

30, 6761-6768

9. Parkes, J. H. and Liebman, P. A. (1984) Biochemistry 23, 5054-5061

10. Arnis, S. and Hofmann, K. P. (1993) Proc. Natl. Acad. Sci. USA 90, 7849-7853

11. Parkes, J. H., Gibson, S. K., and Liebman, P. A. (1999) Biochemistry 38, 6862-

6878

12. Fahmy, K. and Sakmar, T. P. (1993) Biochemistry 32, 7229-7236


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


MS 240300.doc

22

13. Arnis, S., Fahmy, K., Hofmann, K. P., and Sakmar, T. P. (1994) J. Biol. Chem.

269, 23879-23881

14. Scheer, A., Fanelli, F., Costa, T., De Benedetti, P. G., and Cotecchia, S. (1996)

EMBO J. 15, 3566-3578

15. Scheer, A., Fanelli, F., Costa, T., De Benedetti, P. G., and Cotecchia, S. (1997)

Proc. Natl. Acad. Sci. USA 94, 808-813

16. Shieh, T., Han, M., Sakmar, T. P., and Smith, S. O. (1997) J. Mol. Biol. 269,

373-384

17. Szundi, I., Mah, T. L., Lewis, J. W., Jäger, S., Ernst, O. P., Hofmann, K. P., and

Kliger, D. S. (1998) Biochemistry 37, 14237-14244

18. Hofmann, K. P. (1999) Signalling states of photoactivated rhodopsin. In: Rho-

dopsins and Phototransduction, Novartis Found. Symp., 224, Wiley, Chichester,

pp. 158-180

19. Yan, B., Nakanishi, K., and Spudich, J. L. (1991) Proc. Natl. Acad. Sci. USA 88,

9412-9416

20. Robinson, P. R., Cohen, G. B., Zhukovsky, E. A., and Oprian, D. D. (1992) Neu-

ron 9, 719-725

21. Han, M., Groesbeek, M., Sakmar, T. P., and Smith, S. O. (1997) Proc. Natl.

Acad. Sci. USA 94, 13442-13447

22. Han, M., Groesbeek, M., Smith, S. O., and Sakmar, T. P. (1998) Biochemistry

37, 538-545


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


MS 240300.doc

23

23. Ganter, U. M., Schmid, E. D., Perez-Sala, D., Rando, R. R., and Siebert, F.

(1989) Biochemistry 28, 5954-5962

24. Weitz, C. J. and Nathans, J. (1993) Biochemistry 32, 14176-14182

25. Kim, J. M., Altenbach, C., Thurmond, R. L., Khorana, H. G., and Hubbell, W. L.

(1997) Proc. Natl. Acad. Sci. USA 94, 14273-14278

26. Molday, R. S. and MacKenzie, D. (1983) Biochemistry 22, 653-660

27. Sakmar, T. P., Franke, R. R., and Khorana, H. G. (1989) Proc. Natl. Acad. Sci.

USA 86, 8309-8313

28. Oprian, D. D. (1993) Meth. Neurosci. 15, 301-306

29. Oprian, D. D., Molday, R. S., Kaufman, R. J., and Khorana, H. G. (1987) Proc.

Natl. Acad. Sci. USA 84, 8874-8878

30. Heck, M. and Hofmann, K. P. (1993) Biochemistry 32, 8220-8227

31. Van den Tempel, P. J. and Huisman, H. O. (1966) Tetrahedron 22, 293-299

32. Ernst, O. P., Bieri, C., Vogel, H., and Hofmann, K. P. (2000) Meth. Enzymol.

315, 471-489

33. Sakmar, T. P., Franke, R. R., and Khorana, H. G. (1991) Proc. Natl. Acad. Sci.

USA 88, 3079-3083

34. Samama, P., Cotecchia, S., Costa, T., and Lefkowitz, R. J. (1993) J. Biol.

Chem. 268, 4625-4636

35. Arnis, S. and Hofmann, K. P. (1995) Biochemistry 34, 9333-9340


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


MS 240300.doc

24

36. Hofmann, K. P. (in press) Late photoproducts and signaling states of bovine

rhodopsin. In: Hoff, A. J., Stavenga, D. G., de Grip, W. J., and Pugh, Jr. E. N.,

eds. Handbook of Biological Physics, Vol. IV, Elsevier, Amsterdam,

37. Farrens, D. L., Altenbach, C., Yang, K., Hubbell, W. L., and Khorana, H. G.

(1996) Science 274, 768-770

38. Dunham, T. D. and Farrens, D. L. (1999) J. Biol. Chem. 274, 1683-1690

39. Abdulaev, N. G., Ridge, K. D. (1998) Proc. Natl. Acad. Sci. USA 95, 12854-

12859

40. Jäger, F., Jäger, S., Kräutle, O., Friedman, N., Sheves, M., Hofmann, K. P., and

Siebert, F. (1994) Biochemistry 33, 7389-7397

41. DeLange, F., Bovee-Geurts, P. H., VanOostrum, J., Portier, M. D., Verdegem,

P. J., Lugtenburg, J., and DeGrip, W. J. (1998) Biochemistry 37, 1411-1420


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FOOTNOTES

1.) Nomenclature: Pigments consisting of the opsin with the Glu → Gln re-

placement at position 134 are designated by E134Q. Abbreviations: DIBAH: diiso-

propylaluminiumhydride; dm: demethyl; DM: n-dodecyl-β-D-maltoside; DMEM: Dul-

becco’s modified Eagle’s medium; FBS: Fetal bovine serum; G-protein: guanine nu-

cleotide-binding regulatory protein; meta I: metarhodopsin I; meta II: metarhodop-

sin II; Gt: retinal G-protein transducin; PBS: phosphate buffered saline; R, R*: inactive

and active receptor conformations, resp.; SB: Schiff base; TM: transmembrane helix;

wt: wild-type.


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FIGURE LEGENDS

Fig. 1

Photoactivation schemes of rhodopsin and 9-demethyl rhodopsin. a.) Rhodop-

sin (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 inac-

tive forms is shifted in favor of the inactive form, due to the only partial agonism of all-

trans-9-dm-retinal, also leading to reduced Gt-activation. According to our data,

scheme c.) is correct.

Fig. 2

Structural formula of 9-demethyl-11-cis-retinal.

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) 1M

HCl. The inset shows a similar experiment performed on wt rhodopsin bearing nor-

mal retinal (dotted line: spectrum before illumination; solid lines: spectra after illumi-

nation). Samples contained 250 and 350 nM pigment, resp..


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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 “+GTPγS” was taken after

this non-hydrolyzable GTP-analog was added to the illuminated sample. Note the

high absorption around 280 nm due to 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.

Fig. 5

Spectra of the mutant 9-dm E134Q pigment, at pH 5.5, 6.5, 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) 1M HCl.

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 in-

creases in this region indicate SB-deprotonation. Open symbols are simultaneously

measured absorbance decreases at 464 ± 2 nm. Absorbance decreases in this re-

gion 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.


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b.) Titration curve of SB-deprotonation by 9-dm rhodopsin. The plot shows SB-

deprotonation by 9-dm wt as percent of SB-deprotonation by 9-dm E134Q (for details

see text ). At every pH, the 9-dm wt data from panel 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.

Fig. 7

a.) Gt-activation by wild type and 9-dm wild type pigments. The traces show

percent 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, GTPγS 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 pig-

ments. A similar experiment as a.), but with 5 µM GTPγS final concentration in all

samples. Traces generated using the different pigments are marked in the figure.

The scale bars indicate 10% fluorescence increase and 200 seconds, respectively.

Fig. 8

Proposed scaffolding function of retinal in the arrangement of the apoprotein.

In native rhodopsin, steric interaction of retinal’s 9-methyl group with G121 holds

TM3 of opsin in a position where E113 and E134 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.


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TABLES

Table 1:

Relative rates of transducin activation

Rates of Gt activation by 2 nM pigment at 20°C, pH 7.5. All values are relative

to wild type pigment bearing the normal retinal.

Concentration of GTPγγS

5 µM 1 µM 0.2 µM

wild type (wt) 1.00 0.95 0.639-dm-wt 0.22 0.23 0.17E134Q 1.05Pi

gmen

t

9-dm-E134Q 0.97


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Rhodopsin(500)

Lumi(500)

Meta I(478)

Meta II(380)

steric trigger Salt-bridgebreaking

weakly activephotoproduct

9-demethylRhodopsin

(464) Lumi analog

a.)

b.)

Schiff-base protonated speciesSchiff-base

deprotonated

H+

deficientsteric trigger

9-dmMeta Ianalog

9-demethylRhodopsin

(464)Lumi

analogH+

9-dmMeta II(380)

Schiff-basedeprotonated

shiftedequilibrium

c.)

Gt-interactionand activation

Fig. 1


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O

12

34

5

67

8

9

10

1112

1314

15

16 17

18 20

Fig. 2


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Fig. 3

nm250 300 350 400 450 500 550 600

Abs

(mO

D)

0

4

8

12

16

20

pH

7.56.55.5

nm300 400 500 600

Ab

s (m

OD

)0

20pH 5.5 and 7.5


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Fig. 4

nm250 300 350 400 450 500 550 600

dark

illuminated

+ GTPgS

b)

nm250 300 350 400 450 500 550 600

darkilluminated

a)

Abs

(mO

D)

0

4

8

12

16

20

Abs

(mO

D)

0

4

8

12

16

20


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Fig. 5

nm250 300 350 400 450 500 550 600

pH

5.56.57.5

Abs

(mO

D)

0

4

8

12

16

20


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pH3 4 5 6 7 8 9

mO

D

-2

0

2

4

6

8

10

12

pH3 4 5 6 7 8 9SB

dep

rot.

by 9

-dm

wt (

% o

f 9-d

m E

134Q

)

0

20

40

60

80

100

pK = 5.7

Fig. 6

a)

b) by guest on February 17, 2020http://w

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a.)

b.)

Fig. 7

E134Qwt9-dm E134Q

9-dm wt

10%

DFl

uor

200 sec

10%

DFl

uor

200 sec

µM GTPgS

5.01.0

0.2

5.01.00.2

}}

wt

9-dm wt

GTPgS

GTPgS


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native 9-demethyl

SBH+

134

113 121

SBH+

134

113 121

Fig. 8


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Hofmann and Oliver P ErnstChristoph K Meyer, Monika Böhme, Andreas Ockenfels, Wolfgang Gärtner, Klaus P

transfer switchesSignaling states of rhodopsin: Retinal provides a scaffold for activating proton

published online April 17, 2000J. Biol. Chem.

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