The FASEB Journal • Research Communication

Asymmetry of the rhodopsin dimer in complexwith transducin

Beata Jastrzebska,1 Tivadar Orban, Marcin Golczak, Andreas Engel,and Krzysztof Palczewski1

Department of Pharmacology, School of Medicine, Case Western Reserve University, Cleveland,Ohio, USA

ABSTRACT A large body of evidence for G-protein-coupled receptor (GPCR) oligomerization has accumu-lated over the past 2 decades. The smallest of theseoligomers in vivo most likely is a dimer that buries1000-Å2 intramolecular surfaces and on stimulationforms a complex with heterotrimeric G protein in 2:1stoichiometry. However, it is unclear whether each ofthe monomers adopts the same or a different confor-mation and function after activation of this dimer. Withbovine rhodopsin (Rho) and its cognate bovine G-pro-tein transducin (Gt) as a model system, we used theretinoid chromophores 11-cis-retinal and 9-cis-retinal tomonitor each monomer of the dimeric GPCR within astable complex with nucleotide-free Gt. We found thatonly 50% of Rho* in the Rho*-Gt complex is trapped ina Meta II conformation, while 50% evolves toward anopsin conformation and can be regenerated with 9-cis-retinal. We also found that all-trans-retinal can regener-ate chromophore-depleted Rho*e complexed with Gt

and FAK*TSA peptide containing Lys296 with the at-tached all-trans retinoid (m/z of 934.5[MH]�) was iden-tified by mass spectrometry. Thus, our study shows thateach of the monomers contributes unequally to thepentameric (2:1:1:1) complex of Rho dimer and Gtheterotrimer, validating the oligomeric structure of thecomplex and the asymmetry of the GPCR dimer, andrevealing its structural/functional signature. Thisstudy provides a clear functional distinction betweenmonomers of family A GPCRs in their oligomericform.—Jastrzebska, B., Orban, T., Golczak, M., En-gel, A., Palczewski, K. Asymmetry of the rhodopsin

dimer in complex with transducin. FASEB J. 27,1572–1584 (2013). www.fasebj.org

Key Words: G-protein-coupled receptor � heterotrimeric Gprotein � retinoids � retinal isomerization � membrane protein

Insights into the molecular architecture of G-pro-tein-coupled receptors (GPCRs) and their activationmechanisms have been gained steadily over the past 2decades. These were derived initially from secondarystructure predictions of GPCR overall topology (1, 2),through low-resolution 3-dimensonal structures (3)and then structures of native crystallized rhodopsin(Rho; ref. 4), photoactivated Rho (Rho*), and ligand-free and chromophore-bound opsin (5–9). This steadyprogress was complemented recently by various con-structs in the presence of agonists and antagonists ofthermostabilized receptors (10) and the fusion of mod-ified GPCRs and fast folding soluble proteins (11).

GPCRs form homo- and heterodimeric and oligo-meric structures (12) identified by imaging of nativemembranes (13, 14). Moreover, obligatory heterodi-meric functional expression was documented primarilyfor family C GPCRs that contain large N-terminaldomains (15). For the largest family A of GPCRs,however, the situation is more complex. Purified familyA monomeric receptors can activate G proteins, bothalone and when reconstituted into nanodiscs, wherethey apparently retain a monomeric structure (16, 17).However, lack of direct organizational proof compli-cates the interpretation of these last experiments. Thenature of their diffusible ligands and rather crudeassays employed lacked the sensitivity to distinguishbetween different oligomeric states, particularly be-cause oligomers could rapidly form and break apart(18). Nonetheless, transgenic mouse experiments (19,20), electron microscopic studies (21, 22), and somecrystallographic structures (7, 23) support a vast num-ber of different biochemical assays and spectroscopic

1 Correspondence: Department of Pharmacology, Schoolof Medicine, Case Western Reserve University, 10900 EuclidAve, Cleveland, OH 44106-4965, USA. E-mail: K.P., kxp65@case.edu; B.J.,bxj27@case.edu

doi: 10.1096/fj.12-225383

Abbreviations: DDM, n-dodecyl-�-d-maltoside; DTT, dithio-threitol; EM, electron microscopy; GDP, guanosine diphos-phate; GPCR, G-protein-coupled receptor; Gt, G-proteintransducin; GTP, guanosine triphosphate; GTP�S, guanosine5=-3-O-(thio)triphosphate; HPLC, high-performance liquidchromatography; isoRho, isorhodopsin; Meta II, metarho-dopsin II; MS, mass spectrometry; MS2, tandem mass spec-trometry; MS3, triple mass spectrometry; NMWL, normalmolecular weight limit; Rho, rhodopsin; Rho*, photoacti-vated rhodopsin; Rho*e-Gt, chromophore-depleted Rho*-Gt;Rho9-cis-RAL-Gt, Rho*e-Gt regenerated with 9-cis-retinal;Rho*9-cis-RAL-Gt, Rho*-Gt regenerated with 9-cis-retinal;Rho*11-cis-RAL-Gt, Rho*e-Gt regenerated with 11-cis-retinal; ROS, rodouter segment; sConA, succinylated concanavalin A

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approaches (24–26) indicating that family A GPCRshave an intrinsic propensity to dimerize/oligomerize.

The family A GPCR Rho has been a model forG-protein signaling for decades (27). This membraneprotein has also offered opportunities to advance thestructural and functional understanding of GPCR oli-gomerization. Thus, Rho photoactivated states can bereadily monitored spectrophotometrically because in-active Rho with its 11-cis-retinylidene chromophoreabsorbs around 500 nm, whereas Rho*, the so-calledmetarhodopsin II (Meta II) formed in the all-trans-retinylidene configuration, absorbs at �380 nm (28).After chromophore release from Rho*, the resultingopsin can be recharged physiologically with 11-cis-retinal to regenerate light-sensitive Rho (24), or withthe 9-cis-retinal analog to form isorhodopsin (isoRho),which absorbs at 485 nm (29). The isomeric state ofchromophore can also be chemically analyzed afterrapid hydrolysis with hydroxylamine in ethanolic solu-tion and separation of the retinal isomers by high-performance liquid chromatography (HPLC). In nativemembranes, all-trans-retinylidene is hydrolyzed, andall-trans-retinal slowly released from the binding pocketis recycled back to the 11-cis conformation by a series ofenzymatic reactions termed the retinoid cycle. Notably,depletion of guanosine triphosphate (GTP) and guanos-ine diphosphate (GDP) stabilizes the complex betweenRho and its cognate G-protein transducin (Gt) for bio-chemical manipulation (30). Visualization of Rho*-Gtcomplexes with (unpublished results) and without thesuccinylated lectin, succinylated concanavalin A(sConA; ref. 21, 31) unequivocally indicates a pentam-eric assembly of the Rho*-Gt complex in which thephotoactivated Rho dimer serves as a platform forbinding the Gt heterotrimer, as predicted previously(32). The pentameric nature of this Rho*-Gt complexreveals that each Rho monomer in the complex mustbe differently arranged in its interaction with Gt. How-ever, it is unclear whether the Rho monomers haveseparate functions in the pentameric complex. In thisstudy, we demonstrate that photoactivated dimeric Rhois structurally and functionally in an asymmetric state inthe Rho*-Gt complex.

MATERIALS AND METHODS

Chemicals

Guanosine 5=-3-O-(thio)triphosphate (GTP�S) and 9-cis-reti-nal were purchased from Sigma-Aldrich (St. Louis, MO,USA). n-Dodecyl-�-d-maltoside (DDM) was obtained fromAffymetrix (Maumee, OH, USA). Bradford Ultra was pur-chased from Novexin (Cambridge, UK). 11-cis-retinal was agenerous gift from Dr. R. Crouch (Medical University ofSouth Carolina, Charleston, SC, USA). All-trans-retinal waspurchased from Toronto Research Chemicals (Toronto, ON,Canada).

Purification of Rho

Bovine rod outer segment (ROS) membranes were preparedfrom fresh retinas under dim red light, as described previ-

ously (33). These were solubilized in DDM and used for Rhopurification by a ZnCl2-opsin precipitation method (34).ZnCl2 was removed by a 48-h dialysis in the presence of 0.2mM DDM. Rho concentrations were measured with a UV-visible spectrophotometer (Cary 50; Varian, Palo Alto, CA,USA) and quantified by using the absorption coefficientε500nm � 40,600 M�1 cm�1 (35).

Purification of Gt

Gt was purified after extraction with hypotonic buffer fromROS membranes isolated from 200 dark-adapted bovineretinas as described previously (36). Briefly, ROS membraneswere resuspended in 30 ml of isotonic buffer, composed of 20mM HEPES (pH 7.5), 5 mM MgCl2, 1 mM dithiothreitol(DTT), and 100 mM NaCl, and soluble proteins were re-moved by gentle homogenization, followed by centrifugationat 25,000 g at 4°C for 15 min. Gt was extracted from the pelletby 3 washes with 30 ml of hypotonic buffer, composed of 5mM HEPES (pH 7.5), 0.1 mM EDTA, and 1 mM DTT;membranes were pelleted after each wash by centrifugation at25,000 g at 4°C for 30 min. All 3 wash supernatants werecombined and centrifuged at 25,000 g for 60 min to removeremaining ROS membrane contaminants. Then 1 M HEPES(pH 7.5) was added to the final supernatant to reach a finalconcentration of 10 mM, and MgCl2 was added to achieve a 2mM final concentration. The resulting sample in this equili-brating buffer was applied at a flow rate of 15 ml/h to a 10- �100-mm column loaded with 5 ml of preequilibrated pentyl-agarose resin, and the column was washed with 10 column volof equilibrating buffer. Bound proteins were eluted with a50-ml linear gradient of 0 to 0.5 M NaCl in the equilibratingbuffer at a flow rate of 15 ml/h, and 1-ml fractions werecollected. Fractions containing Gt were pooled and concen-trated by 30,000 normal molecular weight limit (NMWL)Centricon devices (Millipore, Billerica, MA, USA). Gt waspurified to homogeneity on a tandem Superdex 200 gelfiltration column equilibrated with buffer composed of 10mM HEPES (pH 7.5), 100 mM NaCl, 2 mM MgCl2, and 1 mMDTT at 4°C (flow rate 0.4 ml/min with collection of 0.5 mlfractions). Fractions containing Gt were combined andconcentrated by 30,000 NMWL Centricon devices (Milli-pore) to �10 mg protein/ml, as determined by the Brad-ford assay (37).

Purification and regeneration of Rho*-Gt and Rho*e-Gtcomplexes

The Rho*-Gt complex was purified by sConA affinity chroma-tography, as described previously (21). Briefly, the sConAaffinity resin was prepared by coupling sConA (Vector Labo-ratories, Burlingame, CA, USA) to CNBr-activated agarose(Santa Cruz Biotechnology, Santa Cruz, CA, USA) at a densityof 8 mg sConA/ml of resin. Purified Rho (200 �g) was dilutedto �0.2 mg/ml and loaded onto the sConA column (400 �l)equilibrated with an equilibrating buffer (20 mM BTP, pH6.9, containing 120 mM NaCl, 1 mM MnCl2, 1 mM CaCl2, 1mM MgCl2, 1 mM DTT, and 0.5 mM DDM). The resin waswashed with 5 column vol of the same buffer and thenilluminated for 10 min with a 150-W fiber light (Dolan JennerIndustries, Boxborough, MA, USA) delivered through a 480-to 520-nm bandpass filter. Purified native Gt (200 �g), dilutedto �0.2 mg/ml with this equilibrating buffer, was applied tothe column immediately after light exposure. Excess Gt waswashed out with 10 column vol of the above buffer. To obtainthe chromophore-depleted Rho*-Gt (Rho*e-Gt) complex, theRho*-Gt complex bound to the column was first washed with40 mM NH2OH in the equilibrating buffer. Excess NH2OH

1573ASYMMETRY OF RHODOPSIN DIMER

was washed out with 10 column vol of the equilibrationbuffer. Then Rho*-Gt or Rho*e-Gt complexes were eithereluted from the column with the elution buffer (equilibrationbuffer containing 200 mM �-methyl-d-mannoside) or regen-erated with a specified retinoid.

Alternatively, Rho itself was bound to the sConA affinityresin, and opsin was generated by light illumination (asdescribed above), followed by a prolonged wash (200 columnvol) with the equilibrating buffer, and eluted with the elutionbuffer. Opsin concentration was determined from its absorp-tion at 280 nm using the extinction coefficient ε � 81,200M�1 cm�1 (38).

Regeneration of Rho*-Gt and Rho*e-Gt complexes andopsin

To regenerate Rho*-Gt, Rho*e-Gt or opsin, a 2-fold molarexcess of either 9-cis-retinal, 11-cis-retinal, or all-trans-retinalwas used (see Results). An ethanolic solution of the selectedretinoid diluted with equilibration buffer (final ethanol con-centration 1%) was loaded on a column with specific boundcomplexes. The column then was closed for 1 h and kept at4°C in the dark. Excess retinal then was washed out with 10column vol of equilibrating buffer, and proteins were eluted.Fractions containing either Rho*-Gt, Rho*e-Gt, or chro-mophore-regenerated complexes were used for spectral anal-yses by UV absorption and/or biochemical analysis of reti-noid components.

UV-visible spectroscopy

UV-visible absorption spectra of purified Rho*-Gt, Rho*e-Gt,or complexes regenerated with either all-trans-retinal, 9-cis-retinal, or 11-cis-retinal were measured at 20°C with a Cary 50Varian spectrophotometer. To determine the presence of aSchiff base linkage vs. free retinal in the Rho*e-Gt complexregenerated with all-trans-retinal, we acidified the sample withH2SO4 to a final pH of 1.9. Retinylidene-lys296 has an absorp-tion peak at 440 nm, whereas maximum absorption of freeretinal is at 365 nm (39, 40).

Fluorescence measurements

A 2-fold molar excess of 11-cis-retinal or all-trans-retinal wasadded to freshly purified Rho*e-Gt or opsin (1 �M), dilutedwith buffer composed of 20 mM BTP (pH 6.9), 120 mM NaCl,1 mM MnCl2, 1 mM CaCl2, 1 mM MgCl2, 1 mM DTT, and 0.5mM DDM, and then quenching of intrinsic tryptophan fluo-rescence was measured with a Perkin Elmer L55 Lumines-cence Spectrophotometer (Perkin Elmer, Wellesley, MA,USA) at 20°C. Emission spectra at 0 and 10 min afterchromophore addition were recorded between 310 and 450nm after excitation at 295 nm with excitation and emissionslit bands set at 5 and 10 nm, respectively. For time-resolvedmeasurements, fluorescence emission spectra were recordedat 330 nm under conditions outlined above.

Dissociation of Rho*e-Gt or chromophore-regeneratedcomplexes by GTP�S

Rho*e-Gt, Rho*all-trans-RAL-Gt, Rho*9-cis-RAL-Gt, or Rho*11-cis-RAL-Gtcomplexes were all dissociated by adding 200 �M GTP�S while theywere still bound to an sConA affinity column.

Retinoid analyses

Rho*-Gt, Rho*e-Gt, opsin, or chromophore-regenerated com-plexes (�100 �g protein) were denatured for 30 min at room

temperature with 50% CH3OH in the presence of 40 mMNH2OH. The resulting retinal oximes were extracted with300 �l of hexane, and their isomeric composition was deter-mined by normal-phase HPLC with an Ultrasphere-Si, 5 �m,4.5- � 250-mm column (Beckman, San Ramon, CA, USA).Retinoids, eluted isocratically with 10% ethyl acetate inhexane at a flow rate of 1.4 ml/min, were detected by theirabsorption at 360 nm (41, 42). Retinoid oximes were quanti-fied based on areas under corresponding chromatographypeaks. Amounts of retinals were calculated based on a stan-dard curve presenting the correlation between areas andpeaks of synthetic standards for each retinal oxime isomer.

NaBH4 reduction of the Schiff base in Rho*all-trans-RAL-Gt orRho*

sConA resin with bound Rho*all-trans-RAL-Gt (3 ml) was trans-ferred from the column to a 50-ml tube as a 50% slurry. Thesecondary amine was produced by reduction of the Schiffbase bond in Rho with excess of NaBH4 by a proceduredescribed previously (43). Briefly, 6 mg of solid NaBH4 wasadded to a tube containing resin with bound regeneratedRho*all-trans-RAL-Gt complex and, following 30 min incubationon ice, the resin was loaded into the column again. NaBH4was washed out with 5 column vol of the equilibrating buffer,and Rho*all-trans-RAL-Gt containing the reduced Schiff basebond was eluted from the resin. Its UV-visible spectrumshowed a peak at 330 nm, indicating formation of secondaryamine, a product of Schiff base chemical reduction. Thisprotein was concentrated to �3 mg/ml and used for massspectrometry (MS) analyses.

Alternatively, the control sample of Rho* with trappedendogenous all-trans-retinal was prepared as follows: ROSmembranes (200 �l with �1 mg/ml Rho) were illuminatedfor 10 min at room temperature, and the Schiff base bondbetween isomerized all-trans-retinal and opsin was reducedwith NaBH4. NaBH4 (40 mg/ml) was added to the mem-branes in 5-�l aliquots 5 times at 5-min intervals. ExcessNaBH4 was removed by overnight dialysis against fresh H2O.Membranes were solubilized with 10 mM DDM and used forMS analyses.

MS identification of a Schiff base linkage in the Rho*e-Gtcomplex regenerated with all-trans-retinal

Rho*all-trans-RAL-Gt (or free Rho*; 20 �g) was digested over-night with pepsin (10 �g; Worthington, Lakewood, NJ, USA)at pH 2.5 and room temperature after the pH had beenlowered from 6.9 to 2.5 with 0.1% formic acid in H2O.Following this overnight procedure, the pH of the sample wasadjusted to 7.4 with 10 �l of 0.5 M Tris-HCl (pH 7.4), andpeptic fragments were acetylated by adding acetic anhydride(99.5%; Sigma) at a rate of 2.5 �l/10 min for 50 min. Theresulting sample was used for MS analysis with an LXQ massspectrometer (Thermo Scientific, Waltham, MA, USA)equipped with an electrospray ionization source that oper-ated in a positive mode with the capillary temperature set to350°C. The mass spectrometer was coupled to a HP 1100HPLC system (Agilent Technologies, Santa Clara, CA, USA).Peptide separation was achieved by a 2-pump controlledreverse phase elution setup with an aqueous phase composedof 0.1% formic acid in H2O (phase A) and an organic phasecomposed of 0.1% formic acid in acetonitrile (phase B). Thesample (100 �l) was loaded onto a Luna 20- � 2.00-mm C18column (Phenomenex, Torrance, CA, USA) equilibrated with98% phase A and 2% phase B for 30 min. Peptides wereeluted with the following gradients: 0 to 4 min, 98% phase Aand 2% phase B; 4 to 38 min, 2% phase A and 98% phase B.

1574 Vol. 27 April 2013 JASTRZEBSKA ET AL.The FASEB Journal � www.fasebj.org

Tandem MS (MS2) and triple MS (MS3) spectra were col-lected by using collision-induced dissociation with the nor-malized collision energy set to 35 kV. Spectra were analyzedand interpreted with Xcalibur software (ver. 2.1.0.1139;Thermo Scientific).

Preparation of retinoid-modified peptide standard

The 6-aa acetyl-FAKTSA peptide standard (the same that wasidentified in both Rho*all-trans-RAL-Gt complex and free R*)was custom ordered (EZBbiolab, Westfield, IN, USA) andconjugated with retinal as follows: 1 mg peptide was dissolvedin acetonitrile-methanol at a 1:1 ratio, mixed with 1 mg ofall-trans-retinal and 1% acetic acid, and incubated 48 h atroom temperature to form the Schiff base linkage, followingreduction with NaHB4. Solid NaHB4 (�2 mg) was added tothe sample, and the mixture was incubated for 30 min on ice.Then 500 �l H20 was added to react with excess NaHB4.Acetyl-FAK*TSA peptide (wherein K* is the retinoid modifiedlysine) was extracted with chloroform, dried in a SpeedVac(Thermo Scientific), and dissolved in 1:1 acetonitrile-metha-nol, and used immediately for MS analysis or stored at �20°C.

RESULTS

Gt prevents all-trans-retinal release fromphotoactivated Rho

Photoactivation of Rho results in sequential steps of11-cis-retinylidene chromophore isomerization to all-trans-retinylidene, followed by Schiff base linkage hy-drolysis and release of all-trans-retinal (44, 45). In itstransient state, Rho* binds to the heterotrimeric Gprotein, transducin, promoting dissociation of GDPfrom Gt� and formation of the transitory Rho*-Gtcomplex. Although short-lived in nature, this Rho*-Gtcomplex can be trapped as a stable intermediate in theabsence of nucleotides. Electron microscopy (EM) andsingle-particle reconstruction of this Rho*-Gt complexrecently revealed its pentameric assembly with a Rho toGt stoichiometry of 2:1 (21). This complex exhibits aUV-visible maximum absorption peak at 380 nm andcontains only all-trans-retinal bound, indicating that Gtstabilizes the activated conformation of Rho and inhib-its retinal release from the retinal-binding pocket.However, the chromophoric stoichiometry of this sta-bilized adduct has not been documented. To deter-mine the effect of Gt on retinal release from thechromophore-binding pocket of photoactivated Rho,we prepared sConA resin that bound either the Rho*dimer or the Rho*-Gt complex. Extensive washing ofresin bound-Rho* with buffer containing DDM resultedin nearly complete depletion of the chromophore (Fig. 1;retinoid analysis plots, left top and bottom panels, alsomodeled yellow-yellow Rho dimer transitioned to gray-grayRho dimer, shown on the left). In contrast, chromophorerelease from the Rho*-Gt complex was only partial, with�50% of all-trans-retinal remaining trapped in thechromophore-binding pocket (Fig. 1; retinoid analysisplots, right top panel and bottom left panel, alsomodeled yellow-yellow Rho dimer coupled to Gt transi-tioned to yellow-gray Rho dimer coupled to Gt shown

on the right), indicating a protective role of bound Gt.To achieve complete chromophore release from theRho*-Gt complex, a wash with NH2OH was applied topromote Schiff base hydrolysis. The resulting chro-mophore-depleted Rho*e-Gt complex remained intact(Fig. 1; model of gray-gray Rho coupled to Gt) butsensitive to dissociation by GTP�S (Fig. 1; SDS-PAGEgels, bottom center, also model of gray-gray Rho cou-pled to Gt transitioned to gray-gray Rho dimer).

Binding of cis-chromophore to the Rho*e-Gt complex

To determine whether the chromophore-depletedRho*e-Gt complex could be regenerated with 11-cis-retinal to form Rho or with 9-cis-retinal to form isoRho,the specific retinoid was added to Rho*e-Gt bound tothe sConA resin and excess retinoid was washed out.Formation of Rho or isoRho still coupled to Gt wasmonitored immediately after their elution from thecolumn by either UV-visible absorption spectroscopy orHPLC analyses of retinoids extracted from the regen-erated complexes. Regeneration of Rho*e was docu-mented by an increased maximum absorption at 485 or500 nm after Rho*e-Gt (Fig. 2; model of gray-gray Rhodimer coupled to Gt) incubation with 9-cis-retinal or11-cis-retinal, respectively (Fig. 2A). (The peak at �380nm observed in both samples could result from adeprotonated form of the Schiff base). HPLC retinoidanalyses of the complex regenerated with 9-cis-retinal(Rho9-cis-RAL-Gt) primarily identified 9-cis-retinal (95%)with only a small fraction of all-trans-retinal (5%),indicating that both Rho molecules of the dimer hadbeen regenerated with 9-cis-retinal (Fig. 2B, light blueline; also model of pink-pink Rho dimer coupled to Gt).However, a �1:1 ratio of 11-cis-retinal (44%) and all-trans-retinal (56%) was identified in the Rho*11-cis-RAL-Gt complex(Fig. 2B, dark blue line). Therefore, only one Rho of thedimer could be regenerated with 11-cis-retinal, whereas thesecond Rho monomer promoted the isomerization ofthe less stable 11-cis-retinal to all-trans-retinal, theconformation present in the active state of Rho (Fig.2; model of yellow-red Rho dimer coupled to Gt).Both Rho*11-cis-RAL-Gt (Fig. 2C) and Rho9-cis-RAL-Gt(not shown) remained functionally active after elu-tion from their respective sConA columns, becauseboth were sensitive to dissociation by GTP�S. Afterdissociation of the Rho*11-cis-RAL-Gt complex and lossof protection ensured by bound Gt, all-trans-retinalwas released from the chromophore binding pocket,resulting in the formation of the Rho*e-Rho dimer,where one Rho monomer is in an opsin-like confor-mation, and the second in a ground, 11-cis-retinalbound state (Fig. 2; model of gray-red Rho dimer).

Quenching of intrinsic tryptophan fluorescence byincorporation of 11-cis-retinal and all-trans-retinal intoRho*e-Gt or opsin

Here interactions between retinal and protein weremonitored by recording changes in the intrinsic fluo-

1575ASYMMETRY OF RHODOPSIN DIMER

rescence of tryptophan. Addition of a 2-fold molarexcess of either 11-cis-retinal or all-trans-retinal to thesample rapidly generated retinoid uptake signals forthe chromophore-depleted samples of opsin (Fig. 3C,D; insets) and Rho*e-Gt complex (Fig. 3), manifested asquenched intrinsic protein fluorescence. These obser-vations indicated formation of a complex betweenRho*e in the Rho*e-Gt complex and either 11-cis-retinalor all-trans-retinal (Fig. 3; models of yellow-red Rhodimer coupled to Gt and yellow-gray Rho dimer cou-pled to Gt, respectively). Remarkably, while the fluores-cence of free opsin was quenched by 11-cis-retinal, onlya minor change in opsin intrinsic fluorescence wasobserved after addition of all-trans-retinal (Fig. 3C, D;insets). These results indicate a structural differencebetween retinal-free Rho*e in the Rho*e-Gt complexand opsin.

Asymmetry of Rho monomers in the Rho*-Gtcomplex

As described above, Gt bound to photoactivated Rhoinhibited release of all-trans-retinal, trapping �50% ofthis isomerized endogenous chromophore in its bind-ing pocket. This uneven protection of Rho monomers

comprising the dimer demonstrates asymmetric prop-erties of Rho molecules in the Rho*-Gt heteropentamer(Fig. 4; model of yellow-gray Rho dimer coupled to Gt).To investigate this phenomenon further, we incubatedthe Rho*-Gt complex with an excess of 9-cis-retinal andmonitored product regeneration by UV-visible absor-bance spectroscopy and retinoid analyses. The Rho*-Gtcomplex exhibited a UV-visible absorption spectrumwith a major peak at �380 nm, whereas peaks at 380and 485 nm were detected in the sample regeneratedwith 9-cis-retinal (Fig. 4B). Though only all-trans-retinalwas detected in the Rho*-Gt complex (Fig. 4A; blackspectrum and model of yellow-gray Rho dimer coupledto Gt), a mixture of all-trans-retinal and 9-cis-retinal witha 1:1 stoichiometry was detected in the Rho*9-cis-RAL-Gtcomplex (Fig. 4A; very dark gray spectrum and modelof yellow-pink Rho dimer coupled to Gt), indicatingthat bound Gt prevents release of all-trans-retinal fromonly one Rho molecule, while the second one eventu-ally loses its chromophore and can be loaded with9-cis-retinal. This result demonstrates functional andstructural asymmetry of Rho dimer triggered by Gtbinding. Regeneration of the Rho*-Gt with 9-cis-retinaldid not affect the integrity of the complex (Fig. 4C).However, exposure of the regenerated Rho*9-cis-RAL-Gt

Wash (column volumes)0 50 100 150 200

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Figure 1. Protection of the chromophore-protein linkage in the Rho*-Gt complex. Activated Rho (Rho*, yellow-yellow dimer onthe left) and the Rho*-Gt complex (yellow-yellow dimer bound to Gt on the right), each bound to sConA resin, and washed withincreasing volumes of equilibrating buffer and used for retinoid analyses. Similar levels of all-trans-retinal were detected in theinitial Rho* and Rho*-Gt samples (top left panel, dark green line; and top right panel, dark blue line). Washing bound Rho*-Gtwith 20 column vol of equilibrating buffer resulted in a decrease of all-trans-retinal to about half the original amount, whichremained unchanged after additional washing with 200 column vol (depicted as a yellow-gray dimer bound to Gt). However,similar extensive washing of bound Rho* resulted in almost complete depletion of all-trans-retinal (left top and bottom panels,green lines; modeled as a gray dimer on the left). Complete chromophore release from Rho*-Gt was obtained only after a washwith NH2OH (right top panel, light blue line; modeled as a gray-gray dimer bound to Gt). Rho*e-Gt eluted from the sConAaffinity resin and separated by SDS-PAGE contained both Rho*e and Gt (bottom central panel, top gel). Asterisk indicates thepeak fraction, containing Rho*e-Gt. Functionality of this complex was evidenced by its dissociation with 200 �M GTP�S (bottomcentral panel, bottom gel). Pound signs indicate fractions containing dissociated Gt; asterisk indicates the peak fractioncontaining mostly Rho*e.

1576 Vol. 27 April 2013 JASTRZEBSKA ET AL.The FASEB Journal � www.fasebj.org

complex to 200 �M GTP�S promoted release of theendogenous all-trans-retinal originally trapped by Gt inthe chromophore-binding pocket (Fig. 4A; dark grayspectrum and model of gray-pink Rho dimer). A signif-icant reduction of all-trans-retinal but not 9-cis-retinal inthe intact Rho*9-cis-RAL-Gt complex was also achieved bytreatment with NH2OH (Fig. 4A; light gray spectrumand model of gray-pink Rho dimer coupled to Gt).

All-trans-retinal forms a Schiff base linkage whenadded to the Rho*-Gt complex

In dark-state Rho, 11-cis-retinal, and, in the Rho-acti-vated state, all-trans-retinal, are covalently attached toLys296 in the chromophore-binding pocket. However,other than this main retinal-binding site, secondarybinding sites exist on the surface of opsin where retinalcan bind noncovalently (46). As previously reported(47), all-trans-retinal added to detergent-solubilized op-sin does not form a Schiff base linkage with Lys296

but, rather, attaches noncovalently. In the presentstudy, we investigated whether a Schiff base bond isformed between added all-trans-retinal and Lys296 ofchromophore-depleted Rho*e in the Rho*e-Gt com-plex. First, the Schiff base bond in the regeneratedRho*all-trans-RAL-Gt complex was stabilized by reductionwith NaBH4, resulting in creation of a stable secondaryamine from n-retinylidene Schiff base exhibiting a UVabsorption peak at 330 nm (Fig. 5A). This sample wasacidified and digested with pepsin, and then the prod-ucts were acetylated and analyzed by MS. This proce-

dure resulted in the identification of an acetyl-FAK*TSA peptide containing Lys296 with the attachedall-trans retinoid (m/z of 934.5 [MH]; Fig. 5B). TheMS2 of this singly charged ion led to the identificationof 2 major ions, one with m/z 269.3, corresponding tothe anhydroretinol moiety, and the other with m/z666.5, corresponding to the peptide alone (Fig. 5C).The same result was obtained with a Rho* sampleprepared by illumination of ROS membranes, which,following NaBH4-Schiff base reduction, carried endog-enous all-trans-retinal (Fig. 5D). Fragmentation of them/z 269.3 product revealed its identity as an anhydro-retinol carbocation (Fig. 5E), whereas fragmentationof the second m/z 666.5 product produced a MS3

spectrum that corresponded to the retinoid-freeacetyl-FAK*TSA peptide (Fig. 5F). To further test thisinterpretation, we generated a synthetic standardacetyl-FAK*TSA peptide. This synthetic peptide exhib-ited a MS identical to that of the proposed acetyl-FAK*TSA peptide (Fig. 6A) as identified peptide. TheMS2 of this singly charged ion also yielded 2 major ions,one with m/z 269.3, corresponding to the retinoid, andthe other with m/z 666.5, corresponding to the peptidealone (Fig. 6B). Fragmentation of the m/z 269.3 ion wasidentical to the same ion in both the acetyl-FAK*TSApeptide standard (Fig. 6C) and the retinylamine reti-noid standard (Fig. 6E, F, respectively). Fragmentationof m/z 666.5 was identical to the same ion present inthe acetyl-FAK*TSA peptide standard (Figs. 5F and 6D,respectively). These results clearly demonstrate that Gt

Time (min)

6 8 10 12

Abs

orba

nce

at 3

60 n

mGTPγS

Gt

11-cis-RAL

UB W 1 2 3 4 5 6 7 UB W 1 2 3 5 7GTP

* # # *tRho* -G11-cis-RALC

Gt

GTPγS

Rho

GtαGtβ

A

B

11-cis-RAL-oxime

all-trans-RAL-oxime

9-cis-RAL-oxime

Wavelength (nm)

300 400 500 600

Abs

orba

nce

(AU

)

0.00

0.05

0.10

0.15

0.20 Rho* -Gte

tRho* -G11-cis-RAL

tRho -G9-cis-RAL

9-cis-RAL

all-trans-RAL-oximeGTPγS

Gt20

mA

U+ all-trans-RAL

Figure 2. Regeneration of the Rho*e-Gt com-plex with either 11-cis-retinal or 9-cis-retinal.Rho*e-Gt was prepared by treatment of theRho*-Gt complex with NH2OH (modeled as gray-gray dimer bound to Gt). While still bound to thecolumn, chromophore-depleted Rho*e-Gt was re-generated with a 2-fold molar excess of either11-cis-retinal (shown as a yellow-red dimerbound to Gt) or 9-cis-retinal (shown as pink-pink dimer bound to Gt). Excess retinoid waswashed out, and regenerated complexes wereeluted and analyzed by UV-visible absorbancemeasurements and retinoid oxime quantifica-tion. A) UV-visible absorption spectra of Rho*e-Gt, Rho*11-cis-RAL-Gt and Rho9-cis-RAL-Gt areshown with black, dark blue, and light bluelines, respectively. B) Isocratic analyses of reti-noid oximes extracted from Rho*11-cis-RAL-Gt(dark blue line) and Rho9-cis-RAL-Gt (light blueline) complexes. Most of the 9-cis-retinal (95%)and a small fraction of all-trans-retinal (5%)were identified in the Rho9-cis-RAL-Gt complex,whereas a mixture of 11-cis-retinal (44%) andall-trans-retinal (56%) were found in theRho*11-cis-RAL-Gt complex. C) SDS-PAGE gel ofRho*11-cis-RAL-Gt purified by sConA affinitychromatography (left panel). Asterisk inciatesthe eluted peak fraction containing both Rhoand Gt. Functionality of this complex was evi-denced by its dissociation with 200 �M GTP�S.

Pound signs indicate fractions containing dissociated Gt. Asterisk indicates peak fraction eluted from the column containingmostly Rho.

1577ASYMMETRY OF RHODOPSIN DIMER

bound to the chromophore-depleted Rho*e dimer sta-bilizes the active conformation of Rho, allowing accessof exogenous all-trans-retinal to the retinal-bindingpocket and reformation of a Schiff base linkage withLys296.

DISCUSSION

Convincing evidence indicates that GPCR dimerizationand activation are intricately associated. The functionalconsequences of GPCR dimerization have been re-vealed by studies showing intercommunication between2 protomers in these dimers (19, 48–50). Whereas suchcooperation can be relatively easily proven for het-erodimers, it is more challenging for homodimericGPCRs.

Here we studied the properties of dimeric Rho, aprototypic member of family A GPCRs. In native mem-branes, Rho exists as densely packed dimers organizedin rows (13). Although the functional relevance ofRho’s dimeric nature is highly debated, we have shownby EM and single-particle reconstruction that, in re-sponse to light, a Rho dimer is required for binding toa single G protein (21). Until now it has been unclearwhether both Rho monomers within the activateddimer are structurally and functionally equivalent.

Therefore, by unequivocally tracing each Rho mono-mer with an isomeric variant of the chromophore, weprobed the symmetry of a Rho dimer within the stableRho*-Gt complex. This strategy indicated that eachmonomer in the Rho dimer coupled to Gt exhibiteddifferent conformational properties (Fig. 7). Only oneRho, most likely the one hosting the C terminus of Gt�,is stabilized in its active Meta II state with its chro-mophore, all-trans-retinal, trapped in the retinal-bind-ing pocket. But at the same time, all-trans-retinal isreleased from the second Rho, which eventually decaysto opsin and free retinal (Fig. 7; yellow-gray Rho dimerbound to Gt). Interestingly, this chromophore-freeRho*e molecule can be regenerated with 9-cis-retinalwithout affecting the integrity and activity of the wholeRho*9-cis-RAL-Gt complex. As evidenced by HPLC reti-noid analysis, such a regenerated complex retainedboth all-trans-retinal and 9-cis-retinal in a 1:1 stoichiom-etry, demonstrating unambiguously that 2 Rho mole-cules are bound to 1 Gt, which influences their func-tional and structural distinction (Fig. 7; yellow-pinkRho dimer bound to Gt). Stabilized by Gt, a Rhomonomer can lose its chromophore only after complexdissociation with GTP�S, or its hydrolysis can be ef-fected by treatment of the intact complex with thestrong nucleophile NH2OH, resulting in formation ofthe chromophore-depleted Rho*e-Gt complex (Fig. 7;

11-cis-RAL

Flu

ores

cenc

e at

330

20

40

60

80

Time (min)

Flu

ores

cenc

e at

330

nm

20

40

60

80

0 10 20 30 40 320 360 400 440

Flu

ores

cenc

e In

tens

ity

0

100

200

300

Wavelength (nm)

Flu

ores

cenc

e In

tens

ity

0

100

200

300

+ 11-cis-RAL

+ all-trans-RAL

Wavelength (nm)320 360 400 440F

luor

esce

nce

Inte

nsity

0

100

200

300

Wavelength (nm)320 360 400 440F

luor

esce

nce

Inte

nsity

0

100

200

300

+ 11-cis-RAL

+ all-trans-RAL

Opsin

Opsin

A

B

C

D

all-trans-RALLLLLLL

0 10 20 30 40 320 360 400 440

Figure 3. Fluorescence changes in Rho*e-Gt complex evoked by addition of either 11-cis-retinal or all-trans-retinal. Quenchingof the intrinsic tryptophan fluorescence was measured on addition of a 2-fold molar excess of either 11-cis-retinal orall-trans-retinal to the chromophore-depleted Rho*e-Gt complex (shown as a gray-gray dimer bound to Gt; complex regeneratedwith 11-cis-retinal is modeled as yellow-red dimer bound to Gt, and the complex regenerated with all-trans-retinal is pictured asyellow-gray dimer bound to Gt). A, B) Time-dependent changes in Rho*e-Gt fluorescence emission were recorded at 330 nmwith �ex � 295 nm. After 10 min of recording, either 11-cis-retinal (A) or all-trans-retinal (B) was added, and the resulting rapiduptake signal was recorded. C, D) Fluorescence quenching of Rho*e-Gt was observed after uptake of either 11-cis-retinal (C) orall-trans-retinal (D), whereas marked fluorescence quenching of opsin itself was observed only after uptake of 11-cis-retinal(compare C, D, insets).

1578 Vol. 27 April 2013 JASTRZEBSKA ET AL.The FASEB Journal � www.fasebj.org

gray-gray Rho dimer bound to Gt). Such a complex isstill active and can be regenerated with different iso-meric variants of chromophore. Interestingly, HPLCanalysis of retinoids extracted from the Rho*e-Gt com-plex after incubation with 11-cis-retinal resulted in amixture of 11-cis-retinal and all-trans-retinal in �1:1ratio, indicating that only one Rho molecule in thedimer was regenerated with 11-cis-retinal, whereas theother was bound to all-trans-retinal (Fig. 7; yellow-redRho dimer bound to Gt). The only possible explanationof this unexpected discovery is that one of the Rhomonomers is stabilized by Gt in its active Meta II-likestate even after chromophore depletion. Consequently,the rigid constraints of this protein force the isomer-ization of 11-cis-retinal to its all-trans conformer, be-cause only the latter can fit the chromophore-bindingpocket of Meta II-like Rho. This protein-forced retinalisomerization, however, was not observed for the morestable 9-cis-retinal, which regenerated both Rho mole-cules of the Rho*e-Gt complex (Fig. 7; pink-pink Rhodimer bound to Gt).

Another key finding here is that chromophore-de-pleted Rho*e bound to Gt could uptake exogenousall-trans-retinal into its empty retinal-binding pocketand reform a Schiff base linkage between this retinaland its reactive lysine K296. But as shown previously(47), all-trans-retinal does not reenter the chro-mophore-binding pocket of free opsin, presumablybecause the conformational equilibrium is shifted to-

ward the inactive conformation. Moreover, all-trans-retinal can enter only one of the two retinal-free Rho*emolecules in the Rho*e-Gt complex, implying that,unlike the Rho monomer in the opsin conformation,only the Rho* monomer stabilized by Gt in the MetaII-like state can provide access to all-trans-retinal (resultsnot shown; Fig. 7; yellow-gray Rho dimer bound to Gt).

Rho’s agonist and antagonist ligands are covalentlylinked to the opsin moiety, thus allowing an excess offree ligand to be washed out (51). Indeed, the chro-mophore-opsin interaction gives rise to specific absorp-tion spectra characteristic of both the chromophoreand the activated state of the receptor. Thus the switchfrom agonistic (cis-retinoid) to antagonistic (all-trans-isomer) retinoid can be readily monitored by UV-visiblespectroscopy, whereas the presence of chromophore inthe binding site can be followed by fluorescence spec-troscopy that reveals the quenching of fluorescenceemanating from the specific Trp residues involved inretinoid binding (47, 52). Moreover, because of thehigh absorption coefficient of the chromophore, itsisomeric nature can be determined by chemical analysisusing HPLC and MS even on small sample sizes. Finally,this methodology can be applied to unmodified Rho/opsin-transducin complexes isolated from nativesources, e.g., bovine eyes. All these advantages overother family A GPCR systems enabled us to study thenature of each Rho monomer within Gt-bound penta-meric complex and the dimeric complex without Gt.

9-cis-RAL

Rho*

GtαGtβ

Wavelength (nm)

300 400 500 600

Abs

orba

nce

(mA

U)

0.0

0.1

0.2

0.3

B

all-trans-RAL-oxime9-cis-RAL-oxime

UB W 1 2 3 4 5 6

* tRho* -G9-cis-RAL

tRho* -G9-cis-RAL

tRho*-G

GTPγS

Gt

NH OH2

C

Time (min)

6 8 10 12

Abs

orba

nce

at 3

60 n

m

A

tRho*-G

+ 9-cis-RAL

+ GTPγS

+ NH OH2

20 m

AU

+ all-trans-RAL

Figure 4. Asymmetry of Rho monomers in theRho*-Gt complex. The Rho*-Gt complex pre-pared on an sConA affinity resin (modeled as ayellow-gray dimer bound to Gt) was regeneratedwith 9-cis-retinal (shown as a yellow-pink dimerbound to Gt), followed by either dissociation withGTP�S (depicted as a gray-pink dimer) or treatmentwith NH2OH (modeled as a gray-pink dimer boundto Gt). UV-visible absorbance spectra and retinoidcomposition of those complexes then were ana-lyzed. A) Isocratic analyses of retinoid oximes ex-tracted from Rho*-Gt (black line), regeneratedwith 9-cis-retinal, Rho*9-cis-RAL-Gt (very dark grayline), Rho*9-cis-RAL-Gt treated with either GTP�S(dark gray line) or with NH2OH (light gray line)are shown. Although only all-trans-retinal was de-tected in the Rho*-Gt complex, a mixture ofall-trans-retinal and 9-cis-retinal with a 1:1 stoichi-ometry was found in the Rho*9-cis-RAL-Gt complex.Treatment of Rho*9-cis-RAL-Gt with GTP�S re-sulted in significant reduction in all-trans-retinal, and an even further reduction wasobtained after a wash with NH2OH. B) UV-visible absorption spectra of Rho*-Gt and regen-erated Rho*9-cis-RAL-Gt are shown by black andlight blue lines, respectively. C) SDS-PAGE gel ofRho*9-cis-RAL-Gt purified by sConA affinity chro-matography. Asterisk indicates the peak fractioncontaining both Rho* and Gt.

1579ASYMMETRY OF RHODOPSIN DIMER

The results also provide direct evidence for a differentconformational arrangement of Rho monomers withinthe dimer after assembly with Gt.

When high-resolution structures of vertebrate (4)and invertebrate (53) Rho molecules and structures ofGPCR-interacting proteins, such as G proteins (54),arrestins (55), and GRKs (56, 57) became available, ittranspired that GPCRs, as well as their partner proteins,share striking structural similarity (average root-mean-

square deviation 2 Å). Along with structural informa-tion, physiological findings also support the functionalhetero-oligomerization of GPCRs. For example, themGluR family of receptors’ activation mechanism in-volves a change in quaternary structure of two obliga-tory monomers coupled to each other by a disulfidebridge (58, 59); the GABAB receptor functional unit isan obligate heterodimer composed of GABAB1 andGABAB2 subunits (60–62); and obligate heterooligom-

m/z300 400 500 600 700

Rel

ativ

e in

tens

ity (

%)

0

20

40

60

80

100

269.3

666.5MS2

m/z 934.5Acetyl-F A K T S A

269.3

666.5

A

m/z200 300 400 500 600 700

Rel

ativ

e in

tens

ity (

%)

0

20

40

60

80

100

934.5 666.5 MS3

m/z 666.5

Acetyl-F A K T S A

648.8

-H2O

648.8

490.2

490.2

577.2

577.2389.2

389.2477.2

477.2

406.2

406.2

648.8

m/z50 100 150 200 250 300

Rel

ativ

e in

tens

ity (

%)

0

20

40

60

80

100

934.5 269.3 MS3

m/z 269.3213.

2

227.

2

CH2+

B

C DTime (min)

14 16 18 20 22 24

Rel

ativ

e in

tens

ity (

%)

0

20

40

60

80

100

Base peak of m/z 934.5Acetyl-F A K T S A

m/z

Rel

ativ

e in

tens

ity (

%)

0

20

40

60

80

100

m/z300 400 500 600 700

Rel

ativ

e in

tens

ity (

%)

0

20

40

60

80

100

269.3

666.5MS2

m/z 934.5Acetyl-F A K T S A

269.3

666.5

E F

Wavelength300 400 500 600

Abs

orba

nce

(mA

U)

0.00

330 nm0.05

0.10

0.15

0.20

0.25

135.

0

189.

1

934.5

935.5

936.5

Figure 5. MS identification of the peptide forming a Schiff base linkage with retinal in the Rho*-Gt complex regenerated withall-trans-retinal. Analysis of peptic, acetylated fragments of the Rho*all-trans-RAL-Gt complex (or free Rho*) with a NaHB4-reducedSchiff base bond identified a retinal-bound acetyl-FAK*TSA peptide containing Lys296 (where K* is lysine modified withretinoid). A) UV-visible absorbance spectrum of the Rho*all-trans-RAL-Gt complex with a NaHB4-reduced Schiff base linkage showsan absorption maximum peak at 330 nm. B) Base peak of m/z 934.5 was detected after loading acetylated peptic fragmentsresulting from digestion of a Rho*all-trans-RAL-Gt sample. This m/z 934.5 single-charged species was eluted from a Luna 20- �2.00-mm C18 column (Phenomenex) in 18 to 19 min. Inset: isotopic distribution in the single-charged peptic peptide ion. C,D) MS2 of the m/z 934.5 singly charged ion identified as acetyl-FAK*TSA in Rho*all-trans-RAL-Gt and free Rho* in ROSmembranes, respectively. Major ions observed at m/z 269.3 (for retinoid) and at m/z 666.5 (for acetyl-FAKTSA peptide) wereidentical with those identified in the acetyl-FAK*TSA peptide standard (Fig. 6B) and retinylamine retinoid standard (Fig. 6E).E) Fragmentation of the m/z 269.3 ion derived from the m/z 934.5 parent ion obtained from a Rho*all-trans-RAL-Gt sampleindicates a retinoid ion (see structural formula). F) Fragmentation of m/z 666.5 ion derived from the m/z 934.5 parent ionobtained from the Rho*all-trans-RAL-Gt complex indicates an acetyl-FAKTSA peptide.

1580 Vol. 27 April 2013 JASTRZEBSKA ET AL.The FASEB Journal � www.fasebj.org

ers of taste receptors exist for sweet and umami re-sponses (63, 64). These were the first examples thatunequivocally established dimerization and its func-tional importance. Mindful that the function of allGPCRs depends on intermolecular interactions with Gproteins, GRKs, and arrestins along with additionalevidence that GPCRs have a propensity to form homo-and heterooligomers, it seems logical that dimerizationis not a unique characteristic of only a few receptors butrather a property of a whole subclass of GPCRs. Mod-eling studies also indicated that the Rho dimer offers ageometrically compatible platform for the binding of

partner proteins, such as Gt or arrestin, because each ofthe latter exhibit a “footprint” larger than that of a Rhomonomer (32, 65, 66). The Rho-Gt-induced fit modelimplies that activation could involve the relaxation of asomewhat more rigid structure constituting the inactivestate of the receptor, following a further increase of itsrigidity on Gt binding (67). This process occurs differ-entially within GPCR monomers, as shown in this work;thus, each Rho* provides a different platform for aninteraction with Gt. This interpretation agrees with therecent structural model for the photoactivated Rho*-Gtcomplex featured in a low-resolution 3-dimensional

Time (min)14 16 18 20 22 24

Rel

ativ

e in

tens

ity (

%)

0

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40

60

80

100

SIM m/z 269.3

m/z200 300 400 500 600 700

Rel

ativ

e in

tens

ity (

%)

0

20

40

60

80

100

934.5 666.5MS

3

m/z 666.5

Acetyl-F A K T S A

648.8

-H2O

648.8

490.2

490.2

577.2

577.2389.2

389.2

477.2 477.2

406.2

406.2

NH2

269.3 Da

Time (min)14 16 18 20 22 24

Rel

ativ

e in

tens

ity (

%)

0

20

40

60

80

100

Base peak of m/z 934.5

Acetyl-F A K T S A

m/z

Rel

ativ

e in

tens

ity (

%)

0

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80

100

m/z200 300 400 500 600 700 800 900

Rel

ativ

e in

tens

ity (

%)

0

20

40

60

80

100 Acetyl-F A K T S A

MS2

m/z 934.5

269.3

666.5

269.3

666.5

490.2

490.2

648.3

-H2O

648.3758.5

758.5

m/z50 100 150 200 250 300

Rel

ativ

e in

tens

ity (

%)

0

20

40

60

80

100

934.5 269.3MS3

m/z 269.3

213.

2

227.

2199.

1CH2

+

A B

C D

m/z50 100 150 200 250 300

Rel

ativ

e in

tens

ity (

%)

0

20

40

60

80

100

MS2

m/z 269.3213.

2

227.

2199.

2

CH2+E F

189.

1

189.

1

95.0

95.0

135.

0

149.

1

135.

0

149.

1

934.5

935.5

936.5

Figure 6. MS characterization of the acetyl-FAK*TSA peptide and retinylamine retinoid standards used to analyze MS data in Fig.5. A) Base peak of m/z 934.5 representing the [MH] ion of the acetylated-retinoid modified peptide standard with theacetyl-FAK*TSA sequence, prepared in order to confirm peptide identification from Rho*all-trans-RAL-Gt and Rho* samples. Inset:isotopic distribution of the m/z 934.5 peak with an elution time of 18 to 19 min. B) MS2 spectra of m/z 934.5. Representativeions resulting from collision-induced differentiation are attributed to the retinoid ion with m/z 269.3 and the acetylated peptideacetyl-FAKTSA at m/z 666.5. Several other ions shown were identified as collision induced dissociation (CID) productsoriginating from the acetyl-FAKTSA peptide fragment. C) Fragmentation of m/z 269.3 originating from the MS2 of m/z 934.5described in B. D) Fragmentation of m/z 666.5 originating from the MS2 of m/z 934.5 described in B. E) Selected ion monitoring(SIM) for m/z 269.3 of the retinoid standard (retinylamine) eluted from Luna 20- � 2.00-mm C18 column (Phenomenex).F) MS2 spectra of m/z 269.3 from the SIM experiment with the retinoid standard shown in E.

1581ASYMMETRY OF RHODOPSIN DIMER

map obtained by EM-single particle reconstruction (21,31). Its molecular envelope accommodated two Rhomolecules together with one Gt heterotrimer, consis-tent with a heteropentameric structure of this complexas shown by the present biochemical approach. Thisevidence sharply contrasts with that derived from crys-tal structures of an active state complex comprised ofagonist-occupied monomeric �2-adrenergic receptor-T4L-lyzosome fusion, nucleotide-free Gs heterotrimer,and a nanobody, which provides the most unexpectedstructure, with a major displacement of the entire�-helical domain of G� relative to the Ras-like GTPasedomain (68). The latter is a rigid body type of displace-ment, where one subdomain dives into the lipid bilayerwith an averaged angle between subdomains of �130°,whereas the distance between the �-helical subdomainsof Gt and Gs was found to be �40 Å (68). Thus, for thetwo domains to superpose, a full �180° rotation of theGs�-�-helical subdomain alone is required. Therefore,the relevance of this monomeric GPCR structuralmodel to general GPCR physiology, in our view, re-mains an open question.

Together with our EM structural data, the resultspresented here unambiguously demonstrate that after

light activation, a single Gt heterotrimer binds to a Rhodimer (21, 31). In the Rho*-Gt complex thus formed,each Rho monomer is structurally different, and onlyone is stabilized in the active Meta II state by bound Gt,while the other evolves into an opsin-like conformation.Although the physiological role of this dimer asymme-try has yet to be clarified, regulation of the clearance ofexcess all-trans-retinal produced under bright light con-ditions, where Rho*-Rho* dimer would be at a signifi-cantly higher level as compared to Rho-Rho* as amechanism protecting rod cells, is an intriguing possi-bility. The concept of Rho dimer asymmetry agrees alsowith possible cross-communication between GPCRprotomers within a dimer. Rho and Rho* share thesame recognition mode for Gt (69). Therefore it ispossible that precoupled Rho-Gt complexes exist in thedark. Taking into account that only one Rho moleculewithin the dimer can provide binding determinants forGt�, a possibility of asymmetric Rho activation arises. Insuch situations, photoexcitation of chromophore andits isomerization in the Rho monomer that does notphysically interact with Gt�, would induce conforma-tional changes in the precoupled Rho monomer, lead-ing to conformational evolution toward the Meta II

GTPγS

Gt

Gt

NH OH2

11-cis-RAL

Gt

Wash

NH OH

2

GTPγS

9-cis-RAL

all-trans-R

AL

9-cis-RAL

NH OH2GTPγS

Gt

Gt

GTPγS

Gt

GTPγS

+ all-trans-RAL

+ all-trans-RAL+ all-trans-RAL

+

LS

RAL

L

Figure 7. Asymmetry of Rho* dimer within Rho*-Gt complex. The Rho*-Gt complex is formed by binding of a fully activated Rhodimer to Gt. After light illumination, 11-cis-retinal of ground state Rho (shown as red-red dimer) isomerizes to all-trans-retinal(yellow-yellow dimer), allowing binding of the heterologous Gt trimer (Gt� is depicted in salmon, Gt� in green, and Gt� in lightblue). This binding prevents chromophore release from one Rho in the dimer, introducing dimer asymmetry in the Rho*-Gtcomplex (shown after a wash as a yellow-gray dimer bound to Gt). Release of chromophore from the Gt-protected Rho moleculecan be promoted by NH2OH, resulting in formation of the chromophore-free Rho*e-Gt complex (modeled as a gray-gray dimerbound to Gt). This complex then can be either dissociated with GTP�S (shown as gray-gray dimer) or regenerated. 9-Cis-retinalregenerates both Rho monomers in the dimer (shown as a pink-pink dimer bound to Gt). However, 11-cis-retinal regeneratesonly one Rho monomer, while the second Rho promotes isomerization of 11-cis-retinal to its all-trans conformer, resulting information of an asymmetric dimer (indicated by a yellow-red dimer bound to Gt). Both complexes regenerated with either 9-cis-or 11-cis-retinal can be dissociated with GTP�S (shown as pink-pink or gray-red models, respectively). But regeneration withall-trans-retinal results in its uptake by only one Rho monomer (shown as a yellow-gray dimer bound to Gt), and the second Rhocan be regenerated with 9-cis-retinal (shown as yellow-pink dimer bound to Gt). All-trans-retinal can be released from suchcomplexes by either NH2OH (modeled as a gray-pink dimer bound to Gt) or GTP�S (imaged as a gray-pink dimer).

1582 Vol. 27 April 2013 JASTRZEBSKA ET AL.The FASEB Journal � www.fasebj.org

state, needed to form the active Rho*-Gt complex (70,71). Therefore, this tandem mechanism could be crit-ical to avoid loss of energy, enhance the efficiency ofreceptor activation, and result in desensitization. Be-cause the in vitro assay of Gt activation is highly aberrantand can result in markedly slower activation rates thanthose observed in vivo, these concepts now can betested experimentally using electrophysiological meth-ods.

In any case, our present findings open a new avenuetoward understanding the functional role of Rhodimerization in complex with transducin as well asGPCR dimerization in complex with G proteins ingeneral.

The authors thank Dr. Leslie T. Webster, Jr. and membersof the K.P. laboratory for helpful comments on this manu-script. This work was supported by U.S. National Institutes ofHealth grants EY008061 and EY019478. K.P. is John HordProfessor of Pharmacology.

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Received for publication November 26, 2012.Accepted for publication December 18, 2012.

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