Signaling states of rhodopsin. Formation of the storage form,metarhodopsin III,from active metarhodopsin II

Vertebrate rhodopsin consists of the apoprotein opsin and the chromophore 11-cis-retinal covalently linked via a protonated Schiff base. Upon photoisomerization of the chromophore to all-trans-retinal, the retinylidene linkage hydrolyzes, and all-trans-retinal dissociates from opsin. The pigment is eventually restored by recombining with enzymatically produced 11-cis-retinal. All-trans-retinal release occurs in parallel with decay of the active form, metarhodopsin (Meta) II, in which the original Schiff base is intact but deprotonated. The intermediates formed during Meta II decay include Meta III, with the original Schiff base reprotonated, and Meta III-like pseudo-photoproducts. Using an intrinsic fluorescence assay, Fourier transform infrared spectroscopy, and UV-visible spectroscopy, we investigated Meta II decay in native rod disk membranes. Up to 40% of Meta III is formed without changes in the intrinsic Trp fluorescence and thus without all-trans-retinal release. NADPH, a cofactor for the reduction of all-trans-retinal to all-trans-retinol, does not accelerate Meta II decay nor does it change the amount of Meta III formed. However, Meta III can be photoconverted back to the Meta II signaling state. The data are described by two quasi-irreversible pathways, leading in parallel into Meta III or into release of all-trans-retinal. Therefore, Meta III could be a form of rhodopsin that is stored away, thus regulating photoreceptor regeneration.     

Ligand channeling within a G-protein-coupled receptor. The entry and exit of retinals in native opsin

Deactivation of light-activated rhodopsin (metarhodopsin II) involves, after rhodopsin kinase and arrestin interactions, the hydrolysis of the covalent bond of all-trans-retinal to the apoprotein. Although the long-lived storage form metarhodopsin III is transiently formed, all-trans-retinal is eventually released from the active site. Here we address the question of whether the release results in a retinal that is freely diffusible in the lipid phase of the photoreceptor membrane. The release reaction is accompanied by an increase in intrinsic protein fluorescence (release signal), which arises from the relief of the fluorescence quenching imposed by the retinal in the active site. An analogous fluorescence decrease (uptake signal) was evoked by exogenous retinoids when they non-covalently bound to native opsin membranes. Uptake of 11-cis-retinal was faster than formation of the retinylidene linkage to the apoprotein. Endogenous all-trans-retinal released from the active site during metarhodopsin II decay did not generate the uptake signal. The data show that in addition to the retinylidene pocket (site I) there are two other retinoidbinding sites within opsin. Site II involved in the uptake signal is an entrance site, while the exit site (site III) is occupied when retinal remains bound after its release from site I. Support for a retinal channeling mechanism comes from the rhodopsin crystal structure, which unveiled two putative hydrophobic binding sites. This mechanism enables a unidirectional process for the release of photoisomerized chromophore and the uptake of newly synthesized 11-cis-retinal for the regeneration of rhodopsin.     

Inactivation of photoexcited rhodopsin in retinal rods: the roles of rhodopsin kinase and 48-kDa protein (arrestin)

The inactivation of excited rhodopsin in the presence of ATP, rhodopsin kinase, and/or arrestin has been studied from its effect on the two subsequent steps in the light-induced enzymatic cascade: metarhodopsin II catalyzed activation of G-protein and G-protein-dependent activation of cGMP phosphodiesterase. The inactivation of G-protein (from light-scattering measurements) and that of phosphodiesterase (from measurements of cGMP hydrolysis) have been studied and compared in reconstituted systems containing various combinations of the proteins involved (rhodopsin, G-protein, phosphodiesterase, kinase, and arrestin). Our results show that rhodopsin kinase alone can terminate the activation of G-protein and that arrestin speeds up the process at a relative concentration similar to that reported in the rod (half-maximal effect at 50 nM for 4.4 microM rhodopsin). Measurements of rhodopsin phosphorylation under identical conditions show that in the presence of arrestin total metarhodopsin II inactivation is achieved when only 0.5-1.4 phosphates are bound per bleached rhodopsin, whereas in the absence of arrestin it requires binding of 12-16 phosphates per bleached rhodopsin. Phosphodiesterase activity can similarly be turned off by kinase, and the process is similarly accelerated by arrestin.     

Role of photoreceptor-specific retinol dehydrogenase in the retinoid cycle in vivo

The retinoid cycle is a recycling system that replenishes the 11-cis-retinal chromophore of rhodopsin and cone pigments. Photoreceptor-specific retinol dehydrogenase (prRDH) catalyzes reduction of all-trans-retinal to all-trans-retinol and is thought to be a key enzyme in the retinoid cycle. We disrupted mouse prRDH (human gene symbol RDH8) gene expression by targeted recombination and generated a homozygous prRDH knock-out (prRDH-/-) mouse. Histological analysis and electron microscopy of retinas from 6- to 8-week-old prRDH-/- mice revealed no structural differences of the photoreceptors or inner retina. For brief light exposure, absence of prRDH did not affect the rate of 11-cis-retinal regeneration or the decay of Meta II, the activated form of rhodopsin. Absence of prRDH, however, caused significant accumulation of all-trans-retinal following exposure to bright lights and delayed recovery of rod function as measured by electroretinograms and single cell recordings. Retention of all-trans-retinal resulted in slight overproduction of A2E, a condensation product of all-trans-retinal and phosphatidylethanolamine. We conclude that prRDH is an enzyme that catalyzes reduction of all-trans-retinal in the rod outer segment, most noticeably at higher light intensities and prolonged illumination, but is not an essential enzyme of the retinoid cycle.     

Monomeric Rhodopsin Is the Minimal Functional Unit Required for Arrestin Binding

We have tested whether arrestin binding requires the G-protein-coupled receptor be a dimer or a multimer. To do this, we encapsulated single-rhodopsin molecules into nanoscale phospholipid particles (so-called nanodiscs) and measured their ability to bind arrestin. Our data clearly show that both visual arrestin and β-arrestin 1 can bind to monomeric rhodopsin and stabilize the active metarhodopsin II form. Interestingly, we find that the monomeric rhodopsin in nanodiscs has a higher affinity for wild-type arrestin binding than does oligomeric rhodopsin in liposomes or nanodiscs, as assessed by stabilization of metarhodopsin II. Together, these results establish that rhodopsin self-association is not required to enable arrestin binding.     

Kinetics of visual pigment regeneration in excised mouse eyes and in mice with a targeted disruption of the gene encoding interphotoreceptor retinoid-binding protein or arrestin.

Photoisomerization of 11-cis-retinal to all-trans-retinal and reduction to all-trans-retinol occur in photoreceptor outer segments whereas enzymatic esterification of all-trans-retinol, isomerization to 11-cis-retinol, and oxidation to 11-cis-retinal occur in adjacent cells. The processes are linked into a visual cycle by intercellular diffusion of retinoids. Knowledge of the mechanistic aspects of the visual cycle is very limited. In this study, we utilize chemical analysis of visual cycle retinoids to assess physiological roles for components inferred from in vitro experiments and to understand why excised mouse eyes fail to regenerate their bleached visual pigment. Flash illumination of excised mouse eyes or eyecups, in which regeneration of rhodopsin does not occur, produced a block in the visual cycle after all-trans-retinal formation; constant illumination of eyecups produced a block in the cycle after all-trans-retinol formation; and constant illumination of whole excised eyes resulted in a block of the cycle after formation of all-trans-retinyl ester. These blocks emphasize the role of cellular metabolism in the visual cycle. Interphotoreceptor retinoid-binding protein (IRBP) has been postulated to play a role in intercellular retinoid transfer in the retina; however, the rates of recovery of 11-cis-retinal and of regeneration of rhodopsin in the dark in IRBP-/- mice were very similar to those found with wild-type (wt) mice. Thus, IRBP is necessary for photoreceptor survival but is not essential for a normal rate of visual pigment turnover. Arrestin forms a complex with activated rhodopsin, quenches its activity, and affects the release of all-trans-retinal in vitro. The rate of recovery of 11-cis-retinal in arrestin-/- mice was modestly delayed relative to wt, and the rate of rhodopsin recovery was approximately 80% of that observed with wt mice. Thus, the absence of arrestin appeared to have a minor effect on the kinetics of the visual cycle.     

Two-step mechanism of interaction of rhodopsin intermediates with the C-terminal region of the transducin alpha-subunit

Rhodopsin is a prototypical G-protein-coupled receptor that contains 11-cis-retinal as a light-absorbing chromophore. Light causes conformational changes in the protein moiety through cis-trans isomerization of the chromophore, which leads to the formation of G-protein-interacting states. Our previous studies indicated that there are two intermediate states of rhodopsin, Meta Ib and Meta II, which interact differently with retinal G-protein transducin (Gt) [S. Tachibanaki, H. Imai, T. Mizukami, T. Okada, Y. Imamoto, T. Matsuda, Y. Fukada, A. Terakita, and Y. Shichida (1997) Biochemistry 36, 14173-14180]. Here we demonstrate that the interactions of Gt with these intermediates in the absence of GTPgammaS can be mimicked by the C-terminus 11-amino acid peptide (340-350) of the alpha-subunit of Gt (Gt(alpha)), suggesting that the C-terminal region of Gt(alpha) plays important roles in the interaction with rhodopsin intermediates. Replacement of either of the two leucine residues (Leu344 and Leu349) in the peptide with alanine caused the loss of the interaction with Meta II. However, the interaction with Meta Ib was abolished only when both residues were replaced. These results indicate that rearrangement of the C-terminal region of Gt(alpha) after the binding of a rhodopsin intermediate is necessary for the GDP-GTP exchange reaction on Gt(alpha).     

Studies on cephalopod rhodopsin. Conformational changes in chromophore and protein during the photoregeneration process

The ultraviolet absorbance of squid and octopus rhodopsin changes reversibly at 234 nm and near 280 nm in the interconversion of rhodopsin and metarhodopsin. The absorbance change near 280 nm is ascribed to both protein and chromophore parts. Rhodopsin is photoregenerated from metarhodopsin via an intermediate, P380, on irradiation with yellow light (λ > 520 nm). The ultraviolet absorbance decreases in the change from rhodopsin to metarhodopsin and recovers in two steps; mostly in the process from metarhodopsin to P380 and to a lesser extent in the process from P380 to rhodopsin. P380 has a circular dichroism (CD) band at 380 nm and its magnitude is the same order as that of rhodopsin. Thus it is considered that the molecular structure of P380 is close to that of rhodopsin and that the chromophore is fixed to opsin as in rhodopsin. In the change from metarhodopsin to P380, the chromophore is isomerized from the all-trans to the 11-cis form, and the conformation of opsin changes to fit 11-cis retinal. In the change from P380 to rhodopsin, a small change in the conformation of the protein part and the protonation of the Schiff base, the primary retinal-opsin link, occur.     

Synthesis of the all-trans-retinal chromophore of retinal G protein-coupled receptor opsin in cultured pigment epithelial cells.

Light-dependent production of 11-cis-retinal by the retinal pigment epithelium (RPE) and normal regeneration of rhodopsin under photic conditions involve the RPE retinal G protein-coupled receptor (RGR) opsin. This microsomal opsin is bound to all-trans-retinal which, upon illumination, isomerizes stereospecifically to the 11-cis isomer. In this paper, we investigate the synthesis of the all-trans-retinal chromophore of RGR in cultured ARPE-hRGR and freshly isolated bovine RPE cells. Exogenous all-trans-[(3)H]retinol is incorporated into intact RPE cells and converted mainly into retinyl esters and all-trans-retinal. The intracellular processing of all-trans-[(3)H]retinol results in physiological binding to RGR of a radiolabeled retinoid, identified as all-trans-[(3)H]retinal. The ARPE-hRGR cells contain a membrane-bound NADPH-dependent retinol dehydrogenase that reacts efficiently with all-trans-retinol but not the 11-cis isomer. The NADPH-dependent all-trans-retinol dehydrogenase activity in isolated RPE microsomal membranes can be linked in vitro to specific binding of the chromophore to RGR. These findings provide confirmation that RGR opsin binds the chromophore, all-trans-retinal, in the dark. A novel all-trans-retinol dehydrogenase exists in the RPE and performs a critical function in chromophore biosynthesis.     

Phosphorylated rhodopsin and heparin induce similar conformational changes in arrestin

Photoactivated rhodopsin is quenched upon its phosphorylation in the reaction catalyzed by rhodopsin kinase and the subsequent binding of a regulatory protein, arrestin. We have found that heparin and other polyanions compete with photoactivated, phosphorylated rhodopsin to bind arrestin (48-kDa protein, S-antigen). This is shown (a) by the suppression of stabilized metarhodopsin II; (b) by changes in the digestion of arrestin in the presence of heparin; and (c) by the restoration of arrestin-quenched phosphodiesterase activity. When bound to arrestin, heparin also mimics phosphorylated rhodopsin by similarly exposing arrestin to limited proteolysis. We conclude that heparin and rhodopsin have similar means of binding to arrestin, and we propose a cationic region of arrestin (beginning with Lys163 of the bovine sequence) as the interaction site. In agreement with previous kinetic data we interpret the results in terms of a binding conformation of arrestin which is stabilized by rhodopsin or heparin and is open to proteolytic attack.     

Metarhodopsin control by arrestin,light-filtering screening pigments,and visual pigment turnover in invertebrate microvillar photoreceptors

The visual pigments of most invertebrate photoreceptors have two thermostable photo-interconvertible states, the ground state rhodopsin and photo-activated metarhodopsin, which triggers the phototransduction cascade until it binds arrestin. The ratio of the two states in photoequilibrium is determined by their absorbance spectra and the effective spectral distribution of illumination. Calculations indicate that metarhodopsin levels in fly photoreceptors are maintained below ~35% in normal diurnal environments, due to the combination of a blue-green rhodopsin, an orange-absorbing metarhodopsin and red transparent screening pigments. Slow metarhodopsin degradation and rhodopsin regeneration processes further subserve visual pigment maintenance. In most insect eyes, where the majority of photoreceptors have green-absorbing rhodopsins and blue-absorbing metarhodopsins, natural illuminants are predicted to create metarhodopsin levels greater than 60% at high intensities. However, fast metarhodopsin decay and rhodopsin regeneration also play an important role in controlling metarhodopsin in green receptors, resulting in a high rhodopsin content at low light intensities and a reduced overall visual pigment content in bright light. A simple model for the visual pigment–arrestin cycle is used to illustrate the dependence of the visual pigment population states on light intensity, arrestin levels and pigment turnover.     

Rhodopsin in dimyristoylphosphatidylcholine-reconstituted bilayers forms metarhodopsin II and activates Gt

The photochemical intermediate metarhodopsin II (meta II; lambda max = 380 nm) is generally identified with rho*, the conformation of photolyzed rhodopsin which binds and activates the visual G-protein, Gt [Emeis, D., & Hoffman, K.P. (1981) FEBS Lett. 136, 201-207]. Purified bovine rhodopsin was incorporated into vesicles consisting of dimyristoylphosphatidylcholine (DMPC), and the rapid formation of a photochemical intermediate absorbing maximally at 380 nm was quantified via both flash photolysis and equilibrium spectral measurements. Kinetic and equilibrium spectral measurements performed above the Tm of DMPC showed that Gt, in the absence of GTP, enhances the production of the 380-nm-absorbing species while reducing the concentration of the 478-nm-absorbing species, metarhodopsin I (meta I), in a manner similar to that observed in the native rod outer segment disk membrane. This Gt-induced shift in the equilibrium concentration of photointermediates indicated that the species with an absorbance maximum at 380 nm was meta II. The presence of rho* in the DMPC bilayer was established via measurements of photolysis-induced exchange of tritiated GMPPNP, a nonhydrolyzable analogue of GTP, on Gt. Above Tm, the metarhodopsin equilibrium is strongly shifted toward meta I relative to the native rod outer segment disk membrane; however, at 37 degrees C, 40% of the photointermediates are in the form of meta II. The formation of meta II above Tm is slowed by a factor of ca. 2 relative to the disk membrane. Below Tm, the equilibrium is shifted still further toward meta I, and meta II forms ca. 7 times slower than in the disk membrane.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural changes in lumirhodopsin and metarhodopsin I studied by their photoreactions at 77 K

The functional process of rhodopsin is initiated by cis-trans photoisomerization of the retinal chromophore. One of the primary intermediates, bathorhodopsin (Batho), is stable at 77 K, and structural changes in Batho are limited around the chromophore. Then, relaxation of Batho leads to helix opening at the cytoplasmic surface in metarhodopsin II (Meta II), which allows activation of a G protein transducin. Two intermediates, lumirhodopsin (Lumi) and metarhodopsin I (Meta I), appear between Batho and Meta II, and can be stabilized at 200 and 240 K, respectively. A photoaffinity labeling experiment reported that formation of Lumi accompanied flip-over of the beta-ionone ring of the retinal chromophore so that the ring portion was attached to Ala169 of helix IV [Borhan, B., Souto, M. L., Imai, H., Shichida, Y., and Nakanishi, K. (2000) Science 288, 2209-2212]. According to the crystal structure of bovine rhodopsin, the distance between the labeled C3 atom of the chromophore and Ala169 was >15 A [Palczewski, K., Kumasaka, T., Hori, T., Behnke, C. A., Motoshima, H., Fox, B. A., Le Trong, I., Teller, D. C., Okada, T., Stenkamp, R. E., Yamamoto, M., and Miyano, M. (2000) Science 289, 739-745]. These facts suggest that global protein structural changes such as helix motions take place in Lumi. In the study presented here, Lumi and Meta I are illuminated at 77 K, and protein structural changes are probed by Fourier transform infrared (FTIR) spectroscopy. We found that Lumi can be photoconverted to rhodopsin at 77 K from the IR spectral analysis of the photoproducts of Lumi. In contrast, more complex spectra were obtained for the photoproducts of Meta I at 77 K, implying that the protein structure of Meta I is considerably altered so as not to be reverted to the original state at 77 K. Thus, these photoreaction experiments with Lumi and Meta I at 77 K suggested the presence of global protein structural changes in the process between them. We concluded that the helix motions do not occur at Lumi, but at Meta I, and the flip-over of the beta-ionone ring reported by the photoaffinity labeling takes place through the specific reaction channel without a change in the global structure.     

Partial agonism in a G Protein-coupled receptor: role of the retinal ring structure in rhodopsin activation

The visual process in rod cells is initiated by absorption of a photon in the rhodopsin retinal chromophore and consequent retinal cis/trans-isomerization. The ring structure of retinal is thought to be needed to transmit the photonic energy into conformational changes culminating in the active metarhodopsin II (Meta II) intermediate. Here, we demonstrate that cis-acyclic retinals, lacking four carbon atoms of the ring, can activate rhodopsin. Detailed analysis of the activation pathway showed that, although the photoproduct pathway is more complex, Meta II formed with almost normal kinetics. However, lack of the ring structure resulted in a low amount of Meta II and a fast decay of activity. We conclude that the main role of the ring structure is to maintain the active state, thus specifying a mechanism of activation by a partial agonist of the G protein-coupled receptor rhodopsin.     

Binding of inositol phosphates to arrestin.

Arrestin binds to phosphorylated rhodopsin in its light-activated form (metarhodopsin II), blocking thereby its interaction with the G-protein, transducin. In this study, we show that highly phosphorylated forms of inositol compete against the arrestin-rhodopsin interaction. Competition curves and direct binding assays with free arrestin consistently yield affinities in the micromolar range; for example, inositol 1,3,4,5-tetrakisphosphate (InP4) and inositol hexakisphosphate (InP6 bind to arrestin with dissociation constants of 12 microM and 5 microM, respectively. Only a small control amount of inositol phosphates is bound, when arrestin interacts with phosphorylated rhodopsin. This argues for a release of bound inositol phosphates by interaction with rhodopsin. Transducin, rhodopsin kinase, or cyclic GMP phosphodiesterase are not affected by inositol phosphates. These observations open a new way to purify arrestin and to inhibit its interaction with rhodopsin. Their physiological significance deserves further investigation.     

Energetics of primary processes in visula escitation: photocalorimetry of rhodopsin in rod outer segment membranes.

A sensitive technique for the direct calorimetric determination of the energetics of photochemical reactions under low levels of illumination, and its application to the study of primary processes in visula excitation, are described. Enthlpies are reported for various steps in the bleaching of rhodopsin in intact rod outer segment membranes, together with the heats of appropriate model reactions. Protonation changes are also determined calorimetrically by use of buffers with differing heats of proton ionization. Bleaching of rhodopsin is accompanied by significant uptake of heat energy, vastly in excess of the energy required for simple isomerization of the retinal chromophore. Metarhodopsin I formation involves the uptake of about 17 kcal/mol and no net change in proton ionization of the system. Formation of metarhodopsin II requires an additional energy of about 10 kcal/mol and involves the uptake on one hydrogen ion from solution. The energetics of the overall photolysis reaction, rhodopsin leads to opsin + all-trans-retinal, are pH dependent and involve the exposure of an additional titrating group on opsin. This group has a heat of proton ionization of about 12 kcal/mal, characteristic of a primary amine, but a pKa in the region of neutrality. We suggest that this group is the Schiff base lysine of the chromophore binding site of rhodopsin which becomes exposed on photolysis. The low pKa for this active lysine would result in a more stable retinal-opsin linkage, and might be induced by a nearby positively charged group on the protein (either arginine or a second lysine residue). This leads to a model involving intramolecular protonation of the Schiff base nitrogen in the retinal-opsin linkage of rhodopsin, which is consistent with the thermodynamic and spectroscopic properties of the system. We further propose that the metarhodopsin I leads to metarhodopsin II step in the bleaching sequence involves reversible hydrolysis of the Schiff base linkage in the chromophore binding site, and that subsequent steps are the result of migration of the chromophore from this site.     

Light-induced conformational changes of rhodopsin probed by fluorescent alexa594 immobilized on the cytoplasmic surface

A novel fluorescence method has been developed for detecting the light-induced conformational changes of rhodopsin and for monitoring the interaction between photolyzed rhodopsin and G-protein or arrestin. Rhodopsin in native membranes was selectively modified with fluorescent Alexa594-maleimide at the Cys(316) position, with a large excess of the reagent Cys(140) that was also derivatized. Modification with Alexa594 allowed the monitoring of fluorescence changes at a red excitation light wavelength of 605 nm, thus avoiding significant rhodopsin bleaching. Upon absorption of a photon by rhodopsin, the fluorescence intensity increased as much as 20% at acidic pH with an apparent pK(a) of approximately 6.8 at 4 degrees C, and was sensitive to the presence of hydroxylamine. These findings indicated that the increase in fluorescence is specific for metarhodopsin II. In the presence of transducin, a significant increase in fluorescence was observed. This increase of fluorescence emission intensity was reduced by addition of GTP, in agreement with the fact that transducin enhances the formation of metarhodopsin II. Under conditions that favored the formation of a metarhodopsin II-Alexa594 complex, transducin slightly decreased the fluorescence. In the presence of arrestin, under conditions that favored the formation of metarhodopsin I or II, a phosphorylated, photolyzed rhodopsin-Alexa594 complex only slightly decreased the fluorescence intensity, suggesting that the cytoplasmic surface structure of metarhodopsin II is different in the complex with arrestin and transducin. These results demonstrate the application of Alexa594-modified rhodopsin (Alexa594-rhodopsin) to continuously monitor the conformational changes in rhodopsin during light-induced transformations and its interactions with other proteins.     

Lack of interaction of rhodopsin chromophore with membrane lipids. An electron-electron double resonance study using 14N:15N pairs.

Electron-electron double resonance (ELDOR) has been applied to the study of specific interactions of 15N-spin-labeled stearic acid with the retinal chromophore of a rhodopsin analogue containing a 14N spin-labeled retinal. Both the 5 and 16 spin-labeled stearic acids were incorporated into the lipid bilayer of rod outer segment membranes containing the spin-labeled pigment. No interaction between the 15N and 14N spin-labels was observed in rhodopsin or the metarhodopsin II state with either of these labeled stearic acids. Therefore in this system the ring portion of the chromophore must be highly sequestered from the phospholipid bilayer in both the rhodopsin and metarhodopsin II forms.     

Location of Trp265 in metarhodopsin II: implications for the activation mechanism of the visual receptor rhodopsin

Isomerization of the 11-cis retinal chromophore in the visual pigment rhodopsin is coupled to motion of transmembrane helix H6 and receptor activation. We present solid-state magic angle spinning NMR measurements of rhodopsin and the metarhodopsin II intermediate that support the proposal that interaction of Trp265(6.48) with the retinal chromophore is responsible for stabilizing an inactive conformation in the dark, and that motion of the beta-ionone ring allows Trp265(6.48) and transmembrane helix H6 to adopt active conformations in the light. Two-dimensional dipolar-assisted rotational resonance NMR measurements are made between the C19 and C20-methyl groups of the retinal and uniformly 13C-labeled Trp265(6.48). The retinal C20-Trp265(6.48) contact present in the dark-state of rhodopsin is lost in metarhodopsin II, and a new contact is formed with the C19 methyl group. We have previously shown that the retinal translates 4-5 A toward H5 in metarhodopsin II. This motion, in conjunction with the Trp-C19 contact, implies that the Trp265(6.48) side-chain moves significantly upon rhodopsin activation. NMR measurements also show that a packing interaction in rhodopsin between Trp265(6.48) and Gly121(3.36) is lost in metarhodopsin II, consistent with H6 motion away from H3. However, a close contact between Gly120(3.35) on H3 and Met86(2.53) on H2 is observed in both rhodopsin and metarhodopsin II, suggesting that H3 does not change orientation significantly upon receptor activation.     

Light-induced interaction between rhodopsin and the GTP-binding protein. Metarhodopsin II is the major photoproduct involved

We have previously described [H, Kühn et al. (1981) Proc. Natl Acad. Sci. USA, 78, 6873-6877] a light-induced scattering change ('binding signal') associated with a stoichiometric binding between photoexcited rhodopsin and a peripheral membrane protein, the GTP-binding protein, in bovine rod outer segment suspensions. We have attempted here to identify the rhodopsin intermediate R* which is responsible for this interaction, by studying its dependence on pH, temperature and ionic strength. The results strongly suggest that the active state is metarhodopsin II (M II). 1. The initial phase of the binding signal is slightly slower than the formation of metarhodopsin II (2-37 degrees C, pH 5.5-9). 2. The kinetics of the decay of the active rhodopsin state are similar to those of the metarhodopsin II leads to metarhodopsin III transition (37 degrees C, pH 7.3). 3. All conditions which lead to light-induced binding of the GTP-binding protein to R* also lead to the formation of M II. At 2 degrees C, pH 8.3, in particular where no M II is formed in the absence of GTP-binding protein, binding signals and light-induced attachment of the GTP-binding protein to the membrane are still observed. Consistently, addition of GTP-binding protein to a suspension of extracted membranes bleached at 2 degrees C (pH 8.3) shifts the metarhodopsin I in equilibrium metarhodopsin II equilibrium towards metarhodopsin II. The shift is reversed by GTP, which dissociates the rhodopsin--GTP-binding protein complex. 4. At low ionic strength, where the GTP-binding protein is soluble in the dark (instead of being associated to the membrane as in the above experiments) M II still induces the binding whereas M I does not, indicating a much lower affinity of the GTP-binding protein for MI.