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Signaling of the prototypical G protein–coupled receptor (GPCR) rhodopsin through its cognate G protein transducin (G(t)) is quenched when arrestin binds to the activated receptor. Although the overall architecture of the rhodopsin/arrestin complex is known, many questions regarding its specificity remain unresolved. Here, using FTIR difference spectroscopy and a dual pH/peptide titration assay, we show that rhodopsin maintains certain flexibility upon binding the “finger loop” of visual arrestin (prepared as synthetic peptide ArrFL-1). We found that two distinct complexes can be stabilized depending on the protonation state of E3.49 in the conserved (D)ERY motif. Both complexes exhibit different interaction modes and affinities of ArrFL-1 binding. The plasticity of the receptor within the rhodopsin/ArrFL-1 complex stands in contrast to the complex with the C terminus of the G(t) α-subunit (GαCT), which stabilizes only one specific substate out of the conformational ensemble. However, G(t) α-subunit binding and both ArrFL-1–binding modes involve a direct interaction to conserved R3.50, as determined by site-directed mutagenesis. Our findings highlight the importance of receptor conformational flexibility and cytoplasmic proton uptake for modulation of rhodopsin signaling and thereby extend the picture provided by crystal structures of the rhodopsin/arrestin and rhodopsin/ArrFL-1 complexes. Furthermore, the two binding modes of ArrFL-1 identified here involve motifs of conserved amino acids, which indicates that our results may have elucidated a common modulation mechanism of class A GPCR–G protein/-arrestin signaling.

Among cyclic nucleotide phosphodiesterases (PDEs), PDE6 is unique in serving as an effector enzyme in G protein-coupled signal transduction. In retinal rods and cones, PDE6 is membrane-bound and activated to hydrolyse its substrate, cGMP, by binding of two active G protein α-subunits (Gα*). To investigate the activation mechanism of mammalian rod PDE6, we have collected functional and structural data, and analysed them by reaction–diffusion simulations. Gα* titration of membrane-bound PDE6 reveals a strong functional asymmetry of the enzyme with respect to the affinity of Gα* for its two binding sites on membrane-bound PDE6 and the enzymatic activity of the intermediary 1 : 1 Gα* · PDE6 complex. Employing cGMP and its 8-bromo analogue as substrates, we find that Gα* · PDE6 forms with high affinity but has virtually no cGMP hydrolytic activity. To fully activate PDE6, it takes a second copy of Gα* which binds with lower affinity, forming Gα* · PDE6 · Gα*. Reaction–diffusion simulations show that the functional asymmetry of membrane-bound PDE6 constitutes a coincidence switch and explains the lack of G protein-related noise in visual signal transduction. The high local concentration of Gα* generated by a light-activated rhodopsin molecule efficiently activates PDE6, whereas the low density of spontaneously activated Gα* fails to activate the effector enzyme.

In PNAS, Yue et al. (1) deal with the question of amplification early in the phototransduction cascade. Although their approach is elegant, we cannot agree with some major points in their analysis and conclusions. The authors suggest that their estimate of “12–14 transducin–PDE effector complexes” per photoisomerization is different from literature estimates that “range from teens to hundreds of GT*s per Rho*.” However, the gain at the stage of the effector protein is an indirect measure of the actual rate (νRG) of transducin (GT*) activation, which is the parameter determined in many previous investigations. From the present data, and the authors’ assumption that … [↵][1]1To whom correspondence should be addressed. Email: twkraft{at}uab.edu. [1]: #xref-corresp-1-1

Rhodopsin, the photoreceptor of rod cells, absorbs light to mediate the first step of vision by activating the G protein transducin (Gt). Several human diseases, such as retinitis pigmentosa or congenital night blindness, are linked to rhodopsin malfunctions. Most of the corresponding in vivo studies and structure-function analyses (e.g. based on protein x-ray crystallography or spectroscopy) have been carried out on murine or bovine rhodopsin. Because these rhodopsins differ at several amino acid positions from human rhodopsin, we conducted a comprehensive spectroscopic characterization of human rhodopsin in combination with molecular dynamics simulations. We show by FTIR and UV-visible difference spectroscopy that the light-induced transformations of the early photointermediates are very similar. Significant differences between the pigments appear with formation of the still inactive Meta I state and the transition to active Meta II. However, the conformation of Meta II and its activity toward the G protein are essentially the same, presumably reflecting the evolutionary pressure under which the active state has developed. Altogether, our results show that although the basic activation pathways of human and bovine rhodopsin are similar, structural deviations exist in the inactive conformation and during receptor activation, even between closely related rhodopsins. These differences between the well studied bovine or murine rhodopsins and human rhodopsin have to be taken into account when the influence of point mutations on the activation pathway of human rhodopsin are investigated using the bovine or murine rhodopsin template sequences.

Conformational equilibria of G-protein–coupled receptors (GPCRs) are intimately involved in intracellular signaling. Here conformational substates of the GPCR rhodopsin are investigated in micelles of dodecyl maltoside (DDM) and in phospholipid nanodiscs by monitoring the spatial positions of transmembrane helices 6 and 7 at the cytoplasmic surface using site-directed spin labeling and double electron–electron resonance spectroscopy. The photoactivated receptor in DDM is dominated by one conformation with weak pH dependence. In nanodiscs, however, an ensemble of pH-dependent conformational substates is observed, even at pH 6.0 where the MIIbH+ form defined by proton uptake and optical spectroscopic methods is reported to be the sole species present in native disk membranes. In nanodiscs, the ensemble of substates in the photoactivated receptor spontaneously decays to that characteristic of the inactive state with a lifetime of ∼16 min at 20 °C. Importantly, transducin binding to the activated receptor selects a subset of the ensemble in which multiple substates are apparently retained. The results indicate that in a native-like lipid environment rhodopsin activation is not analogous to a simple binary switch between two defined conformations, but the activated receptor is in equilibrium between multiple conformers that in principle could recognize different binding partners.

Rhodopsin, a seven transmembrane helix (TM) receptor, binds its ligand 11-cis-retinal via a protonated Schiff base. Coupling to the G-protein transducin (G(t)) occurs after light-induced cis/trans-retinal isomerization, which leads through photoproducts into a sequence of metarhodopsin (Meta) states: Meta I ⇌ Meta IIa ⇌ Meta IIb ⇌ Meta IIbH(+). The structural changes behind this three-step activation scheme are mediated by microswitch domains consisting of conserved amino acids. Here we focus on Tyr223(5.58) as part of the Y(5.58)X(7)K(R)(5.66) motif. Mutation to Ala, Phe, or Glu results in specific impairments of G(t)-activation measured by intrinsic G(t) fluorescence. UV-vis/FTIR spectroscopy of rhodopsin and its complex with a C-terminal G(t)α peptide allows the assignment of these deficiencies to specific steps in the activation path. Effects of mutation occur already in Meta I but do not directly influence deprotonation of the Schiff base during formation of Meta IIa. Absence of the whole phenol ring (Y223A) allows the activating motion of TM6 in Meta IIb but impairs the coupling to G(t). When only the hydroxyl group is lacking (Y223F), Meta IIb does not accumulate, but the activity toward G(t) remains substantial. From the FTIR features of Meta IIbH(+) we conclude that proton uptake to Glu134(3.49) is mandatory for Tyr223(5.58) to engage in the interaction with the key player Arg135(3.50) predicted by X-ray analysis. This polar interaction is partially recovered in Y223E, explaining its relatively high activity. Only the phenol side chain of tyrosine provides all characteristics for accumulation of the active state and G-protein activation.

Structural studies of active states of the visual pigment rhodopsin, a G protein-coupled receptor, have previously been limited to apoprotein or opsin forms that do not contain the agonist all-trans-retinal. Two groups now report structures that reveal more details of the transformations involved in rhodopsin activation. Choe et al. solve the X-ray crystal structure of the metarhodopsin II intermediate of the photoreceptor rhodopsin, and Standfuss et al. determine the structure of a constitutively active mutant of rhodopsin bound to a peptide derived from the C-terminus of the G protein transducin. Here the X-ray crystal structure of the metarhodopsin II intermediate of the photoreceptor rhodopsin is solved. Comparison of this structure with previously published structures enabled the proposal of how retinal translocation and rotation induce the conformational changes observed in this structure. G-protein-coupled receptors (GPCRs) are seven transmembrane helix (TM) proteins that transduce signals into living cells by binding extracellular ligands and coupling to intracellular heterotrimeric G proteins (Gαβγ)1. The photoreceptor rhodopsin couples to transducin and bears its ligand 11-cis-retinal covalently bound via a protonated Schiff base to the opsin apoprotein2. Absorption of a photon causes retinal cis/trans isomerization and generates the agonist all-trans-retinal in situ. After early photoproducts, the active G-protein-binding intermediate metarhodopsin II (Meta II) is formed, in which the retinal Schiff base is still intact but deprotonated. Dissociation of the proton from the Schiff base breaks a major constraint in the protein and enables further activating steps, including an outward tilt of TM6 and formation of a large cytoplasmic crevice for uptake of the interacting C terminus of the Gα subunit3,4,5. Owing to Schiff base hydrolysis, Meta II is short-lived and notoriously difficult to crystallize. We therefore soaked opsin crystals with all-trans-retinal to form Meta II, presuming that the crystal’s high concentration of opsin in an active conformation (Ops*)6,7 may facilitate all-trans-retinal uptake and Schiff base formation. Here we present the 3.0 A and 2.85 A crystal structures, respectively, of Meta II alone or in complex with an 11-amino-acid C-terminal fragment derived from Gα (GαCT2). GαCT2 binds in a large crevice at the cytoplasmic side, akin to the binding of a similar Gα-derived peptide to Ops* (ref. 7). In the Meta II structures, the electron density from the retinal ligand seamlessly continues into the Lys 296 side chain, reflecting proper formation of the Schiff base linkage. The retinal is in a relaxed conformation and almost undistorted compared with pure crystalline all-trans-retinal. By comparison with early photoproducts we propose how retinal translocation and rotation induce the gross conformational changes characteristic for Meta II. The structures can now serve as models for the large GPCR family.

Extracellular signals prompt G protein-coupled receptors (GPCRs) to adopt an active conformation (R*) and catalyze GDP/GTP exchange in the α-subunit of intracellular G proteins (Gαβγ). Kinetic analysis of transducin (G t αβγ) activation shows that an intermediary R*·G t αβγ·GDP complex is formed that precedes GDP release and formation of the nucleotide-free R*·G protein complex. Based on this reaction sequence, we explore the dynamic interface between the proteins during formation of these complexes. We start from the R* conformation stabilized by a G t α C-terminal peptide (GαCT) obtained from crystal structures of the GPCR opsin. Molecular modeling allows reconstruction of the fully elongated C-terminal α-helix of G t α (α5) and shows how α5 can be docked to the open binding site of R*. Two modes of interaction are found. One of them – termed stable or S-interaction – matches the position of the GαCT peptide in the crystal structure and reproduces the hydrogen-bonding networks between the C-terminal reverse turn of GαCT and conserved E(D)RY and NPxxY(x) 5,6 F regions of the GPCR. The alternative fit – termed intermediary or I-interaction – is distinguished by a tilt (42°) and rotation (90°) of α5 relative to the S-interaction and shows different α5 contacts with the NPxxY(x) 5,6 F region and the second cytoplasmic loop of R*. From the 2 α5 interactions, we derive a “helix switch” mechanism for the transition of R*·G t αβγ·GDP to the nucleotide-free R*·G protein complex that illustrates how α5 might act as a transmission rod to propagate the conformational change from the receptor-G protein interface to the nucleotide binding site.

Visual arrestin terminates the signal amplification cascade in photoreceptor cells by blocking the interaction of light activated phosphorylated rhodopsin with the G-protein transducin. Although crystal structures of arrestin and rhodopsin are available, it is still unknown how the complex of the two proteins is formed. To investigate the interaction sites of arrestin with rhodopsin various surface regions of recombinant arrestin were sterically blocked by different numbers of fluorophores (Alexa 633). The binding was recorded by time-resolved light scattering. To accomplish site-specific shielding of protein regions, in a first step all three wild-type cysteines were replaced by alanines. Nevertheless, regarding the magnitude and specificity of rhodopsin binding, the protein is still fully active. In a second step, new cysteines were introduced at selected sites to allow covalent binding of fluorophores. Upon attachment of Alexa 633 to the recombinant cysteines we observed that these bulky labels residing in the concave area of either the N- or the C-terminal domain do not perturb the activity of arrestin. By simultaneously modifying both domains with one Alexa 633 the binding capacity was reduced. The presence of two Alexa 633 molecules in each domain prevented binding of rhodopsin to arrestin. This observation indicates that both concave sites participate in binding.

Dim-light vision is mediated by retinal rod cells. Rhodopsin (R), a G-protein-coupled receptor, switches to its active form (R(∗)) in response to absorbing a single photon and activates multiple copies of the G-protein transducin (G) that trigger further downstream reactions of the phototransduction cascade. The classical assumption is that R and G are uniformly distributed and freely diffusing on disk membranes. Recent experimental findings have challenged this view by showing specific R architectures, including RG precomplexes, nonuniform R density, specific R arrangements, and immobile fractions of R. Here, we derive a physical model that describes the first steps of the photoactivation cascade in spatiotemporal detail and single-molecule resolution. The model was implemented in the ReaDDy software for particle-based reaction-diffusion simulations. Detailed kinetic in vitro experiments are used to parametrize the reaction rates and diffusion constants of R and G. Particle diffusion and G activation are then studied under different conditions of R-R interaction. It is found that the classical free-diffusion model is consistent with the available kinetic data. The existence of precomplexes between inactive R and G is only consistent with the data if these precomplexes are weak, with much larger dissociation rates than suggested elsewhere. Microarchitectures of R, such as dimer racks, would effectively immobilize R but have little impact on the diffusivity of G and on the overall amplification of the cascade at the level of the G protein.

In the early steps of visual signal transduction, light-activated rhodopsin (R*) catalyzes GDP/GTP exchange in the heterotrimeric G protein (Galphabetagamma) transducin. We recently reported that the catalytic interaction involves two sequential steps. An initial docking between R* and Gbetagamma leads to conformational changes which make the C-terminus of Galpha (CTalpha) available for binding to R*. Binding of CTalpha by R* then triggers GDP/GTP exchange in the Galpha subunit. To further study this two-step mechanism, we investigated different single amino acid substitutions within CTalpha and discuss the effects of high affinity mutations on nucleotide exchange catalysis.

Catalysis of nucleotide exchange in heterotrimeric G proteins (Gαβγ) is a key step in cellular signal transduction mediated by G protein-coupled receptors. The Gα N terminus with its helical stretch is thought to be crucial for G protein/activated receptor (R*) interaction. The N-terminal fatty acylation of Gα is important for membrane targeting of G proteins. By applying biophysical techniques to the rhodopsin/transducin model system, we studied the effect of N-terminal truncations in Gα. In Gαβγ, lack of the fatty acid and Gα truncations up to 33 amino acids had little effect on R* binding and R*-catalyzed nucleotide exchange, implying that this region is not mandatory for R*/Gαβγ interaction. However, when the other hydrophobic modification of Gαβγ, the Gγ C-terminal farnesyl moiety, is lacking, R* interaction requires the fatty acylated Gα N terminus. This suggests that the two hydrophobic extensions can replace each other in the interaction of Gαβγ with R*. We propose that in native Gαβγ, these two terminal regions are functionally redundant and form a microdomain that serves both to anchor the G protein to the membrane and to establish an initial docking complex with R*. Accordingly, we find that the native fatty acylated Gα is competent to interact with R* even in the absence of Gβγ, whereas nonacylated Gα requires Gβγ for interaction. Experiments with N-terminally truncated Gα subunits suggest that in the second step of the catalytic process, the receptor binds to the αN/β1-loop region of Gα to reduce nucleotide affinity and to make the Gα C terminus available for subsequent interaction with R*.

In the phototransduction pathway of rhodopsin, the metarhodopsin (Meta) III retinal storage form arises from the active G-protein binding Meta II by a slow spontaneous reaction through the Meta I precursor or by light absorption and photoisomerization, respectively. Meta III is a side product of the Meta II decay path and holds its retinal in the original binding site, with the Schiff base bond to the apoprotein reprotonated as in the dark ground state. It thus keeps the retinal away from the regeneration pathway in which the photolyzed all-trans-retinal is released. This study was motivated by our recent observation that Meta III remains stable for hours in membranes devoid of regulatory proteins, whereas it decays much more rapidly in situ. We have now explored the possibility of regulated formation and decay of Meta III, using intrinsic opsin tryptophan fluorescence and UV-visible and Fourier transform infrared spectroscopy. We find that a rapid return of Meta III into the regeneration pathway is triggered by the G-protein transducin (Gt). Depletion of the retinal storage is initiated by a novel direct bimolecular interaction of Gt with Meta III, which was previously considered inactive. Gt thereby induces the transition of Meta III into Meta II, so that the retinylidene bond to the apoprotein can be hydrolyzed, and the retinal can participate again in the normal retinoid cycle. Beyond the potential significance for retinoid metabolism, this may provide the first example of a G-protein-catalyzed conversion of a receptor.

Guanine nucleotide exchange in heterotrimeric G proteins catalyzed by G protein-coupled receptors (GPCRs) is a key event in many physiological processes. The crystal structures of the GPCR rhodopsin and two G proteins as well as binding sites on both catalytically interacting proteins are known, but the temporal sequence of events leading to nucleotide exchange remains to be elucidated. We employed time-resolved near infrared light scattering to study the order in which the Galpha and Ggamma C-terminal binding sites on the holo-G protein interact with the active state of the GPCR rhodopsin (R*) in native membranes. We investigated these key binding sites within mass-tagged peptides and G proteins and found that their binding to R* is mutually exclusive. The interaction of the holo-G protein with R* requires at least one of the lipid modifications of the G protein (i.e. myristoylation of the Galpha N terminus and/or farnesylation of the Ggamma C terminus). A holo-G protein with a high affinity Galpha C terminus shows a specific change of the reaction rate in the GDP release and GTP uptake steps of catalysis. We interpret the data by a sequential fit model where (i) the initial encounter between R* and the G protein occurs with the Gbetagamma subunit, and (ii) the Galpha C-terminal tail then interacts with R* to release bound GDP, thereby decreasing the affinity of R* for the Gbetagamma subunit. The mechanism limits the time in which both C-terminal binding sites of the G protein interact simultaneously with R* to a short lived transitory state.

In the G protein-coupled receptor rhodopsin, light-induced cis/trans isomerization of the retinal ligand triggers a series of distinct receptor states culminating in the active Metarhodopsin II (Meta II) state, which binds and activates the G protein transducin (Gt). Long before Meta II decays into the aporeceptor opsin and free all-trans-retinal, its signaling is quenched by receptor phosphorylation and binding of the protein arrestin-1, which blocks further access of Gt to Meta II. Although recent crystal structures of arrestin indicate how it might look in a precomplex with the phosphorylated receptor, the transition into the high affinity complex is not understood. Here we applied Fourier transform infrared spectroscopy to monitor the interaction of arrestin-1 and phosphorylated rhodopsin in native disc membranes. By isolating the unique infrared signature of arrestin binding, we directly observed the structural alterations in both reaction partners. In the high affinity complex, rhodopsin adopts a structure similar to Gt-bound Meta II. In arrestin, a modest loss of β-sheet structure indicates an increase in flexibility but is inconsistent with a large scale structural change. During Meta II decay, the arrestin-rhodopsin stoichiometry shifts from 1:1 to 1:2. Arrestin stabilizes half of the receptor population in a specific Meta II protein conformation, whereas the other half decays to inactive opsin. Altogether these results illustrate the distinct binding modes used by arrestin to interact with different functional forms of the receptor.

Despite the growing structural information on receptors and G proteins, the information on affinities and kinetics of protein-protein and protein-nucleotide interactions is still not complete. In this study on photoactivated rhodopsin (R*) and the rod G protein, Gt, we have used kinetic light scattering, backed by direct biochemical assays, to follow G protein activation. Our protocol includes the following: (i) to measure initial rates on the background of rapid depletion of the GtGDP substrate; (ii) to titrate GtGDP, GTP, and GDP; and (iii) to apply a double displacement reaction scheme to describe the results. All data are simultaneously fitted by one and the same set of parameters. We obtain values of K m = 2200 Gt/μm2 for GtGDP andK m = 230 μm for GTP; dissociation constants are K d = 530 Gt/μm2 for R*-GtGDP dissociation and K d = 270 μm for GDP release from R*GtGDP, once formed. Maximal catalytic rates per photoexcited rhodopsin are 600 Gt/s at 22 °C and 1300 Gt/s at 34 °C. The analysis provides a tool to allocate and quantify better the effects of chemical or mutational protein modifications to individual steps in signal transduction.

Arrestin blocks the interaction of rhodopsin with the G protein transducin (G(t)). To characterize the sites of arrestin that interact with rhodopsin, we have utilized a spectrophotometric peptide competition assay. It is based on the stabilization of the active intermediates metarhodopsin II (MII) and phosphorylated MII by G(t) and arrestin, respectively (extra MII monitor). The protocol involves native disc membranes and three sets of peptides 10-30 amino acids in length spanning the arrestin sequence. In the absence of arrestin, not one of the peptides by itself had an effect on the amount of MII formed. However, inhibition of arrestin-dependent extra MII was found for the peptides at residues 11-30 and 51-70 (IC(50) < 100 microm) and residues 231-260 (IC(50) < 200 microm). A similar pattern of inhibition by arrestin peptides was seen when arrestin was replaced by G(t) or the farnesylated G(t)gamma C-terminal peptide. Only arrestin-(11-30) inhibited MII.G(t) less (IC(50) = 300 microm) than phosphorylated MII.arrestin. We interpreted the data by competition of the arrestin peptides for interaction sites at rhodopsin, exposed in the MII conformation and specific for both arrestin and G(t). The arrestin sites are located in both the C- and N-terminal domains of the arrestin structure.

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

Metarhodopsin II (MII) provides the active conformation of rhodopsin for interaction with the G-protein, Gt. Fourier transform infrared spectra from samples prepared by centrifugation reflect the pH dependent equilibrium between MII and inactive metarhodopsin I. C-terminal synthetic peptides (Gtalpha(340-350) and Gtgamma(60-71)farnesyl) stabilize MII. We find that both peptides cause similar spectral changes not seen with control peptides (Gtalpha (K341R, L349A) and non-farnesylated Gtgamma). The spectra reflect all the protonation dependent bands normally observed when MII is formed at acidic pH. Beside the protonation dependent bands, additional features, similar with both peptides, appear in the amide I and II regions.

The role of the putative fourth cytoplasmic loop of rhodopsin in the binding and catalytic activation of the heterotrimeric G protein, transducin (G(t)), is not well defined. We developed a novel assay to measure the ability of G(t), or G(t)-derived peptides, to inhibit the photoregeneration of rhodopsin from its active metarhodopsin II state. We show that a peptide corresponding to residues 340-350 of the alpha subunit of G(t), or a cysteinyl-thioetherfarnesyl peptide corresponding to residues 50-71 of the gamma subunit of G(t), are able to interact with metarhodopsin II and inhibit its photoconversion to rhodopsin. Alteration of the amino acid sequence of either peptide, or removal of the farnesyl group from the gamma-derived peptide, prevents inhibition. Mutation of the amino-terminal region of the fourth cytoplasmic loop of rhodopsin affects interaction with G(t) (Marin, E. P., Krishna, A. G., Zvyaga T. A., Isele, J., Siebert, F., and Sakmar, T. P. (2000) J. Biol. Chem. 275, 1930-1936). Here, we provide evidence that this segment of rhodopsin interacts with the carboxyl-terminal peptide of the alpha subunit of G(t). We propose that the amino-terminal region of the fourth cytoplasmic loop of rhodopsin is part of the binding site for the carboxyl terminus of the alpha subunit of G(t) and plays a role in the regulation of betagamma subunit binding.

Functional coupling of the human thrombin receptor PAR1 (protease-activated receptor 1) with the retinal rod G-protein transducin (Gt, a member of the Gi family) was studied in a reconstituted system of membranes from Sf9 cells expressing the thrombin receptor and purified Gt from bovine rod outer segments. TRAP6-agonist-activated PAR1 interacts productively with the distant G-protein. Agonist-dependent Gt activation was measured using a real-time fluorimetric GTP[S]-binding assay and membranes from Sf9 cells. To characterize nucleotide-exchange catalysis by PAR1, we analyzed dependence on nucleotides, temperature and pH. Activation was inhibited by low GDP concentrations (IC50 = 5.2 +/- 1.5 microM at 5 microM GTP[S]), indicating that receptor-Gt coupling, followed by instantaneous GDP release, is rate limiting under the conditions (25 degrees C). Arrhenius plots of the temperature dependence reflect an apparent Ea of 60 +/- 3.5 kJ.mol-1. Evaluation of the pH/rate profiles of Gt activation indicates that the activating conformation of the receptor is determined by protonation of a titratable group with an apparent pKa of 6.4. This supports the idea that the active state of agonist-bound PAR1 depends on forced protonation, indicating possible analogies to the scheme established for rhodopsin.

Transducin is a heterotrimer formed by a fatty acylated alpha-subunit and a farnesylated betagamma-subunit. The role of these two covalent modifications and of adjacent hydrophobic and charged amino acid residues in reversible anchoring at disk model membranes is investigated at different pH values, salt concentrations, and lipid packing densities using the monolayer expansion technique and CD spectroscopy. The heterotrimer only binds if the acetylated alpha-subunit is transformed into its surface-active form by divalent cations. In the presence of salts the alpha(GDP)-subunit, the betagamma-complex, and the heterotrimer bind to POPC monolayers at 30 mN/m, estimated to mimic the lateral packing density of disk membranes, with apparent binding constants of Kapp = (1.1 +/- 0.3) x 10(6) M-1 (reflecting the penetration of the fatty acyl chain together with approximately three adjacent hydrophobic amino acid residues), Kapp = (3.5 +/- 0.5) x 10(6) M-1 (reflecting the penetration of the farnesyl chain), and Kapp = (1.6 +/- 0.3) x 10(6) M-1 (reflecting a major contribution of the alpha(GDP)-subunit with only a minor contribution from the betagamma-complex). The apparent binding constant of the alpha(GTP)-subunit is distinctly smaller than that of the alpha(GDP)-subunit. Binding to negatively charged POPC/POPG (75/25 mole/mole) monolayers is reinforced by 2-3 cationic residues for the betagamma-complex. The alpha-subunit shows no electrostatic attraction and the heterotrimer shows even a slight electrostatic repulsion which becomes the dominating force in the absence of salts.

We propose a model for the recovery of the retinal rod photoresponse after a short stimulus. The approach describes the enzymatic deactivation of the photoactivated receptor, rhodopsin, by simple enzyme kinetics. An important feature of this description is that the R* deactivation obeys different time laws, depending on the numbers of R* formed per disc membrane and available enzyme molecules. If the enzyme works below substrate saturation, the rate of deactivation depends linearly on the number of R*, whereas for substrate saturation a hyperbolic relation--the well-known Michaelis-Menten equation--applies. This dichotomy is used to explain experimental finding that the relation between the saturation time of the photoresponse after short illumination and the flash strength has two sharply separated branches for low and high flash intensities (up to approximately 10% bleaching). By relating both branches to properties of the enzymatic rhodopsin deactivation, the new model transcends the classical notion of a constant characteristic lifetime of activated rhodopsin. With parameters that are plausible in the light of the available data and the additional information that the deactivating enzyme, rhodopsin kinase, and the signaling G-protein, transducin, compete for the active receptor, the slopes of the saturation function are correctly reproduced.

The G protein coupled receptor (GPCR) rhodopsin activates the heterotrimeric G protein transducin (Gt) to transmit the light signal into retinal rod cells. The rhodopsin activity is virtually zero in the dark and jumps by more than one billion fold after photon capture. Such perfect switching implies both high fidelity and speed of rhodopsin/Gt coupling. We employed Fourier transform infrared (FTIR) spectroscopy and supporting all-atom molecular dynamics (MD) simulations to study the conformational diversity of rhodopsin in membrane environment and extend the static picture provided by the available crystal structures. The FTIR results show how the equilibria of inactive and active protein states of the receptor (so-called metarhodopsin states) are regulated by the highly conserved E(D)RY and Yx7K(R) motives. The MD data identify an intrinsically unstructured cytoplasmic loop region connecting transmembrane helices 5 and 6 (CL3) and show how each protein state is split into conformational substates. The C-termini of the Gtγ- and Gtα-subunits (GαCT and GγCT), prepared as synthetic peptides, are likely to bind sequentially and at different sites of the active receptor. The peptides have different effects on the receptor conformation. While GγCT stabilizes the active states but preserves CL3 flexibility, GαCT selectively stabilizes a single conformational substate with largely helical CL3, as it is found in crystal structures. Based on these results we propose a mechanism for the fast and precise signal transfer from rhodopsin to Gt, which assumes a stepwise and mutual reduction of their conformational space. The mechanism relies on conserved amino acids and may therefore underlie GPCR/G protein coupling in general.

On rod disc membranes, single photoactivated rhodopsin (R*) molecules catalytically activate many copies of the G-protein (Gt), which in turn binds and activates the effector (phosphodiesterase). We have performed master equation simulations of the underlying diffusional protein interactions on a rectangular 1-micron2 model membrane, divided into 15 x 15 cells. Mono- and bimolecular reactions occur within cells, and diffusional transitions occur between (neighboring) cells. Reaction and diffusion constants yield the related probabilities for the stochastic transitions. The calculated kinetics of active effector form a response that is essentially determined by the stochastic lifetime distribution of R* (with characteristic time tau R*) and the reaction constants of Gt activation. Only a short tau R* (approximately 0.3 s) and a high catalytic rate (3000–4000 Gt s-1 R*-1) are consistent with electrophysiological data. Although R* shut-off limits the rise of the response, the lifetime distribution of free R* is not translated into a corresponding variability of the response peaks, because 1) the lifetime distribution of catalytically engaged R* is distorted, 2) small responses are enlarged by an overshoot of active effector, and 3) larger responses tend to undergo saturation. Comparison of these results to published photocurrent waveforms may open ways to understand the relative uniformity of the rod response.

After cis/trans isomerization of retinal and early photoproducts, activated rhodopsin (R*) develops signaling states for different proteins in a time-ordered sequence. Rhodopsin kinase binds all Meta forms, including the early Meta I, while interaction with transducin (Gt) or arrestin requires the deprotonated Schiff base form, Meta II (MII). Gt recognizes a specific conformation, termed MIIb, which arises from an additional, spectrally silent conversion, linked to proton uptake. Collisional coupling with the GDP-bound Gt holoprotein induces the release of GDP and formation of a stable R*—Gt complex, in which the nucleotide binding site of Gt is empty. The empty site complex, once formed, remains stable, even if the retinal is re-isomerized to the cis configuration. The cytoplasmic surface of rhodopsin appears to provide the majority of interaction sites for other proteins. Physical analyses and mutagenesis have emphasized the loops connecting helices C/D and E/F of the seven-helix structure. Any interaction between R* and Gt depends on a conserved charge pair at the interface between the helix C and the adjacent loop CD. Replacements or deletions in loops CD and EF were found to cause more specific functional defects, including slow release of GDP or failure of GTP-induced complex dissociation. In the dark, signaling states with low activity can be generated by reversible binding of all-trans-retinal to opsin. These light-independent signaling states are different from the Meta forms.

The photoreactions of rhodopsin regenerated with three 9-cis retinal analogs, modified at or in the vicinity of the beta-ionone ring (namely 5,6-epoxy, 7,8-diH, diethyl-acyclic) have been investigated by UV-vis and FTIR difference spectroscopy. In parallel, the ability to catalyze the GDP-->GTP exchange of G-protein (transducin) has been monitored by time-dependent fluorescence spectroscopy. The first photoproduct obtained with all three pigments at liquid nitrogen temperature is a blue-shifted intermediate (BSI), followed by a lumi-like intermediate at 170 K. For the 5,6-epoxy-ISO and 7,8-diH-ISO pigment we obtain two further intermediates similar to the META-I and META-II states of native RHO. For the diethyl-acyclic-ISO pigment only one further intermediate can be stabilized at 280 K. As compared to META-II the respective photoproduct exhibits striking differences. The latter two pigments have also been investigated in the solubilized lipid-free state (detergent: dodecyl maltoside) at 280 K. For the 5,6-epoxy-ISO pigment, the UV-vis, FTIR, and activation data agree with the formation of a META-II-like photoproduct (81% activation). Less META-II formation is observed for the 7,8-dihydro-ISO pigment in membranes (65% activation), but full formation in detergent (100% activation). Neither the membrane-bound nor the solubilized diethyl-acyclic-ISO pigment forms a META-II-like intermediate (18% and 0% activation, respectively). Therefore, we conclude that the substitution of the beta-ionone ring by two ethyl groups abolishes steric interactions with the protein, which are essential for META-II formation.(ABSTRACT TRUNCATED AT 250 WORDS)

We describe a real-time assay for the interaction of the G-protein of retinal rods, transducin (Gt), with its effector, cGMP phosphodiesterase (PDE). The purified proteins were recombined with isolated rod disk membranes. Reactions were triggered by flash photolysis of the receptor rhodopsin, and monitored by changes of near-infrared light scattering (LS signals). PDE-related LS signals from membrane preparations have been described by others [Caretta, A., & Stein, P. J. (1986) Biochemistry 25, 2335-2341; Bennett, N., & Clerc, A. (1992) Biochemistry 31, 1858-1866]. In the present study, the previous overall light-scattering (turbidity) approach was improved by a specific optical setup and millisecond time resolution. This allowed isolation of a fast "PDE signal" which monitors Gt-PDE interaction on the membrane and in real time. The assignment to the reaction is made by the following observations: (i) preactivation of PDE by purified G alpha GTP gamma S gradually suppresses the signal; (ii) the peak amplitude of the signal is stoichiometric with respect to both Gt and PDE; and (iii) the rise and delay time fit into a kinetic model for a reaction of activated Gt (G*). A comparative investigation, relating the PDE signal with Gt activation and PDE enzymatic activity, led to the following results: (i) the apparent dissociation constant for the interaction of G* with the first binding site on membrane-bound PDE is less than 2.5 nM; (ii) the time interval between formation of G* and its interaction with PDE is less than 5 ms; (iii) membrane-bound PDE, even in its preactivated form, slows the release of G* from the membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

Activation of the G protein-coupled receptor rhodopsin involves both the motion of transmembrane helix 6 (TM6) and proton exchange events. To study how these activation steps relate to each other, spin-labeled rhodopsin in solutions of dodecyl maltoside was used so that time-resolved TM6 motion and proton exchange could each be monitored as a function of pH and temperature after an activating light flash. The results reveal that the motion of TM6 is not synchronized with deprotonation of the Schiff base that binds the chromophore to the protein but is an order of magnitude slower at 30°C. However, TM6 motion and the uptake of a proton from solution in the neutral pH range follow the same time course. Importantly, the motion of TM6 is virtually independent of pH, as is Schiff base deprotonation under the conditions used, whereas proton uptake titrates with a pK of 6.5. This finding shows that proton uptake is a consequence rather than a cause of helix motion. Activated rhodopsin binds to and subsequently activates the cognate G protein, transducin. It has been shown that peptides derived from the C terminus of the transducin α-subunit mimic in part binding of the intact G protein. These peptides are found to bind to rhodopsin after TM6 movement, resulting in the release of protons. Collectively, the data suggest the following temporal sequence of events involved in activation: ( i ) internal Schiff base proton transfer; ( ii ) TM6 movement; and ( iii ) proton uptake from solution and binding of transducin.

In microseconds after photoexcitation, rhodopsin forms the Meta I intermediate from lumirhodopsin. In this conversion, contacts between retinal and the apoprotein are formed, which result in a defined arrangement of donor and acceptor groups for proton translocations. A system of protonation-dependent coupled equilibria is now adopted, comprising Meta intermediates I, II and III, and their isospectral subforms. Some Meta states were identified as signalling states, in which the receptor interacts with transducin (Gt), rhodopsin kinase (RK) and arrestin. The binding of Gt or arrestin shifts the equilibrium to Meta II, while RK does not, indicating exposure of the RK binding site(s) before Meta II is formed. On contact with the activated receptor, each signalling protein responds with a conformational change, which transforms it into a functionally active state. The bell-shaped pH/rate profiles which are seen for the activation of both the G protein and the receptor kinase, indicate the necessary protonation and deprotonation of groups with different pKa. The right wing of the profile reflects the formation of the protonated subconformation (termed MIIb) of Meta II. For the interaction with Gt, recent work suggests a 'sequential fit' mechanism, involving the recognition of the C-terminal peptide of the Gt alpha subunit and of the farnesylated C-terminus of the gamma subunit. Isolated peptides derived from these portions of the G protein mimic the left wing of the pH/rate profile. We discuss the sequential fit as a time-ordered sequence of microscopic recognition and conformational interlocking in the interaction with the G protein.

G protein-coupled receptors mediate biological signals by stimulating nucleotide exchange in heterotrimeric G proteins (Gαβγ). Receptor dimers have been proposed as the functional unit responsible for catalytic interaction with Gαβγ. To investigate whether a G protein-coupled receptor monomer can activate Gαβγ, we used the retinal photoreceptor rhodopsin and its cognate G protein transducin (G t ) to determine the stoichiometry of rhodopsin/G t binding and the rate of catalyzed nucleotide exchange in G t . Purified rhodopsin was prepared in dodecyl maltoside detergent solution. Rhodopsin was monomeric as concluded from fluorescence resonance energy transfer, copurification studies with fluorescent labeled and unlabeled rhodopsin, size exclusion chromatography, and multiangle laser light scattering. A 1:1 complex between light-activated rhodopsin and G t was found in the elution profiles, and one molecule of GDP was released upon complex formation. Analysis of the speed of catalytic rhodopsin/G t interaction yielded a maximum of ≈50 G t molecules per second and molecule of activated rhodopsin. The bimolecular rate constant is close to the diffusion limit in the diluted system. The results show that the interaction of G t with an activated rhodopsin monomer is sufficient for fully functional G t activation. Although the activation rate in solution is at the physically possible limit, the rate in the native membrane is still 10-fold higher. This is likely attributable to the precise orientation of the G protein to the membrane surface, which enables a fast docking process preceding the actual activation step. Whether docking in membranes involves the formation of rhodopsin dimers or oligomers remains to be elucidated.

Photoreceptor cGMP Phsophodiesterase 6 (PDE6) is the effector molecule of visual signal transduction and mediates fast response of light signals. The rod holo-PDE6 comprises catalytic (α, β; each ~ 90 kDa) and two identical inhibitiory (γ; ~ 10 kDa) subunits. The catalytic subunits comprise N-terminal tandem GAF domains followed by C-terminal catalytic domains and isoprenylations for membrane-association. Contrary to activation of other tandem GAF comprising PDEs, PDE6 activation does not occur via cGMP-induced concerted conformational changes. Rather two copies of the α-subunit of retinal G-Protein (Gα*), transducin, activate PDE6 by partially displacing the inhibitory subunits. The activation of PDE6 has therefore been described as a “de-inhibition”. The affinity of Gα* to PDE6 and the enzymatic activity of the intermediary 1:1 complex is highly disputed, therefore a conclusive activation model is lacking so far. Our combined structural, enzymatic and computational investigations deal with the activation-mechanism of PDE6. Our cryo electron microscopy (EM) structure of PDEαβ catalytic core shows an elongated bell-shaped structure with symmetric side-by-side arrangement of the two subunits with flexible membrane-binding domains. A comparison with nearly full-length inactive PDE2A structure [1] suggests that less compaction of both subunits and higher degree of conformational freedom of the catalytic domains result in constitutive activation of PDE6αβ, which is kept inactive by the inhibitory γ subunits. Furthermore, the structure of PDE6 suggests Gα* binding-sites pointing to opposing faces. The enzymatic characterization using Gα* titration of the PDE6 however reveal striking asymmetry of the two catalytic subunits with a high and a low affinity binding site for Gα*. Occupancy of the PDE6 with one Gα* induces negligible activity, whereas occupancy with two copies of Gα* leads to full enzyme activity. Such an activation mechanism constitutes a “coincidence switch” that allows noise filtering (i.e., spontaneously produced Gα* do not activate PDE6). Our spatiotemporal simulation work indeed confirms that spontaneously generated Gα* lead to the formation of singly occupied PDE6 and only a high local concentration of Gα*, as produced by an active receptor (rhodopsin), leads to doubly Gα* occupied effector complex. Therefore the localized large concentration of Gα* combined with the asymmetric properties of PDE6 constitutes a “density switch” that allows suppression effector level noise and reliable reporting of single quantum events in rod photoreceptor cells.

Catalysis of nucleotide exchange in heterotrimeric G proteins (Galphabetagamma) is a key step in cellular signal transduction mediated by G protein-coupled receptors. The Galpha N terminus with its helical stretch is thought to be crucial for G protein/activated receptor (R(*)) interaction. The N-terminal fatty acylation of Galpha is important for membrane targeting of G proteins. By applying biophysical techniques to the rhodopsin/transducin model system, we studied the effect of N-terminal truncations in Galpha. In Galphabetagamma, lack of the fatty acid and Galpha truncations up to 33 amino acids had little effect on R(*) binding and R(*)-catalyzed nucleotide exchange, implying that this region is not mandatory for R(*)/Galphabetagamma interaction. However, when the other hydrophobic modification of Galphabetagamma, the Ggamma C-terminal farnesyl moiety, is lacking, R(*) interaction requires the fatty acylated Galpha N terminus. This suggests that the two hydrophobic extensions can replace each other in the interaction of Galphabetagamma with R(*). We propose that in native Galphabetagamma, these two terminal regions are functionally redundant and form a microdomain that serves both to anchor the G protein to the membrane and to establish an initial docking complex with R(*). Accordingly, we find that the native fatty acylated Galpha is competent to interact with R(*) even in the absence of Gbetagamma, whereas nonacylated Galpha requires Gbetagamma for interaction. Experiments with N-terminally truncated Galpha subunits suggest that in the second step of the catalytic process, the receptor binds to the alphaN/beta1-loop region of Galpha to reduce nucleotide affinity and to make the Galpha C terminus available for subsequent interaction with R(*).

Photoisomerization of rhodopsin activates a heterotrimeric G-protein cascade leading to closure of cGMP-gated channels and hyperpolarization of photoreceptor cells. Massive translocation of the visual G-protein transducin, Gt, between subcellular compartments contributes to long term adaptation of photoreceptor cells. Ca(2+)-triggered assembly of a centrin-transducin complex in the connecting cilium of photoreceptor cells may regulate these transducin translocations. Here we demonstrate expression of all four known, closely related centrin isoforms in the mammalian retina. Interaction assays revealed binding potential of the four centrin isoforms to Gtbetagamma heterodimers. High affinity binding to Gtbetagamma and subcellular localization of the centrin isoforms Cen1 and Cen2 in the connecting cilium indicated that these isoforms contribute to the centrin-transducin complex and potentially participate in the regulation of transducin translocation through the photoreceptor cilium. Binding of Cen2 and Cen4 to Gbetagamma of non-visual G-proteins may additionally regulate G-proteins involved in centrosome and basal body functions.

In the phototransduction pathway of rhodopsin, the metarhodopsin (Meta) III retinal storage form arises from the active G-protein binding Meta II by a slow spontaneous reaction through the Meta I precursor or by light absorption and photoisomerization, respectively. Meta III is a side product of the Meta II decay path and holds its retinal in the original binding site, with the Schiff base bond to the apoprotein reprotonated as in the dark ground state. It thus keeps the retinal away from the regeneration pathway in which the photolyzed all-trans-retinal is released. This study was motivated by our recent observation that Meta III remains stable for hours in membranes devoid of regulatory proteins, whereas it decays much more rapidly in situ. We have now explored the possibility of regulated formation and decay of Meta III, using intrinsic opsin tryptophan fluorescence and UV-visible and Fourier transform infrared spectroscopy. We find that a rapid return of Meta III into the regeneration pathway is triggered by the G-protein transducin (G(t)). Depletion of the retinal storage is initiated by a novel direct bimolecular interaction of G(t) with Meta III, which was previously considered inactive. G(t) thereby induces the transition of Meta III into Meta II, so that the retinylidene bond to the apoprotein can be hydrolyzed, and the retinal can participate again in the normal retinoid cycle. Beyond the potential significance for retinoid metabolism, this may provide the first example of a G-protein-catalyzed conversion of a receptor.

Rhodopsin bears 11-cis-retinal covalently bound by a protonated Schiff base linkage. 11-cis/all-trans isomerization, induced by absorption of green light, leads to active metarhodopsin II, in which the Schiff base is intact but deprotonated. The subsequent metabolic retinoid cycle starts with Schiff base hydrolysis and release of photolyzed all-trans-retinal from the active site and ends with the uptake of fresh 11-cis-retinal. To probe chromophore-protein interaction in the active state, we have studied the effects of blue light absorption on metarhodopsin II using infrared and time-resolved UV-visible spectroscopy. A light-induced shortcut of the retinoid cycle, as it occurs in other retinal proteins, is not observed. The predominantly formed illumination product contains all-trans-retinal, although the spectra reflect Schiff base reprotonation and protein deactivation. By its kinetics of formation and decay, its low temperature photointermediates, and its interaction with transducin, this illumination product is identified as metarhodopsin III. This species is known to bind all-trans-retinal via a reprotonated Schiff base and forms normally in parallel to retinal release. We find that its generation by light absorption is only achieved when starting from active metarhodopsin II and is not found with any of its precursors, including metarhodopsin I. Based on the finding of others that metarhodopsin III binds retinal in all-trans-C(15)-syn configuration, we can now conclude that light-induced formation of metarhodopsin III operates by Schiff base isomerization ("second switch"). Our reaction model assumes steric hindrance of the retinal polyene chain in the active conformation, thus preventing central double bond isomerization.

In retinal rod photoreceptor cells, transducin (Gt) and cyclic GMP phosphodiesterase (PDE) are peripherally anchored to the cytoplasmic surface of the disk saccules. We have examined the role of specific phospholipids in the interaction of these proteins with native osmotically intact disk vesicles, employing spin-labeled phospholipid analogues (2% of total phospholipids) and bovine serum albumin back-exchange assay. Inactive GDP-bound transducin exclusively reduced the extraction of negatively charged phosphatidylserine. The effect disappeared upon activation of the G-protein with guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS). PDE affected the extraction of the zwitterionic phosphatidylcholine and, to a smaller extent, of phosphatidylethanolamine. When active GtGTPgammaS interacted with the PDE to form the active effector, the interaction with phosphatidylcholine was specifically enhanced. Each copy of the G-protein bound 3 +/- 1 molecules of phosphatidylserine, whereas the PDE bound a much larger amount (70 +/- 10) of a mixture of phosphatidylcholine and ethanolamine. The results are interpreted as a head group-specific and state-dependent interaction of the signaling proteins with the phospholipids of the photoreceptor membrane.

The transbilayer redistribution of spin-labeled phospholipid analogues (SL-PL) with choline, serine, and ethanolamine head groups (PC, PS, and PE, respectively) was studied on intact disc vesicles of bovine rod outer segment membranes in the dark and after illumination. Redistribution was measured by the extraction of spin-labeled lipid analogues from the outer leaflet of membrane using the bovine serum albumin back-exchange assay. In the dark, PS was distributed asymmetrically, favoring the outer leaflet, whereas PC and PE showed small if any asymmetry. Green illumination for 1 min caused lipid head group-specific reorganization of SL-PL. Extraction of SL-PS by bovine serum albumin showed a fast transient (10 min) enhancement, which was further augmented by a peptide stabilizing the active metarhodopsin II conformation. The data suggest a direct release of 1 molecule of bound PS per rhodopsin into the outer leaflet and subsequent redistribution between the two leaflets. SL-PE and SL-PC showed more complex kinetics, in both cases consistent with a prolonged period of reduced extraction (2 phospholipids per rhodopsin in each case). The different phases of SL-PL reorganization after illumination may be related to the formation and decay of the active rhodopsin species and to their subsequent regeneration process.

In rhodopsin's function as a photoreceptor, 11-cis-retinal is covalently bound to Lys(296) via a protonated Schiff base. 11-cis/all-trans photoisomerization and relaxation through intermediates lead to the metarhodopsin II photoproduct, which couples to transducin (G(t)). Here we have analyzed a different signaling state that arises from noncovalent binding of all-trans-retinal (atr) to the aporeceptor opsin and enhances the very low opsin activity by several orders of magnitude. Like with metarhodopsin II, coupling of G(t) to opsin-atr is sensitive to competition by synthetic peptides from the COOH termini of both G(t)alpha and G(t)gamma. However, atr does not compete with 11-cis-retinal incorporation into the Lys(296) binding site and formation of the light-sensitive pigment. Blue light illumination fails to photorevert opsin-atr to the ground state. Thus noncovalently bound atr has no access to the light-dependent binding site and reaction pathway. Moreover, in contrast to light-dependent signaling, removal of the palmitoyl anchors at Cys(322) and Cys(323) in the rhodopsin COOH terminus impairs the atr-stimulated activity. Repalmitoylation by autoacylation with palmitoyl-coenzyme A restores most of the original activity. We hypothesize that the palmitoyl moieties are part of a second binding pocket for the chromophore, mediating hydrophobic interactions that can activate a large part of the catalytic receptor/G-protein interface.

Information on the temporal order, the kinetics, and the energetics of each of the reaction intermediates is required in order to fully elucidate the mechanism of catalytic interaction of G proteins with their receptors. Intrinsic monitors, such as light scattering, absorption, and fluorescence, exploit endogenous properties of proteins, and therefore offer the advantage of leaving the system under investigation undisturbed. Such assays currently available for G proteins include the well-known intrinsic fluorescence changes of a tryptophan residue located near the G protein's active center. In addn., the visual system offers UV-visible spectrophotometric assays in which the retinal chromophore itself serves as an intrinsic reporter group. These assays include monitoring the transducin sensitive formation of the active metarhodopsin-II (MII) conformation, as well as the photoregeneration of dark state rhodopsin from MII. A third technique is kinetic light scattering, which makes use of the unique activation-dependent soly. of the rod G protein. While these monitors are highly specialized, more generally applicable monitors have been developed in recent years and successfully tested with the visual system. This family of techniques is based on the evanescent field in the vicinity of a reflecting optical surface, and includes surface plasmon resonance and resonant mirror spectroscopy. Applications and limitations of these techniques are described. (c) 2000 Academic Press. [on SciFinder (R)]

Visual signal transduction in the dim-light sensitive rod cell begins with light-induced isomerization of the covalently-attached chromophore ligand of rhodopsin, 11-cis retinal, to all-trans retinal. This event results in formation of the active species Metarhodopsin II (Meta II), which binds and activates several copies of the G-protein transducin, leading to signaling to nearby nerve cells. Metarhodopsin II is also the substrate for rhodopsin kinase, which phosphorylates the receptor to allow binding of the signal-quenching protein arrestin. Metarhodopsin II is not stable and decays within minutes into late photoproducts and ultimately the apoprotein opsin. In a rod cell exposed to the bright light associated with daytime, nearly all rhodopsin photoreceptors are “bleached” and exist as a heterogeneous population of these various late-photoproducts. Intriguingly, arrestin has been observed to translocate to the photoreceptor-rich disc membranes of the rod outer segment upon exposure to light, and arrestin remains there for the duration of light exposure.To better understand the molecular mechanisms involved in this phenomenon, we have studied the interactions of arrestin with phosphorylated opsin (opsin-P) and the effects of all-trans retinal on this interaction. We find that, although arrestin has poor affinity for opsin-P (Kd ∼ 40 μM) compared to Meta II-P (Kd ∼ 20 nM), the addition of all-trans retinal to opsin-P increases its affinity for arrestin by approximately two orders of magnitude. Given that all rhodopsin photoproducts eventually decay to opsin and free all-trans retinal, this result could explain the persistence of arrestin in the outer segment during constant light-exposure. Finally, we find that arrestin binds opsin-P differently than Meta II-P but undergoes a similar conformational change upon the addition of all-trans retinal. The structural and physiological implications of our results will be discussed.

The assembly of signalling molecules into macromolecular complexes (transducisomes) provides specificity, sensitivity and speed in intracellular signalling pathways. Rod photoreceptors in the eye contain an unusual set of glutamic-acid-rich proteins (GARPs) of unknown function. GARPs exist as two soluble forms, GARP1 and GARP2, and as a large cytoplasmic domain (GARP' part) of the beta-subunit of the cyclic GMP-gated channel. Here we identify GARPs as multivalent proteins that interact with the key players of cGMP signalling, phosphodiesterase and guanylate cyclase, and with a retina-specific ATP-binding cassette transporter (ABCR), through four, short, repetitive sequences. In electron micrographs, GARPs are restricted to the rim region and incisures of discs in close proximity to the guanylate cyclase and ABCR, whereas the phosphodiesterase is randomly distributed. GARP2, the most abundant splice form, associates more strongly with light-activated than with inactive phosphodiesterase, and GARP2 potently inhibits phosphodiesterase activity. Thus, the GARPs organize a dynamic protein complex near the disc rim that may control cGMP turnover and possibly other light-dependent processes. Because there are no similar GARPs in cones, we propose that GARPs may prevent unnecessary cGMP turnover during daylight, when rods are held in saturation by the relatively high light levels.

Photoactivation of the retinal photoreceptor rhodopsin proceeds through a cascade of intermediates, resulting in protein-protein interactions catalyzing the activation of the G-protein transducin (Gt). Using stabilization and photoregeneration of the receptor's signaling state and Gt activation assays, we provide evidence for a two-site sequential fit mechanism of Gt activation. We show that the C-terminal peptide from the Gt gamma-subunit, Gtgamma(50-71)farnesyl, can replace the holoprotein in stabilizing rhodopsin's active intermediate metarhodopsin II (MII). However, the peptide cannot replace the Gtbeta gamma complex in direct activation assays. Competition by Gtgamma(50-71)farnesyl with Gt for the active receptor suggests a pivotal role for Gtbeta gamma in signal transfer from MII to Gt. MII stabilization and competition is also found for the C-terminal peptide from the Gt alpha-subunit, Gtalpha(340-350), but the capacity of this peptide to interfere in MII-Gt interactions is paradoxically low compared with its activity to stabilize MII. Besides this disparity, the pH profiles of competition with Gt are characteristically different for the two peptides. We propose a two-site sequential fit model for signal transfer from the activated receptor, R*, to the G-protein. In the center of the model is specific recognition of conformationally distinct sites of R* by Gtalpha(340-350) and Gtgamma(50-71)farnesyl. One matching pair of domains on the proteins would, on binding, lead to a conformational change in the G-protein and/or receptor, with subsequent binding of the second pair of domains. This process could be the structural basis for GDP release and the formation of a stable empty site complex that is ready to receive the activating cofactor, GTP.

Publisher Summary Membrane-bound protein-protein interactions are key processes in biological signal transduction. The biomembrane reduces the dimensionality of the interactions and provides a barrier for the soluble components involved. With the vertebrate rod outer segment, nature has provided a model system for the study of such interactions. It contains the relevant proteins in abundance, and is accessible to biophysical as well as biochemical techniques. The analysis is simpler than in most related systems, because the rod photoreceptive apparatus is specialized to just one purpose, the monochromatic detection of low light levels. Besides the high quantum yield of the receptor protein, the fidelity of signal transduction via protein-protein interactions is most important for the high sensitivity achieved. Absorption of light initiates a cascade of biochemical reactions in the rod outer segment that finally leads to the hydrolysis of intracellular cyclic GMP, hyperpolarization of the cell and synaptic signaling. Many biochemical reactions can be assigned to specific steps of the physiological signal transduction pathway. Interactions of the receptor protein, rhodopsin, of the G-protein, transducin, and of the effector, cyclic GMP phosphodiesterase, have been analyzed in detail. In this chapter, aspects of signal transduction and regulation are discussed with emphasis on the role of these membrane bound interactions.

Electroretinographic data obtained from human subjects show that bright test flashes of increasing intensity induce progressively longer periods of apparent saturation of the rod-mediated electroretinogram (ERG) alpha wave. A prominent feature of the saturation function [the function that relates the saturation period T with the natural logarithm of flash intensity (ln I(f)] is its two-branched character. At relatively low flash intensities (I(f) below approximately 4 x 10(4) scotopic troland second), T increases approximately in proportion to ln I(f) with a slope [delta T/delta (ln I(f)] of approximately 0.3 s. At higher flash intensities, a different linear relation prevails, in which [deltaT/delta(ln I(f) is approximately 2.3 s [Invest. Ophthalmol. Vis. Sci. 36, 1603 (1995)]. Based on a model for photocurrent recovery in isolated single rods [Vis. Neurosci. 8, 9 (1992)], it was suggested that the upper-branch slope of approximately 2.3 s represents tau R*, the lifetime of photoactivated rhodopsin (R*). Here we show that a modified version of this model provides an explanation for the lower branch of the alpha-wave saturation function. In this model, tau E* is the exponential lifetime of an activated species (E*) within the transducin or guanosine 3', 5'-cyclic monophosphate (cGMP) phosphodiesterase stages of rod phototransduction; the generation of E* by a single R* occurs within temporally defined, elemental domains of disk membrane; and Ex, the immediate product of E* deactivation, is converted only slowly (time constant tau Ex) to E, the form susceptible to reactivation by R*. The model predicts that the decay of flash-activated cGMP phosphodiesterase (PDE*) is largely independent of the deactivation kinetics of R* at early postflash times (i.e., at times preceding or comparable with the lifetime tau E*) and that the lower-branch slope (approximately 0.3s) of the a-wave saturation function represent tau E*. The predicted early-stage independence of PDE* decay and R* deactivation furthermore suggests a basis for the relative constancy of the single-photon response observed in studies of isolated rods. Numerical evaluation of the model yields a value of approximately 6.7s for the time constant tau Ex.

Centrins are members of the family of Ca2+-binding EF-hand proteins. In photoreceptor cells, centrin isoform 1 is specifically localized in the non-motile cilium. This connecting cilium links the light-sensitive outer segment with the biosynthetic active inner segment of the photoreceptor cell. All intracellular exchanges between these compartments have to occur through this cilium. Three-dimensional structures of centrins from diverse organisms are known, showing that the EF-hand motifs of the N-terminal domains adopt closed conformations, while the C-terminal EF-hand motifs have open conformations. The crystal structure of an N-terminally extended mouse centrin 1 (MmCen1-L) resembles the overall structure of troponin C in its two Ca2+ bound form. Within the N-terminal extension in MmCen1-L, residues W24 and R25 bind to the C-terminal domain of centrin 1 in a target-protein-like geometry. Here, we discuss this binding mode in connection with putative interaction sites of the target-protein transducin and the self-assembly of centrins.

The so-called AT-signal described here is a transient light-induced increase of the near-infrared scattering from isolated bovine rod outer segments (ROS). Freshly prepared ROS are permeabilized with 0.01% Triton X-100 immediately before measurement in the presence of 1 mM GTP. The signal amplitude is saturated when approximately 2 rhodopsin molecules out of 30 000 are photo-excited. The signal recovers rapidly (approximately 90 s) and can be repeated in a succession of flashes. The AT-signal can be prevented by pre-activation of the phosphodiesterase (PDE) enzyme cascade at various levels: either at the level of G-protein, using ALF4- in darkness or GTP gamma S plus light; or at the level of the PDE catalytic unit, using protamine as an activator. The light sensitivity and kinetics of the AT-signal are similar to published parameters of PDE activation. These data suggest that light-induced activation of the PDE is the key reaction for the generation of the signal. On the other hand, blocking of the catalytic cGMP binding site by isobutylmethylxanthine only slightly affects the signal. We propose that the AT-signal reflects a structural change linked to the transient removal of the PDE inhibitory subunit from the catalytic unit.

In vertebrate rod cells absorption of a light quantum by rhodopsin is followed by intramolecular reactions of rhodopsin (photoproducts batho to meta II, ps to ms (1,2)). After milliseconds, photoexcited rhodopsin is able to trigger membrane bound enzymatic reactions. The first step thereby is stoichiometric association of the G-unit (G-protein, “transducin”) to the activated rhodopsin molecule (3).

On detergent-solubilised bovine rhodopsin, we have studied the formation of the active photoproduct, metarhodopsin II (MII), and its interaction with the rod G-protein, transducin (Gt). The measured rate of flash-induced MII formation decreases by a factor of 300 from n-dodecyl-β-D-maltoside (k = 4 × 103 at 18°C, pH 6.5), over (3-(lauroyloxy)propyl)-phosphorylcholine (deoxylysolecithin), n-octyl-β-D-glucopyranoside, sodium cholate to Chapso. For the two last agents. MII formation is similarly slow as in the native disc membrane; however, the micellar system does not display the very large decrease of the rate with lowering temperature, as is characteristic for the membrane. This points to entropic factors determining the rate in the micellar systems. An admixture of rigid steroid molecules (11-deoxycorticosterone) to lysolecithin micelles (‘doped micelles’) slows MII formation and shifts the MI/MII equilibrium to values typical for detergents of rigid structure. The observation gives further support to the surface free energy concept of MII formation outlined in previous studies. The free adjustment of the MI/MII equilibrium in these doped micelles allows Gt-induced formation of extra-MII to be measured, providing a convenient monitor of rhodopsin-Gt interaction in solution.Rhodopsin; Metarhodopsin II; Transducin; G-protein; (Detergent micelle)