Search

The light sensor of vertebrate scotopic (low-light) vision, rhodopsin, is a G-protein-coupled receptor comprising a polypeptide chain with bound chromophore, 11-cis-retinal, that exhibits remarkable physicochemical properties. This photopigment is extremely stable in the dark, yet its chromophore isomerises upon photon absorption with 70% efficiency, enabling the activation of its G-protein, transducin, with high efficiency. Rhodopsin's photochemical and biochemical activities occur over very different time-scales: the energy of retinaldehyde's excited state is stored in1 ps in retinal-protein interactions, but it takes milliseconds for the catalytically active state to form, and many tens of minutes for the resting state to be restored. In this review, we describe the properties of rhodopsin and its role in rod phototransduction. We first introduce rhodopsin's gross structural features, its evolution, and the basic mechanisms of its activation. We then discuss light absorption and spectral sensitivity, photoreceptor electrical responses that result from the activity of individual rhodopsin molecules, and recovery of rhodopsin and the visual system from intense bleaching exposures. We then provide a detailed examination of rhodopsin's molecular structure and function, first in its dark state, and then in the active Meta states that govern its interactions with transducin, rhodopsin kinase and arrestin. While it is clear that rhodopsin's molecular properties are exquisitely honed for phototransduction, from starlight to dawn/dusk intensity levels, our understanding of how its molecular interactions determine the properties of scotopic vision remains incomplete. We describe potential future directions of research, and outline several major problems that remain to be solved.

Current submillisecond time resolved broad band infrared spectrosco py, one of the most frequently used techniques for studying structure amp; 8722;functionrelationships in life sciences, is typically limited to fast cycling reactions that can berepeated thousands of times with high frequency. Notably, a majority of chemical andbiological processes do not comply with this requirement. For example, the activationof vertebrate rhodopsin, a prototype of many protein receptors in biological organismsthat mediate basic functions of life, including vision, smell, and taste, is irreversible.Here we present a dispersive single shot Fe amp; 769;ry spectrometer setup that extends suchspectroscopy to irreversible and slow cycling systems by exploiting the uniqueproperties of brilliant synchrotron infrared light combined with an advanced focalplane detector array embedded in a dispersive optical concept. We demonstrate oursingle shot method on microbial actinorhodopsin with a slow photocycle and onvertebrate rhodopsin with irreversible activation

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.

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.

A Mid-Infrared spectrometer specifically suitable for non-cyclic or slow cycling processes was developed for microsecond time-resolved measurements. The spectrometer overcomes the limit of conventional time-resolved Fourier-transform techniques such as step-scan FTIR which require cyclic systems where the reaction cascade is to be triggered thousands of times and in quick succession. In our approach, a combination of a fast linear detector array with highly brilliant synchrotron radiation source and a dispersive spectrometer core allows investigations of irreversible reaction cascades with high signal-to-noise ratio. We discuss first measurements to estimate the performance achieved so far.

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.

Receptor-ligand interaction: Olfactory receptors (ORs) are G-protein-coupled receptors (GPCRs), which detect signaling molecules such as hormones and odorants. The structure of opsin, the GPCR employed in vision, with a detergent molecule bound deep in its orthosteric ligand-binding pocket provides a template for OR homology modeling, thus enabling investigation of the structural basis of the mechanism of odorant-receptor recognition.

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.

In the rod cell of the retina, arrestin is responsible for blocking signaling of the G-protein-coupled receptor rhodopsin. The general visual signal transduction model implies that arrestin must be able to interact with a single light-activated, phosphorylated rhodopsin molecule (Rho*P), as would be generated at physiologically relevant low light levels. However, the elongated bi-lobed structure of arrestin suggests that it might be able to accommodate two rhodopsin molecules. In this study, we directly addressed the question of binding stoichiometry by quantifying arrestin binding to Rho*P in isolated rod outer segment membranes. We manipulated the “photoactivation density,” i.e. the percentage of active receptors in the membrane, with the use of a light flash or by partially regenerating membranes containing phosphorylated opsin with 11-cis-retinal. Curiously, we found that the apparent arrestin-Rho*P binding stoichiometry was linearly dependent on the photoactivation density, with one-to-one binding at low photoactivation density and one-to-two binding at high photoactivation density. We also observed that, irrespective of the photoactivation density, a single arrestin molecule was able to stabilize the active metarhodopsin II conformation of only a single Rho*P. We hypothesize that, although arrestin requires at least a single Rho*P to bind the membrane, a single arrestin can actually interact with a pair of receptors. The ability of arrestin to interact with heterogeneous receptor pairs composed of two different photo-intermediate states would be well suited to the rod cell, which functions at low light intensity but is routinely exposed to several orders of magnitude more light.

Phytochromes, which regulate many biological processes in plants, bacteria, and fungi, can exist in two stable states, Pr and Pfr, that can be interconverted by light, via a number of intermediates such as meta-Rc. Herein we employ FTIR spectroscopy to study the Pr-to-Pfr conversion of the bacteriophytochrome Agp1 from Agrobacterium tumefaciens. Static FTIR Pfr/Pr and meta-Rc/Pr difference spectra are disentangled in terms of cofactor and protein structural changes. Guided by DFT calculations on cofactor models, the chromophore conformational changes can be grouped into structural adjustments of the cofactor-protein interactions localized in the C-D dipyrrole moiety, that is, the photoisomerisation site, and in the A-B dipyrrole moiety including the protein attachment site. Whereas changes at the C and D rings appear to be largely completed in the meta-Rc state, the structural changes in the A-B unit occur during the transition from meta-Rc to Pfr, concomitant with the main protein structural changes, as demonstrated by static and time-resolved FTIR difference spectroscopy. We employ this technique to monitor, for the first time, the dynamics of the photocycle of phytochrome on the millisecond timescale. By extending the studies to genetically engineered protein variants of Agp1, we further demonstrate that H250 and D197 as well as the PHY domain are essential for formation of the Pfr state. Based on the IR spectroscopic and available crystallographic data we discuss the role of critical amino acid residues for the protein-cofactor interactions during the photoinduced reaction cycle.

Enveloped viruses and cellular transport vesicles share obvious morphological and functional properties. Both are composed of a closed membrane, which is lined with coat proteins and encases cargo. Transmembrane proteins inserted into the membrane define the target membrane area with which the vesicle or virus is destined to fuse. Here we discuss recent insight into the functioning of enveloped viruses in the framework of the “functional module” concept. Vesicular transport is an exemplary case of a functional module, as defined as a part of the proteome that assembles to perform a specific autonomous function in a living cell. Cellular vesicles serve to transport cargo between membranous organelles inside the cell, while enveloped viruses can be seen as carriers of the viral genome delivering their cargo from an infected to an uninfected cell. The turnover of both vesicles and viruses involves an analogous series of submodular events. This comprises assembly of elements, budding from the donor compartment, uncoating and/or maturation, docking to and finally fusion with the target membrane to release the cargo. This modular perception enables us to define submodular building blocks so that mechanisms and elements can be directly compared. It will be analyzed where viruses have developed their own specific strategy, where they share functional schemes with vesicles, and also where they even have “hijacked” complete submodular schemes from the cell. Such a perspective may also include new and more specific approaches to pharmacological interference with virus function, which could avoid some of the most severe side effects.

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.

Rhodopsin is the best-understood member of the large G protein-coupled receptor (GPCR) superfamily. The G-protein amplification cascade is triggered by poorly understood light-induced conformational changes in rhodopsin that are homologous to changes caused by agonists in other GPCRs. We have applied the "antibody imprint" method to light-activated rhodopsin in native membranes by using nine monoclonal antibodies (mAbs) against aqueous faces of rhodopsin. Epitopes recognized by these mAbs were found by selection from random peptide libraries displayed on phage. A new computer algorithm, FINDMAP, was used to map the epitopes to discontinuous segments of rhodopsin that are distant in the primary sequence but are in close spatial proximity in the structure. The proximity of a segment of the N-terminal and the loop between helices VI and VIII found by FINDMAP is consistent with the X-ray structure of the dark-adapted rhodopsin. Epitopes to the cytoplasmic face segregated into two classes with different predicted spatial proximities of protein segments that correlate with different preferences of the antibodies for stabilizing the metarhodopsin I or metarhodopsin II conformations of light-excited rhodopsin. Epitopes of antibodies that stabilize metarhodopsin II indicate conformational changes from dark-adapted rhodopsin, including rearrangements of the C-terminal tail and altered exposure of the cytoplasmic end of helix VI, a portion of the C-3 loop, and helix VIII. As additional antibodies are subjected to antibody imprinting, this approach should provide increasingly detailed information on the conformation of light-excited rhodopsin and be applicable to structural studies of other challenging protein targets.

Opsin, the ligand-free form of the G-protein-coupled receptor rhodopsin, at low pH adopts a conformationally distinct, active G-protein-binding state known as Ops*. A synthetic peptide derived from the main binding site of the heterotrimeric G protein-the carboxy terminus of the alpha-subunit (GalphaCT)-stabilizes Ops*. Here we present the 3.2 A crystal structure of the bovine Ops*-GalphaCT peptide complex. GalphaCT binds to a site in opsin that is opened by an outward tilt of transmembrane helix (TM) 6, a pairing of TM5 and TM6, and a restructured TM7-helix 8 kink. Contacts along the inner surface of TM5 and TM6 induce an alpha-helical conformation in GalphaCT with a C-terminal reverse turn. Main-chain carbonyl groups in the reverse turn constitute the centre of a hydrogen-bonded network, which links the two receptor regions containing the conserved E(D)RY and NPxxY(x)(5,6)F motifs. On the basis of the Ops*-GalphaCT structure and known conformational changes in Galpha, we discuss signal transfer from the receptor to the G protein nucleotide-binding site.

Rhodopsin is the visual pigment of rod cells and a prototypical G protein-coupled receptor. It is activated by cis-->trans photoisomerization of the covalently bound chromophore 11-cis-retinal, which acts in the cis configuration as an inverse agonist. Light-induced formation of the full agonist all-trans-retinal in situ triggers conformational changes in the protein moiety. Partial agonists of rhodopsin include a retinal analog lacking the methyl group at C-9, termed 9-demethyl-retinal (9-dm-retinal). Rhodopsin reconstituted with this retinal (9-dm-rhodopsin) activates G protein poorly. Here we investigated the molecular nature of the partial agonism in 9-dm-rhodopsin using site-directed spin labeling. Earlier site-directed spin labeling studies of rhodopsin identified a rigid-body tilt of the cytoplasmic segment of [corrected] transmembrane helix 6 (TM6) by approximately 6A as a central event in rhodopsin activation. Data presented here provide additional evidence for this mechanism. Only a small fraction of photoexcited 9-dm pigments reaches the TM6-tilted conformation. This fraction can be increased by increasing proton concentration or [corrected] by anticipation of the activating protonation step by the mutation E134Q in 9-dm-rhodopsin. These results on protein conformation are in complete accord with previous findings regarding the biological activity of the 9-dm pigments. When the proton concentration is further increased, a new state arises in 9-dm pigments that is linked to direct proton uptake at the retinal Schiff base. This state apparently has a conformation distinguishable from the active state.

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.

GPCRs catalyze GDP/GTP exchange in the a-subunit of heterotrimeric G proteins (G alpha beta gamma) through displacement of the G alpha C-terminal alpha 5 helix, which directly connects the interface of the active receptor (R*) to the nucleotide binding pocket of G. Hydrogen-deuterium exchange mass spectrometry and kinetic analysis of R* catalysed G protein activation have suggested that displacement of a5 starts from an intermediate GDP bound complex (R*center dot G(GDP)). To elucidate the structural basis of receptor-catalysed displacement of alpha 5, we modelled the structure of R*center dot G(GDP). A flexible docking protocol yielded an intermediate R*center dot G(GDP) complex, with a similar overall arrangement as in the X-ray structure of the nucleotide free complex (R*center dot G(empty)), however with the alpha 5 C-terminus (G alpha CT) forming different polar contacts with R*. Starting molecular dynamics simulations of G alpha CT bound to R* in the intermediate position, we observe a screw-like motion, which restores the specific interactions of alpha 5 with R* in R*center dot G(empty). The observed rotation of alpha 5 by 60 degrees is in line with experimental data. Reformation of hydrogen bonds, water expulsion and formation of hydrophobic interactions are driving forces of the alpha 5 displacement. We conclude that the identified interactions between R* and G protein define a structural framework in which the alpha 5 displacement promotes direct transmission of the signal from R* to the GDP binding pocket.

Signaling by G-protein–coupled receptors is often considered a uniform process, whereby a homogeneously activated proportion of randomly distributed receptors are activated under equilibrium conditions and produce homogeneous, steady-state intracellular signals. While this may be the case in some biologic systems, the example of rhodopsin with its strictly local single-quantum mode of function shows that homogeneity in space and time cannot be a general property of G-protein–coupled systems. Recent work has now revealed many other systems where such simplicity does not prevail. Instead, a plethora of mechanisms allows much more complex patterns of receptor activation and signaling: different mechanisms of protein-protein interaction; temporal changes under nonequilibrium conditions; localized receptor activation; and localized second messenger generation and degradation—all of which shape receptor-generated signals and permit the creation of multiple signal types. Here, we review the evidence for such pleiotropic receptor signaling in space and time.

In this chapter, we describe a set of complementary techniques that we use to study the activation of rhodopsin, a G protein-coupled receptor (GPCR), and its functional interactions with G protein and arrestin. The protein reagents used for these studies come from native disc membranes or heterologous expression, and G protein and arrestin are often replaced with less complex synthetic peptides derived from key interaction sites of these binding partners (BPs). We first report on our approach to protein X-ray crystallography and describe how protein crystals from native membranes are obtained. The crystal structures provide invaluable resolution, but other techniques are required to assess the dynamic equilibria characteristic for active GPCRs. The simplest approach is "Extra Meta II," which uses UV/Vis absorption spectroscopy to monitor the equilibrium of photoactivated states. Site-specific information about the BPs (e.g., arrestin) is added by fluorescence techniques employing mutants labeled with reporter groups. All functional changes in both the receptor and interacting proteins or peptides are seen with highest precision using Fourier transform infrared (FTIR) difference spectroscopy. In our approach, the lack of site-specific information in FTIR is overcome by parallel molecular dynamics simulations, which are employed to interpret the results and to extend the timescale down to the range of conformational substates.

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*.

BET3 is a component of TRAPP, a complex involved in the tethering of transport vesicles to the cis-Golgi membrane. The crystal structure of human BET3 has been determined to 1.55-A resolution. BET3 adopts an alpha/beta-plait fold and forms dimers in the crystal and in solution, which predetermines the architecture of TRAPP where subunits are present in equimolar stoichiometry. A hydrophobic pocket within BET3 buries a palmitate bound through a thioester linkage to cysteine 68. BET3 and yeast Bet3p are palmitoylated in recombinant yeast cells, the mutant proteins BET3 C68S and Bet3p C80S remain unmodified. Both BET3 and BET3 C68S are found in membrane and cytosolic fractions of these cells; in membrane extractions, they behave like tightly membrane-associated proteins. In a deletion strain, both Bet3p and Bet3p C80S rescue cell viability. Thus, palmitoylation is neither required for viability nor sufficient for membrane association of BET3, which may depend on protein-protein contacts within TRAPP or additional, yet unidentified modifications of BET3. A conformational change may facilitate palmitoyl extrusion from BET3 and allow the fatty acid chain to engage in intermolecular hydrophobic interactions.

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.

To regenerate light-sensitive rhodopsin in rods from active metarhodopsin II (Meta II), all-trans-retinal must be removed from the retinal binding pocket and metabolically supplied 11-cis-retinal has to form a new retinylidene bond in the active site. Recent work from this laboratory has focused on Meta II decay and release and uptake of retinals in opsin employing intrinsic protein fluorescence. Here we summarize the results in the retinal channeling hypothesis, which describes a passage of the chromophore through the protein. 11-cis-retinal is taken up into an entrance site, and photolyzed all-trans-retinal is released from the active site into an exit site.

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.

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 storaged away, thus regulating photoreceptor regeneration.

Deactivation of G-protein-coupled receptors relies on a timely blockade by arrestin. However, under dim light conditions, virtually all arrestin is in the rod inner segment, and the splice variant p(44) (Arr(1-370A)) is the stop protein responsible for receptor deactivation. Using size exclusion chromatography and biophysical assays for membrane-bound protein-protein interaction, membrane binding, and G-protein activation, we have investigated the interactions of Arr(1-370A) and proteolytically truncated Arr(3-367) with rhodopsin. We find that these short arrestins do not only interact with the phosphorylated active receptor but also with inactive phosphorylated rhodopsin or opsin in membranes or solution. Because of the latter interaction they are not soluble (like arrestin) but membrane-bound in the dark. Upon photoexcitation, Arr(3-367) and Arr(1-370A) interact with prephosphorylated rhodopsin faster than arrestin and start to quench G(t) activation on a subsecond time scale. The data indicate that in the course of rhodopsin deactivation, Arr(1-370A) is handed over from inactive to active phosphorylated rhodopsin. This mechanism could provide a new aspect of receptor shutoff in the single photon operating range of the rod cell.

Phototransduction is initiated by the photoisomerization of rhodopsin (Rho) chromophore 11-cis-retinylidene to all-trans-retinylidene. Here, using Rho regenerated with retinal analogs with different ring sizes, which prevent isomerization around the C(11)=C(12) double bond, the activation mechanism of this G-protein-coupled receptor was investigated. We demonstrate that 11-cis-7-ring-Rho does not activate G-protein in vivo and in vitro, and that it does not isomerize along other double bonds, suggesting that it fits tightly into the binding site of opsin. In contrast, bleaching 11-cis-6-ring-Rho modestly activates phototransduction in vivo and at low pH in vitro. These results reveal that partial activation is caused by isomerization along other double bonds in more rigid 6-locked retinal isomers and protonation of key residues by lowering pH in 11-cis-6-ring-Rhos. Full activation is not achieved, because isomerization does not induce a complete set of conformational rearrangements of Rho. These results with 6- and 7-ring-constrained retinoids provide new insights into Rho activation and suggest a potential use of locked retinals, particularly 11-cis-7-ring-retinal, to inactivate opsin in some retinal degeneration diseases.

Low viscosity of the surface of alveolar fluid is mandatory for undisturbed surfactant function. Based on the known reduction of the viscosity of surfactant-like phospholipid (PL-) mixtures by plasmalogens, the effect of cholesterol and surfactant protein (SP-) B on surface viscosity of these lipid mixtures has been studied. Surface viscosity at the corresponding surface tension was measured with the oscillating drop surfactometer. We found that the viscosity was lowest in cholesterol-, followed by plasmalogen- and SP-B containing samples. Addition of SP-B to a plasmalogen-containing PL-mixture caused a further decrease in viscosity. However, in cholesterol containing mixtures, addition of SP-B led to a significant increase in viscosity, and the effect was reversed by further addition of plasmalogens. We conclude that SP-B, plasmalogens and cholesterol all affect the surface viscosity, thus synergistically regulate monolayer stability. This suggests that they are all needed in vivo for fine tuning of surface properties of pulmonary surfactant.

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.

G-protein-coupled receptors (GPCRs) transmit extracellular signals to activate intracellular heterotrimeric G proteins (Gαβγ) and arrestins. For G protein signalling, the Gα C-terminus (GαCT) binds to a cytoplasmic crevice of the receptor that opens upon activation. A consensus motif is shared among GαCT from the Gi/Gt family and the ‘finger loop’ region (ArrFL1–4) of all four arrestins. Here we present a 2.75 Å crystal structure of ArrFL-1, a peptide analogue of the finger loop of rod photoreceptor arrestin, in complex with the prototypical GPCR rhodopsin. Functional binding of ArrFL to the receptor was confirmed by ultraviolet-visible absorption spectroscopy, competitive binding assays and Fourier transform infrared spectroscopy. For both GαCT and ArrFL, binding to the receptor crevice induces a similar reverse turn structure, although significant structural differences are seen at the rim of the binding crevice. Our results reflect both the common receptor-binding interface and the divergent biological functions of G proteins and arrestins., G-protein-coupled receptors (GPCRs) transmit signals through intracellular heterotrimeric G proteins and arrestins. Here, Szczepek et al. present the structure of a common binding interface for Gα and arrestin on rhodopsin to shed light on key interactions that mediate transduction of specific signals through a single GPCR.

G protein coupled receptors (GPCRs) transmit extracellular signals into the cell by binding and activating different intracellular signaling proteins, such as G proteins (Gαβγ, families Gi, Gs, Gq, G12/13) or arrestins. To address the issue of Gs vs Gi coupling specificity, we carried out molecular dynamics simulations of lipid-embedded active β2-adrenoceptor (β2AR*) in complex with C-terminal peptides derived from the key interaction site of Gα (GαCT) as surrogate of Gαβγ. We find that GiαCT and GsαCT exploit distinct cytoplasmic receptor conformations that coexist in the uncomplexed β2AR*. The slim GiαCT stabilizes a β2AR* conformation, not accessible to the bulkier GsαCT, which requires a larger TM6 outward tilt for binding. Our results suggest that the TM6 conformational heterogeneity regulates the catalytic activity of β2AR* toward Gi or Gs.

Absorption of light in rhodopsin leads through 11-cis- and all-trans-retinal isomerization, proton transfers, and structural changes to the active G-protein binding meta-II state. When meta-II is photolysed by blue light absorption, the activating pathway is apparently reverted, and rhodopsin is photoregenerated. However, the product formed, a P subspecies with A(max) = 500 nm (P(500)), is different from the ground state based on the following observations: (i) the ground state fingerprint of 11-cis-retinal does not appear in the infrared spectra, although the proton transfers and structural changes are reverted; (ii) extraction of the retinal from P(500) does not yield the expected stoichiometric amount of 11-cis-retinal but predominantly yields all-trans-retinal; (iii) the infrared spectrum of P(500) is similar to the classical meta-III intermediate, which arises from meta-II by thermal decay; and (iv) both P(500) and meta-III can be photoconverted to meta-II with the same changes in the infrared spectrum and without a significant change in the isomerization state of the extracted chromophore. The data indicate the presence of a "second switch" between active and inactive conformations that operates by photolysis but without isomerization around the C(11)-C(12) double bond. This emphasizes the exclusivity of the ground state, which is only accessible by the metabolic regeneration with 11-cis-retinal.

G-protein-coupled receptors (GPCRs) are involved in a vast variety of cellular signal transduction processes from visual, taste and odor perceptions to sensing the levels of many hormones and neurotransmitters. As a result of agonist-induced conformation changes, GPCRs become activated and catalyze nucleotide exchange within the G proteins, thus detecting and amplifying the signal. GPCRs share a common heptahelical transmembrane structure as well as many conserved key residues and regions. Rhodopsins are prototypical GPCRs that detect photons in retinal photoreceptor cells and trigger a phototransduction cascade that culminates in neuronal signaling. Biophysical and biochemical studies of rhodopsin activation, and the recent crystal structure determination of bovine rhodopsin, have provided new information that enables a more complete mechanism of vertebrate rhodopsin activation to be proposed. In many aspects, rhodopsin might provide a structural and functional template for other members of the GPCR family.

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.

The oscillating drop surfactometer (ODS) measures surface tension (gamma) and energy dissipation (damping constant b) of surfactant on a 1 microl sample. gamma is obtained from the period of oscillation and b from its free decay or from the phase shift slope in resonance. After calibration with substances with different gamma, corrections were made for capillary fixation and loss of mass by evaporation. Surface active substances are delivered from liposomes in the interior (subphase) or injected from outside, with microdrops (180 pl each) of solution. As an application example, we have investigated surfactant extract and pure phospholipid. In minutes after formation of a drop containing a diluted Survanta suspension, gamma, decreases by 20 mN/m, while b increases three-fold. This effect, assigned to spontaneous adsorption from liposomes to the surface, is not seen with pure dipalmitoylphosphatidylcholine (DPPC) under our conditions. However, microdrop injection of DPPC triggers a rapid decrease of gamma and a delayed strong increase in b. The effect is modulated by DPPC in the subphase and by cholesterol. Investigations with L-alpha-lysophosphatidylcholine show the high sensitivity of the ODS technique in the determination of the energy dissipation at air-liquid boundary surfaces. Although the ODS is limited to applications with gamma15 mN m(-1), it offers the advantage to give, with small samples and within seconds, a simultaneous readout of both Surface tension gamma and the parameter b, as a measure of surface viscosity.

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.