Rhodopsin mutants that bind but fail to activate transducin.

Rhodopsin is a member of a family of receptors that contain seven transmembrane helices and are coupled to G proteins. The nature of the interactions between rhodopsin mutants and the G protein, transduction (Gt), was investigated by flash photolysis in order to monitor directly Gt binding and dissociation. Three mutant opsins with alterations in their cytoplasmic loops bound 11-cis-retinal to yield pigments with native rhodopsin absorption spectra, but they failed to stimulate the guanosine triphosphatase activity of Gt. The opsin mutations included reversal of a charged pair conserved in all G protein-coupled receptors at the cytoplasmic border of the third transmembrane helix (mutant CD1), replacement of 13 amino acids in the second cytoplasmic loop (mutant CD2), and deletion of 13 amino acids from the third cytoplasmic loop (mutant EF1). Whereas mutant CD1 failed to bind Gt, mutants CD2 and EF1 showed normal Gt binding but failed to release Gt in the presence of guanosine triphosphate. Therefore, it appears that at least the second and third cytoplasmic loops of rhodopsin are required for activation of bound Gt.

Site of G protein binding to rhodopsin mapped with synthetic peptides from the alpha subunit.

The interaction between receptors and guanine nucleotide binding (G) proteins leads to G protein activation and subsequent regulation of effector enzymes. The molecular basis of receptor-G protein interaction has been examined by using the ability of the G protein from rods (transducin) to cause a conformational change in rhodopsin as an assay. Synthetic peptides corresponding to two regions near the carboxyl terminus of the G protein alpha subunit, Glu311-Val328 and Ile340-Phe350, compete with G protein for interaction with rhodopsin. Amino acid substitution studies show that Cys321 is required for this effect. Ile340-Phe350 and a modified peptide, acetyl-Glu311-Lys329-amide, mimic G protein effects on rhodopsin conformation, showing that these peptides bind to and stabilize the activated conformation of rhodopsin.

Activation of rhodopsin: new insights from structural and biochemical studies.

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.

Micropatterned immobilization of a G protein-coupled receptor and direct detection of G protein activation.

G protein-coupled receptors (GPCRs) constitute an abundant family of membrane receptors of high pharmacological interest. Cell-based assays are the predominant means of assessing GPCR activation, but are limited by their inherent complexity. Functional molecular assays that directly and specifically report G protein activation by receptors could offer substantial advantages. We present an approach to immobilize receptors stably and with defined orientation to substrates. By surface plasmon resonance (SPR), we were able to follow ligand binding, G protein activation, and receptor deactivation of a representative GPCR, bovine rhodopsin. Microcontact printing was used to produce micrometer-sized patterns with high contrast in receptor activity. These patterns can be used for local referencing to enhance the sensitivity of chip-based assays. The immobilized receptor was stable both for hours and during several activation cycles. A ligand dose-response curve with the photoactivatable agonist 11-cis-retinal showed a half-maximal signal at 120 nM. Our findings may be useful to develop novel assay formats for GPCRs based on receptor immobilization to solid supports, particularly to sensor surfaces.

Effect of GTP on the rhodopsin-G-protein complex by transient formation of extra metarhodopsin II.

The light-induced transient interaction between rhodopsin and G-protein in the presence of GTP has been measured by the formation of extra metarhodopsin II. Disc membranes were recombined with the hypotonic extract containing the G-protein. Without GTP, a flash induces stable rhodopsin-G-protein complexes which dissociate upon addition of GTP. In low GTP (less than 10 microM) transient rhodopsin X G-protein interaction is observed. Rhodopsin X G-protein dissociates the faster, the more GTP is present (rate of dissociation, 0.3/s at 5 microM GTP; T = 3.5 degrees C). The results corroborate that the uptake of GTP terminates the rhodopsin-G-protein complex and allow an estimation of the rhodopsin X G-protein lifetime.

Interplay between hydroxylamine, metarhodopsin II and GTP-binding protein in bovine photoreceptor membranes.

The decay reactions of metarhodopsin II and the dissociation of the complex between rhodopsin (in the metarhodopsin II state) and the GTP-binding protein (G-protein) (in its inactive, GDP-binding form) have been compared at various concentrations of hydroxylamine. The reactions of the chromophore were measured by absorption changes in the visible range, the complex dissociation by changes in the near-infrared scattering. An additional monitor of the complex was given by the G-protein-dependent equilibrium between metarhodopsin I and metarhodopsin II. For all measurements, fragments of isolated bovine rod outer segments in suspension were used. In the absence of hydroxylamine, the rhodopsin-G-protein complex dissociated within 20-30 min at room temperature. The presence of hydroxylamine greatly accelerated (e.g., 5-fold at 1 mM NH2OH) the dissociation. Under all conditions, the free, dissociated G-protein can reassociate to metarhodopsin II produced by subsequent bleaching. Dissociation of the metarhodopsin II-G-protein complex required the decay of photoproducts with a maximal absorbance of 380 nm, but was not affected by the simultaneous presence of metarhodopsin III or metarhodopsin III - like photoproducts with a maximal absorbance between 450 and 470 nm. Despite the acceleration of metarhodopsin II-G-protein dissociation by NH2OH, metarhodopsin II-G-protein was relatively stabilized as compared to free metarhodopsin II. The ratio of the decay rates of free metarhodopsin II and metarhodopsin III-G-protein was increased as much as 10-fold in the presence of 25 mM NH2OH. The results indicate a mutual interdependence of retinal, opsin and G-protein.

Two distinct rhodopsin molecules within the disc membrane of vertebrate rod outer segments.

The kinetics of the metarhodopsin I-II reaction have been measured over a wide range of temperatures (1-37C ) and pH values (4.5-8) with suspensions containing fragments of bovine rod outer segments. It was found that for all conditions the occurrence of metarhodopsin II could be described by two independent first-order processes. The fast component: slow component amplitude ratio depends upon pH and temperature. The kinetics of the lumi-metarhodopsin I reaction show the same pH dependence for the fast component: slow component amplitude ratio as the one observed for the metarhodopsin II signals. All the results observed could be described with the assumption that rhodopsin itself exists in two conformational states before bleaching which are in a pH and temperature-dependent equilibrium. This hypothesis is confirmed by its ability to explain some apparently anomalous observations in the literature.

Interaction of pulmonary surfactant protein A with phospholipid liposomes: a kinetic study on head group and fatty acid specificity.

Recent work on surfactant protein A (SP-A) has shown that Ca(2+) induces an active conformation, SP-A, which binds rapidly to liposomes and mediates their aggregation. Employing sensitive real time assays, we have now studied the lipid binding characteristics of the SP-A liposome interaction. From the final equilibrium level of the resonant mirror binding signal, an apparent dissociation constant of ca. K(d)=5 microM is obtained for the complex between SP-A and dipalmitoylphosphatidylcholine (DPPC) liposomes. At nanomolar SP-A concentrations, this complex is formed with a subsecond (0.3 s) reaction time, as measured by light-scattering signals evoked by photolysis of caged Ca(2+). With palmitoyloleoylphosphatidylcholine (POPC), the complex formation proceeds at half the rate, compared to DPPC, leading to a lower final equilibrium level of SP-A lipid interaction. Distearoylphosphatidylcholine (DSPC) shows a stronger interaction than DPPC. Regarding the phospholipid headgroups, phosphatidylinositol (PI) and sphingomyelin (SM) interact comparable to DPPC, while less interaction is seen with phosphatidylethanolamine (PE) or with phosphatidylglycerol (PG). Thus both headgroup and fatty acid composition determine SP-A phospholipid interaction. However, the protein does not exhibit high specificity for either the polar or the apolar moiety of phospholipids.

Palmitoylation of rhodopsin with S-protein acyltransferase: enzyme catalyzed reaction versus autocatalytic acylation.

Protein palmitoylation in vitro was studied using bovine rhodopsin as the substrate and a partially purified acylating enzymatic activity (PAT) from placental membranes. PAT incorporates fatty acid into rhodopsin with higher efficiency (10 times higher initial rate), as compared to autoacylation. The activity is sensitive to heat and trypsin, indicating a protein-mediated enzymatic process and requires the native conformation of rhodopsin. The presence of deacylated, free cysteine residues in dark-adapted rhodopsin increases palmitoylation via PAT. The sites for non-enzymatic and enzymatic palmitoylation could not be distinguished by peptide mapping. The reversible palmitoylation described here will provide a tool for the study of the role of palmitoylation in photoreceptor function.

ATP can promote activation and deactivation of the rod cGMP-phosphodiesterase. Kinetic light scattering on intact rod outer segments.

The AT (amplified transient) signal is a flash-induced increase of the near-infrared light scattering from isolated bovine rod outer segments and is interpreted as a monitor of cGMP-phosphodiesterase activation [(1985) FEBS Lett. 188, 15-20]. We have investigated the effects of ATP and cyclic GMP on this signal. It has been found that ATP enhances the AT signal, the relative effect being the largest for low photoexcitation (approximately 1 rhodopsin per disc membrane). At a high rhodopsin turnover, which saturates the AT amplitude, the effect of ATP is to accelerate the rise of the signal. ATP can also accelerate the falling phase of the signal. This deactivating effect depends on the simultaneous presence of cyclic GMP. The results indicate that ATP acts on the phosphodiesterase activation cycle, promoting activation as well as deactivation, dependent on cGMP as a cofactor.

Sulfhydryl group modification of photoreceptor G-protein prevents its light-induced binding to rhodopsin.

The effect of sulfhydryl modification on the light-induced interaction between rhodopsin and the peripheral GTP-binding protein of the photoreceptor membrane (G-protein) has been investigated by time-resolved near-infrared light-scattering and polyacrylamide gel electrophoresis. It has been found that the modification of rhodopsin with the alkylating agent N-ethylmaleimide (NEM) does not affect its light-induced interaction with the G-protein. Modification of G-protein with NEM or other sulfhydryl agents prevents any light-induced binding to rhodopsin. Dark-association of G to the membrane as well as the light-induced complex with rhodopsin (once formed) is insensitive to NEM.

Photoactivation of rhodopsin and interaction with transducin in detergent micelles. Effect of 'doping' with steroid molecules.

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 (G1). The measured rate of flash-induced MII formation decreases by a factor of 300 from n-dodecyl-beta-D-maltoside (k = 4 x 10(3) at 18 degrees C, pH 6.5), over (3-(lauroyloxy)propyl)-phosphorylcholine (deoxylysolecithin), n-octyl-beta-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.

FTIR spectroscopy of complexes formed between metarhodopsin II and C-terminal peptides from the G-protein alpha- and gamma-subunits.

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.

Light-induced activation of the rod phosphodiesterase leads to a rapid transient increase of near-infrared light scattering.

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.

Binding of inositol phosphates to arrestin.

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

Assays for activation of opsin by all-trans-retinal.

The data collected with the techniques discussed in this chapter suggest significant differences between the active conformation(s) of the opsin/atr complex, which are reversibly formed in the dark, and the active conformation (R*) of the meta-II photoproduct. First, there is good evidence for noncovalent opsin/atr complexes with considerable activity (although covalent binding of atr is found in mutant opsins. Even more intriguing, all-trans-retinal in an amount that saturates the activity of the opsin/atr complex toward Gt does not measurably inhibit the access of 11-cis-retinal to the light-sensitive binding site during regeneration (Fig. 2C). On the other hand, forced protonation at or near Glu-134 appears to be an integral mechanism for both the meta-II and the opsin-like activities (Fig. 4). Thus, it is not inconceivable that these two activities of the receptor arise from two fundamentally different conformations, one meta-II-like and one opsin-like. They would be similar with respect to the Gt (or RK) protein-protein interaction but different in their mode of retinal-protein interaction.

Signalling states of photoactivated rhodopsin.

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.

Time-resolved angular dependent measurement of triggered light scattering changes in biological suspensions.

We describe a photometer for time-resolved measurements of small changes in light scattering suited for suspensions of biological material. The time resolution is 35 mus, the amplitude resolution for bovine rod outer segments is typically delta I/I = 5 X 10(-4) at a scattering angle of = 20 degrees. The use of the apparatus is demonstrated by recording the near infrared scattering of bovine rod outer segments after excitation with flashes of green light. Semiconductor detector arrays are arranged centrosymmetrically around a hemispherical cuvette. The optical characteristics of a hemispherical cuvette and the resulting geometry of cuvette and detection are discussed. Calculations of optimal signal transfer and noise of the detectors led to the following arrangement for each scattering angle: pairs of parallel connected photodiodes are fed into several current-to-voltage converters, whose output voltages are summed up by a summing amplifier. For the test of the device so-called N signals of fresh and liquid N2-frozen and thawed ROS samples were measured at four scattering angles simultaneously. A strong angular dependence (difference scattering curve) of the relative light scattering change is seen for fresh ROS which is transformed into a flat curve by freezing and thawing. It is concluded that the competence of the fresh sample to extend the light-induced local events - presumably rhodopsin conformational changes - into the gross-structural range is terminated by freezing.

Differential light detector.

A sensitive differential light detector is described which consists of two antiparallel silicon photovoltaic diodes and a current-to-voltage converter. The device is optimized to the detection of small differences in intensity. The noise and signal transfer features are considered, and an application example is given.

[To see from light--biophysics of visual signal transduction]. / Vom Licht zum Sehen--Biophysik der visuellen Signaltransduktion.

To perform their functions within an organism, or to adapt to the environment as single cells, living cells react to signals detected by highly specialized receptor proteins. These include the G-protein coupled receptors (GPCRs), a receptor family, which comprises more than 1000 members, and is of outstanding significance in basic research and medical application. An archetype of a GPCR is the visual pigment rhodopsin, the photoreceptor of the retinal rod cell. Biophysical methods have largely contributed to the elucidation of rhodopsin structure and function, as well as of the corresponding signal cascade. This article discusses some of the more recent developments.