The amino terminus of the fourth cytoplasmic loop of rhodopsin modulates rhodopsin-transducin interaction.

Rhodopsin is a seven-transmembrane helix receptor that binds and catalytically activates the heterotrimeric G protein transducin (G(t)). This interaction involves the cytoplasmic surface of rhodopsin, which comprises four putative loops and the carboxyl-terminal tail. The fourth loop connects the carboxyl end of transmembrane helix 7 with Cys(322) and Cys(323), which are both modified by membrane-inserted palmitoyl groups. Published data on the roles of the fourth loop in the binding and activation of G(t) are contradictory. Here, we attempt to reconcile these conflicts and define a role for the fourth loop in rhodopsin-G(t) interactions. Fluorescence experiments demonstrated that a synthetic peptide corresponding to the fourth loop of rhodopsin inhibited the activation of G(t) by rhodopsin and interacted directly with the alpha subunit of G(t). A series of rhodopsin mutants was prepared in which portions of the fourth loop were replaced with analogous sequences from the beta(2)-adrenergic receptor or the m1 muscarinic receptor. Chimeric receptors in which residues 310-312 were replaced could not efficiently activate G(t). The defect in G(t) interaction in the fourth loop mutants was not affected by preventing palmitoylation of Cys(322) and Cys(323). We suggest that the amino terminus of the fourth loop interacts directly with G(t), particularly with Galpha(t), and with other regions of the intracellular surface of rhodopsin to support G(t) binding.

Chromophore structural changes in rhodopsin from nanoseconds to microseconds following pigment photolysis.

Rhodopsin is a prototypical G protein-coupled receptor that is activated by photoisomerization of its 11-cis-retinal chromophore. Mutant forms of rhodopsin were prepared in which the carboxylic acid counterion was moved relative to the positively charged chromophore Schiff base. Nanosecond time-resolved laser photolysis measurements of wild-type recombinant rhodopsin and two mutant pigments then were used to determine reaction schemes and spectra of their early photolysis intermediates. These results, together with linear dichroism data, yielded detailed structural information concerning chromophore movements during the first microsecond after photolysis. These chromophore structural changes provide a basis for understanding the relative movement of rhodopsin's transmembrane helices 3 and 6 required for activation of rhodopsin. Thus, early structural changes following isomerization of retinal are linked to the activation of this G protein-coupled receptor. Such rapid structural changes lie at the heart of the pharmacologically important signal transduction mechanisms in a large variety of receptors, which use extrinsic activators, but are impossible to study in receptors using diffusible agonist ligands.

Time-resolved spectroscopy of the early photolysis intermediates of rhodopsin Schiff base counterion mutants.

Time-resolved absorption difference spectra of COS-cell expressed rhodopsin and rhodopsin mutants (E113D, E113A/A117E, and G90D), solubilized in detergent, were collected from 20 ns to 510 ms after laser photolysis with 7 ns pulses (lambda(max) = 477 nm). The data were analyzed using a global exponential fitting procedure following singular value decomposition (SVD). Over the entire time range excellent agreement was achieved between results for COS-cell and rod outer segment rhodopsin both in kinetics and in the lambda(max) values of the intermediates. The Schiff base counterion mutant E113D showed strong similarities to rhodopsin up to lumi, following the established scheme: batho <==> bsi --> lumi. Including late delay times (past 1 micros), the mutant E113D lumi decayed to metarhodopsin II (MII), showing that the detergent strongly favors MII over metarhodopsin I (MI). However, a back-reaction from MII to lumi was observed that was not seen for rhodopsin. The kinetic schemes for the mutants E113A/A117E and G90D were significantly different from that of rhodopsin. In both mutants batho decay into an equilibrium with bsi was too fast to resolve (<20 ns). The batho/bsi mixtures decayed with the following reaction scheme: batho/bsi <==> lumi <==> MI-like <==> MII-like. However, the back-reaction from MI-like to lumi was not seen in G90D. MI-like spectral intermediates absorbing around 460 nm appeared in both mutants. They have been shown to be the transducin-activating species (R*). These data, interpreted in the context of previous NMR, FTIR, and Raman data, are consistent with a picture in which the kinetics of batho decay is dependent on a protein-induced perturbation near C12-C13 of the retinal chromophore. The lambda(max) values of the bsi and lumi intermediates in the mutant pigments are interpreted in terms of movement of the Schiff base relative to its counterion.

Spectroscopic evidence for altered chromophore--protein interactions in low-temperature photoproducts of the visual pigment responsible for congenital night blindness.

The replacement of Gly90 by Asp in human rhodopsin causes congenital night blindness. It has been suggested that the molecular origin for the trait is an altered electrostatic environment of the protonated retinal Schiff base chromophore. We have investigated the corresponding recombinant bovine rhodopsin mutant G90D, as well as the related mutants E113A and G90D/E113A, using spectroscopy at low temperature. This allows the assessment of chromophore-protein interactions under conditions where conformational changes are mainly restricted to the retinal-binding site. Each of the mutant pigments formed bathorhodopsin- and isorhodopsin-like intermediates, but the concomitant visible absorption changes reflected differences in the electrostatic environment of the protonated Schiff base in each pigment. Fourier transform infrared-difference spectroscopy revealed effects on the chromophore fingerprint and hydrogen-out-of-plane vibrational modes, which were indicative of the removal of an electrostatic perturbation near C12 of the retinal chromophore in all three mutants. A comparison of the UV-visible and infrared-difference spectra of the mutant pigments strongly suggests that Glu113 is stably protonated in G90D. The corresponding carbonyl-stretching mode is assigned to a band at 1727 cm-1. In contrast to the case in native bathorhodopsin, the all-trans-retinal chromophores in the primary photoproducts of the mutant pigments are essentially relaxed. The peptide carbonyl vibrational changes in mutants G90D and G90D/ E113A suggest that this is due to a more flexible retinal-binding site. Therefore, the steric strain exerted on the chromophore in native bathorhodopsin may be caused by electrostatic forces that specifically involve glutamate 113.

Rhodopsin activation blocked by metal-ion-binding sites linking transmembrane helices C and F.

A large superfamily of receptors containing seven transmembrane (TM) helices transmits hormonal and sensory signals across the plasma membrane to heterotrimeric G proteins at the cytoplasmic face of the membrane. To investigate how G-protein-coupled receptors work at the molecular level, we have engineered metal-ion-binding sites between TM helices to restrain activation-induced conformational change in specific locations. In rhodopsin, the photoreceptor of retinal rod cells, we substituted histidine residues for natural amino acids at the cytoplasmic ends of the TM helices C and F. The resulting mutant proteins were able to activate the visual G protein transducin in the absence but not in the presence of metal ions. These results indicate that the TM helices C and F are in close proximity and suggest that movements of these helices relative to one another are required for transducin activation. Thus a change in the orientations of TM helices C and F is likely to be a key element in the mechanism for coupling binding of ligands (or isomerization of retinal) to the activation of G-protein-coupled receptors.

Characterization of the mutant visual pigment responsible for congenital night blindness: a biochemical and Fourier-transform infrared spectroscopy study.

A mutation in the gene for the rod photoreceptor molecule rhodopsin causes congenital night blindness. The mutation results in a replacement of Gly90 by an aspartic acid residue. Two molecular mechanisms have been proposed to explain the physiology of affected rod cells. One involves constitutive activity of the G90D mutant opsin [Rao, V. R., Cohen, G. B., & Oprian, D. D. (1994) Nature 367, 639-642]. A second involves increased photoreceptor noise caused by thermal isomerization of the G90D pigment chromophore [Sieving, P. A., Richards, J. E., Naarendorp F., Bingham, E. L., Scott, K., & Alpern, M. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 880-884]. Based on existing models of rhodopsin and in vitro biochemical studies of site-directed mutants, it appears likely that Gly90 is in the immediate proximity of the Schiff base chromophore linkage. We have studied in detail the mutant pigments G90D and G90D/E113A using biochemical and Fourier-transform infrared (FTIR) spectroscopic methods. The photoproduct of mutant pigment G90D, which absorbs maximally at 468 nm and contains a protonated Schiff base linkage, can activate transducin. However, the active photoproduct decays rapidly to opsin and free all-trans-retinal. FTIR studies of mutant G90D show that the dark state of the pigment has several structural features of metarhodopsin II, the active form of rhodopsin. These include a protonated carboxylic acid group at position Glu113 and increased hydrogen-bond strength of Asp83. Additional results, which relate to the structure of the active G90D photoproduct, are also reported. Taken together, these results may be relevant to understanding the molecular mechanism of congenital night blindness caused by the G90D mutation in human rhodopsin.

Characterization of rhodopsin-transducin interaction: a mutant rhodopsin photoproduct with a protonated Schiff base activates transducin.

Rhodopsin, a G protein-coupled seven-transmembrane helix receptor, contains an 11-cis-retinal chromophore covalently linked to opsin apoprotein by a protonated Schiff base. Photoisomerization of the chromophore followed by Schiff base deprotonation forms metarhodopsin II (MII, lambda max = 380 nm), the active state (R*) that catalyzes guanine nucleotide exchange in transducin, the G protein of the photoreceptor cell. Schiff base deprotonation is required for R* formation. The Schiff base positive charge in rhodopsin is stabilized by a carboxylic acid counterion, Glu113. The position of the carboxylate counterion was moved by one helix turn to position 117 by site-specific mutagenesis. Photolysis of the mutant pigment E113A/A117E (lambda max = 491 nm) resulted in a mixture of two photoproducts: (1) an MII-like form with an unprotonated Schiff base (lambda max = 382 nm) favored at alkaline pH; and (2) a photoproduct with a protonated Schiff base (lambda max = 474 nm), spectroscopically similar to metarhodopsin I, favored at acidic pH. Here, we have studied the interactions between the mutant E113A/A117E photoproducts and transducin in detail. Transducin slowed down thermal conversion of the 474 nm form to the 382 nm form by stabilizing the 474 nm photoproduct. This effect was maximal at the pH optimum of transducin activation by the mutant R* and was abolished in the presence of GTP gamma S. In addition, the amount of the 474 nm species correlated with transducin activation rates during the thermal conversion of the photoproduct mixture. Thus, the 474 nm photoproduct of the mutant pigment, which contained a protonated Schiff base, activated transducin.(ABSTRACT TRUNCATED AT 250 WORDS)

Protonation states of membrane-embedded carboxylic acid groups in rhodopsin and metarhodopsin II: a Fourier-transform infrared spectroscopy study of site-directed mutants.

A method was developed to measure Fourier-transform infrared (FTIR) difference spectra of detergent-solubilized rhodopsin expressed in COS cells. Experiments were performed on native bovine rhodopsin, rhodopsin expressed in COS cells, and three expressed rhodopsin mutants with amino acid replacements of membrane-embedded carboxylic acid groups: Asp-83-->Asn (D83N), Glu-122-->Gln (E122Q), and the double mutant D83N/E122Q. Each of the mutant opsins bound 11-cis-retinal to yield a visible light-absorbing pigment. Upon illumination, each of the mutant pigments formed a metarhodopsin II-like species with maximal absorption at 380 nm that was able to activate guanine nucleotide exchange by transducin. Rhodopsin versus metarhodopsin II-like photoproduct FTIR-difference spectra were recorded for each sample. The COS-cell rhodopsin and mutant difference spectra showed close correspondence to that of rhodopsin from disc membranes. Difference bands (rhodopsin/metarhodopsin II) at 1767/1750 cm-1 and at 1734/1745 cm-1 were absent from the spectra of mutants D83N and E122Q, respectively. Both bands were absent from the spectrum of the double mutant D83N/E122Q. These results show that Asp-83 and Glu-122 are protonated both in rhodopsin and in metarhodopsin II, in agreement with the isotope effects observed in spectra measured in 2H2O. A photoproduct band at 1712 cm-1 was not affected by either single or double replacements at positions 83 and 122. We deduce that the 1712 cm-1 band arises from the protonation of Glu-113 in metarhodopsin II.

Characterization of mutant rhodopsins responsible for autosomal dominant retinitis pigmentosa. Mutations on the cytoplasmic surface affect transducin activation.

Rhodopsin mutants responsible for autosomal dominant retinitis pigmentosa (ADRP) were prepared by site-directed mutagenesis and characterized. The aim was to evaluate ADRP mutations that occur at three locations on the cytoplasmic surface of rhodopsin: Thr-58 near the cytoplasmic border of helix A, the tetrapeptide Leu-68 to Pro-71 in the first cytoplasmic loop, and Arg-135 at the cytoplasmic border of helix C. It was hypothesized that amino acid changes at these sites would result in mutant rhodopsins with normal spectral properties but defects in their ability to interact with the rod outer segment G protein, transducin. A set of 12 mutant opsin genes was prepared. Four of the mutants were known to cause ADRP: Thr-58 replaced by Arg, a four-amino acid deletion (Leu-68/Arg-69/Thr-70/Pro-71), Arg-135 replaced by Leu, and Arg-135 replaced by Trp. Eight additional mutants were prepared to provide complementary structure-function information. The four-amino acid deletion mutant failed to bind 11-cis-retinal. However, each of the Thr-58 and Arg-135 mutants bound 11-cis-retinal to form a pigment with a visible absorbance maximum (lambda max) of 500 nm. Upon illumination, each pigment was converted to a metarhodopsin II-like spectral form (lambda max = 380 nm). However, each of these spectrally normal ADRP mutants was defective in activating guanine nucleotide exchange by transducin. These results identify a defect in the signal transduction pathway in spectrally normal mutant rhodopsins that cause ADRP.

Movement of the retinylidene Schiff base counterion in rhodopsin by one helix turn reverses the pH dependence of the metarhodopsin I to metarhodopsin II transition.

The environment of the retinylidene Schiff base in bovine rhodopsin has been studied by movement of its carboxylic acid counterion from position 113 to position 117 by site-specific mutagenesis. Replacement of the counterion at position 113 by a neutral amino acid residue has been shown to produce a lowering of the Schiff base acidity constant (pKa) from > 8.5 to about 6. The aim of the present work was to change the position of the counterion without causing a significant effect on the Schiff base pKa. A triple replacement mutant (Glu113-->Ala/Ala117-->Glu/Glu122-->Gln) was designed to move the position of the counterion by one helix turn in the third putative transmembrane helix (helix C). The mutant bound 11-cis-retinal to form a chromophore with a visible absorbance maximum (lambda max) of 490 nm which was independent of pH in the range of about 5-8.5. Upon illumination under conditions in which rhodopsin was converted to the active metarhodopsin II (MII) photoproduct, the mutant was converted to a metarhodopsin I (MI)-like species (lambda max = 475 nm). Furthermore, the effect of pH on the photobleaching behavior of the mutant was the reverse of that reported for rhodopsin. In the mutant, acidic pH favored the formation of the MI-like photoproduct, and basic pH favored the formation of an MII-like photoproduct (lambda max = 380 nm). The MII-like photoproduct of the mutant pigment was able to activate the guanine nucleotide-binding protein, transducin. We conclude that the Schiff base counterion in rhodopsin can be repositioned to form a pigment with an apparently unperturbed Schiff base pKa. Furthermore, a specific amino acid residue that acts as a Schiff base proton acceptor is not strictly required for photoconversion of rhodopsin to its active MII form.

Functional expression in vitro of bovine visual rhodopsin.

Expression of functionally active bovine visual rhodopsin was achieved by sequential transcription and translation in vitro of rhodopsin gene cDNA with co-translational insertion of the protein into phosphatidylcholine liposomes. The recombinant rhodopsin has functional, spectral and immunochemical properties similar to those of natural rhodopsin from bovine retina. Two mutant rhodopsins, Cys316----Ser and Asp330----Asn, Asp331----Asn, were produced by oligonucleotide-directed mutagenesis. The first mutation does not affect rhodopsin's ability to stimulate transducin GTPase and visual cGMP phosphodiesterase activities, while the second double mutation leads to a sharp decrease in rhodopsin activity.