The Magnitude of the Light-induced Conformational Change in Different Rhodopsins Correlates with Their Ability to Activate G Proteins

Light converts rhodopsin, the prototypical G protein-coupled receptor, into a form capable of activating G proteins. Recent work has shown that the light-activated state of different rhodopsins can possess different molecular properties, especially different abilities to activate G protein. For example, bovine rhodopsin is ∼20-fold more effective at activating G protein than parapinopsin, a non-visual rhodopsin, although these rhodopsins share relatively high sequence similarity. Here we have investigated possible structural aspects that might underlie this difference. Using a site-directed fluorescence labeling approach, we attached the fluorescent probe bimane to cysteine residues introduced in the cytoplasmic ends of transmembrane helices V and VI in both rhodopsins. The fluorescence spectra of these probes as well as their accessibility to aqueous quenching agents changed dramatically upon photoactivation in bovine rhodopsin but only moderately so in parapinopsin. We also compared the relative movement of helices V and VI upon photoactivation of both rhodopsins by introducing a bimane label and the bimane-quenching residue tryptophan into helices VI and V, respectively. Both receptors showed movement in this region upon activation, although the movement appears much greater in bovine rhodopsin than in parapinopsin. Together, these data suggest that a larger conformational change in helices V and VI of bovine rhodopsin explains why it has greater G protein activation ability than other rhodopsins. The different amplitude of the helix movement may also be responsible for functional diversity of G protein-coupled receptors.Rhodopsin, the photosensitive G protein-coupled receptor (GPCR),³ is responsible for transmitting a light signal into an intracellular signaling cascade through activation of G protein in visual and non-visual photoreceptor cells. Rhodopsin consists of a protein moiety (opsin, comprising seven transmembrane α-helical segments) combined with a chromophore (11-cis retinal) that acts as the light-sensitive ligand. Photoisomerization of the 11-cis retinal to the all-trans form induces structural changes in the protein moiety that then enable it to couple with and activate the G protein.The crystal structure of inactive bovine rhodopsin has been extensively investigated (1–3). Recently, a crystal structure of inactive invertebrate squid rhodopsin was also solved (4), and crystal structures of the inactive form of β-adrenergic receptors and A2 adenosine receptor have been reported (5–7). Remarkably, all of these crystal structures exhibit a very similar arrangement for the seven transmembrane helices (4, 8). Together, these facts suggest that the architecture for the inactive form is conserved among rhodopsin-like GPCRs.The structural features of an activated GPCR are much less defined. Thus, a variety of biochemical and biophysical methods, including cross-linking methods (9, 10) and site-directed spin and fluorescence labeling methods (10–13), have been employed to identify the dynamic and structural changes involved in forming the activated state. The data from these studies consistently suggest that some kind of movement of helix VI is involved in the formation of the active state of the rhodopsins. In particular, the cytoplasmic end of helix VI has been proposed to rotate and/or tilt toward helix V (10–13). Remarkably, the recent crystal structures of bovine opsin are consistent with the widely accepted helix motion model. Both the structures of opsin (the ligand-free form of rhodopsin that has partial G protein activation ability) and a complex of opsin with a peptide derived from the G protein C terminus show a movement of helix VI toward helix V, compared with the dark state rhodopsin structure (14, 15). Studies of β-adrenergic and muscarinic receptors also show that agonist binding promotes movement of helix VI toward helix V in these receptors (16, 17). Because the region between the cytoplasmic ends of helices V and VI in various GPCRs is a main site of interaction with G proteins (18), it is possible that movement of helices V and VI leads to formation of a conformation capable of interacting with G protein (19).Together, these studies imply that the active state conformation of GPCRs may be similar. However, a detailed comparison of the active-state conformation for two different GPCRs has never been precisely undertaken in the same laboratory using the same methods.In this context we have been investigating rhodopsins with different functional properties to determine whether their active states have different conformations. Our goal was to determine whether any functional or structural differences in the active states of these GPCRs could be detected under the exact same experimental conditions.Previously, we have found that several rhodopsins, such as an invertebrate rhodopsin and a vertebrate non-visual rhodopsin parapinopsin (20, 21), can be activated not only by light but also by exogenous all-trans retinal acting as a full agonist (22). This is in contrast to vertebrate visual rhodopsins, including bovine rhodopsin, which cannot fully form the active state by direct binding of all-trans retinal (23), although all-trans retinal can fully activate some rhodopsin mutants (24). Other invertebrate rhodopsin (25) and the circadian photoreceptor melanopsin (26) can also bind all-trans retinal directly.Interestingly, the active form of the all-trans retinal-activated rhodopsins exhibit some striking differences in their spectroscopic and biochemical properties compared with vertebrate visual rhodopsins (27). In particular, the efficiency of bovine rhodopsin for activating G protein is ∼20∼50-fold higher than that of parapinopsin and invertebrate rhodopsin. This difference could be related to the difference in position of a specific amino acid residue counterion that is essential for rhodopsin to absorb visible light, namely one at position 113 or 181 (28).⁴ Further biochemical analyses using chimeric mutants combining rhodopsins with lower and higher G protein activation abilities suggested that the difference in G protein activation ability was because of a structural difference in transmembrane helices in the active states but not because of difference in amino acid sequence of G protein interaction site (29) (Fig. 1, A–C). In addition, the active states of parapinopsin and the invertebrate rhodopsin are thermally stable and can be reconverted to the inactive state by subsequent light absorption, showing photo-regenerable or bistable nature (21, 28), unlike the active state of bovine rhodopsin, which is thermally unstable and cannot revert to the inactive state by subsequent light absorption (30).Open in a separate windowFIGURE 1.Molecular properties and sites of fluorescent probe attachment for bovine rhodopsin and parapinopsin. A, sequence alignment of bovine rhodopsin and parapinopsin. Amino acid residues to which cysteine and fluorescence label were introduced are marked with red. The amino acid residues identical and similar between bovine rhodopsin and parapinopsin are shown with white characters with black and gray background, respectively. Bovine rhodopsin and parapinopsin show 41% sequence identity and 61% similarity. In this paper the residue number of parapinopsin is described by using the bovine rhodopsin numbering system. B and C, comparison of G protein activation ability of rhodopsin and parapinopsin wild type (WT) proteins and loop-replaced mutants. In these mutants the second and/or third cytoplasmic loop was swapped between the two receptors. ParaL2 and ParaL3 indicate mutants of bovine rhodopsin in which second and third loops were replaced with the corresponding loop of parapinopsin, respectively. RhoL2 and RhoL3 indicate mutants of parapinopsin in which the second and third loops were replaced with the corresponding loops of bovine rhodopsin, respectively. ParaL2L3 and RhoL2L3 are mutants of bovine rhodopsin and parapinopsin in which both the second and third loops were swapped, respectively. See Terakita et al. (29) for more details. Data are presented as the means ± S.E. of three separate experiments except for mutants RhoL3, RhoL2L3, and ParaL2L3 (n = 2). D, model of bovine rhodopsin. Amino acid residues which were mutated to cysteine to enable attachment of the fluorescent probe bimane or mutated to tryptophan are indicated. Positions 226, 227, 244, 250, and 251 in the crystal structure of the dark state of bovine rhodopsin (PDB code 1GZM) are shown. E, reaction of the mBBr label with a sulfhydryl group. The mutants labeled with mBBr are named by the number of the residue and the suffix B₁. F, reaction of the PDT-bimane with a sulfhydryl group. The mutants labeled with PDT-bimane are named by the number of the residue and the suffix B₂. The disulfide linkage between the label and protein can be cleaved using Tris(2-carboxyethyl)phosphine (32).In this study we used site-directed fluorescence labeling (13, 31) to compare the structural features of active states of bovine rhodopsin with lamprey parapinopsin, a UV-sensitive non-visual pigment in the pineal organs (21). Parapinopsin shows relatively high sequence similarity (∼60%) to bovine rhodopsin, yet it has a greatly reduced ability to activate G protein (see Fig. 1, A–C) (21, 28). Using established protocols, we introduced cysteine residues into the cytoplasmic ends of helices V and VI, the region proposed to rearrange upon activation in GPCRs (11, 12, 14, 18). We then site-specifically labeled these cysteines with the small, non-polar fluorescent probe, bimane, and used the spectral properties of these bimane probes to act as reporter groups for environmental changes around their site of attachment upon formation of the photoactivated state for both rhodopsins.In addition, we measured changes in the relative proximity of the cytoplasmic ends of helix VI to helix V in both rhodopsin and parapinopsin using the tryptophan-induced-quenching of bimane (TrIQ-bimane) fluorescence method (31, 32). TrIQ-bimane measures the efficiency of intramolecular fluorescence quenching of bimane caused by tryptophan (Trp), which occurs in a distance-dependent manner. The goal of this study was to determine whether the helices in both receptors moved in the same way during formation of the active state. Our results show that whereas movement of helix VI relative to helix V occurs during formation of the active state for both parapinopsin and bovine rhodopsin, the “amplitude” of the movement is markedly different between the two rhodopsins.     

The chromosomal mazEF locus of Streptococcus mutans encodes a functional type II toxin-antitoxin addiction system

Type II chromosomal toxin-antitoxin (TA) modules consist of a pair of genes that encode two components: a stable toxin and a labile antitoxin interfering with the lethal action of the toxin through protein complex formation. Bioinformatic analysis of Streptococcus mutans UA159 genome identified a pair of linked genes encoding a MazEF-like TA. Our results show that S. mutans mazEF genes form a bicistronic operon that is cotranscribed from a σ70-like promoter. Overproduction of S. mutans MazF toxin had a toxic effect on S. mutans which can be neutralized by coexpression of its cognate antitoxin, S. mutans MazE. Although mazF expression inhibited cell growth, no cell lysis of S. mutans cultures was observed under the conditions tested. The MazEF TA is also functional in E. coli, where S. mutans MazF did not kill the cells but rather caused reversible cell growth arrest. Recombinant S. mutans MazE and MazF proteins were purified and were shown to interact with each other in vivo, confirming the nature of this TA as a type II addiction system. Our data indicate that MazF is a toxic nuclease arresting cell growth through the mechanism of RNA cleavage and that MazE inhibits the RNase activity of MazF by forming a complex. Our results suggest that the MazEF TA module might represent a cell growth modulator facilitating the persistence of S. mutans under the harsh conditions of the oral cavity.     

Anti-monocyte chemoattractant protein-1 gene therapy inhibits vascular remodeling in rats: blockade of MCP-1 activity after intramuscular transfer of a mutant gene inhibits vascular remodeling induced by chronic blockade of NO synthesis.

Monocyte chemoattractant protein-1 (MCP-1) may play an essential part in the formation of arteriosclerosis by recruiting monocytes into the arterial wall. Thus, we devised a new strategy for anti-MCP-1 gene therapy against arteriosclerosis by transfecting an amino-terminal deletion mutant (missing the amino-terminal amino acids 2 to 8) of the human MCP-1 gene into a remote organ (skeletal muscles). Intramuscular transduction with the mutant MCP-1 gene blocked monocyte recruitment induced by a subcutaneous injection of recombinant MCP-1. In a rat model in which the chronic inhibition of endothelial nitric oxide synthesis induces early vascular inflammation as well as subsequent coronary vascular remodeling, this strategy suppressed monocyte recruitment into the coronary vessels and the development of vascular medial thickening, but did not reduce perivascular fibrosis. Thus, MCP-1 is necessary for the development of medial thickening but not for fibrosis in this model. This new strategy may be a useful and feasible gene therapy against arteriosclerosis.     

Examination of the method for measuring soil respiration in cultivated land: Effect of carbon dioxide concentration on soil respiration

An acceleration of soil respiration with decreasing CO₂ concentration was suggested in the field measurements. The result supporrs that obtained in laboratory experiments in our previous study. The CO₂ concentrations in a chamber of the alkali absorption method (the AA-method) were about 150–250 parts/10⁶ lower than that in the atmosphere (about 350 parts/10⁶), while those observed in the open-flow IRGA method (the OF-method) were nearly equal to the soil surface CO₂ levels. The AA-method at such low CO₂ levels in the chamber appears to overestimate the soil respiration. Our results showed that the rates obtained by the AA-method were about twice as large as those by the OF-method in field and laboratory measurements. This finding has important consequences with respect to the validity of the existing data obtained by the AA-method and the estimation of changes in the terrestrial carbon flow with elevated CO₂     

Retinal Attachment Instability Is Diversified among Mammalian Melanopsins

Melanopsins play a key role in non-visual photoreception in mammals. Their close phylogenetic relationship to the photopigments in invertebrate visual cells suggests they have evolved to acquire molecular characteristics that are more suited for their non-visual functions. Here we set out to identify such characteristics by comparing the molecular properties of mammalian melanopsin to those of invertebrate melanopsin and visual pigment. Our data show that the Schiff base linking the chromophore retinal to the protein is more susceptive to spontaneous cleavage in mammalian melanopsins. We also find this stability is highly diversified between mammalian species, being particularly unstable for human melanopsin. Through mutagenesis analyses, we find that this diversified stability is mainly due to parallel amino acid substitutions in extracellular regions. We propose that the different stability of the retinal attachment in melanopsins may contribute to functional tuning of non-visual photoreception in mammals.     

Proxisome proliferator-activated receptor-α mediates high-fat, diet-enhanced daily oscillation of plasminogen activator inhibitor-1 activity in mice

Acute thrombotic events frequently occur in the early morning among hyperlipidemic patients. The activity of plasminogen activator inhibitor-1 (PAI-1), a potent inhibitor of the fibrinolytic system, oscillates daily, and this is considered one mechanism that underlies the morning onset of acute thrombotic events in hyperlipidemia. Although several studies have reported the expression of the PAI-1 gene is under the control of the circadian clock system, the molecular mechanism of the circadian transactivation of PAI-1 gene under hyperlipidemic conditions remains to be elucidated. Here, the authors investigated whether hyperlipidemia induced by a high-fat diet (HFD) enhances the daily oscillation of plasma PAI-1 activity in mice. The mRNA levels of the PAI-1 gene were increased and rhythmically fluctuated with high-oscillation amplitude in the livers of wild-type mice fed with the HFD. Circadian expression of proxisome proliferator-activated receptor-α (PPARα) mRNA was also augmented as well as that of PAI-1. Chromatin immunoprecipitation showed the HFD-induced hyperlipidemia significantly increased the binding of PPARα to the PAI-1 promoter. Luciferase reporter analysis using primary hepatocytes revealed CLOCK/BMAL1-mediated PAI-1 promoter activity was synergistically enhanced by cotransfection with PPARα/retinoid X receptor-α (RXRα), and this synergistic transactivation was repressed by negative limbs of the circadian clock, PERIOD2 and CRYPTOCHROME1. As expected, HFD-induced PAI-1 mRNA expression was significantly attenuated in PPARα-null mice. These results suggest a molecular link between the circadian clock and lipid metabolism system in the regulation of PAI-1 gene expression, and provide an aid for understanding why hyperlipidemia increases the risk of acute thrombotic events in the morning.     

Regression by ACE inhibition of arteriosclerotic changes induced by chronic blockade of NO synthesis in rats

We previously reported that chronic inhibition of nitric oxide (NO) synthesis with N(omega)-nitro-L-arginine methyl ester (L-NAME) induces vascular inflammation at week 1 and produces subsequent arteriosclerosis at week 4 and that cotreatment with an angiotensin-converting enzyme (ACE) inhibitor prevents such changes. In the present study, we tested the hypothesis that treatment with an ACE inhibitor after development of vascular inflammation could inhibit arteriosclerosis in rats. Wistar-Kyoto rats were randomized to four groups: the control group received no drugs, the 4wL-NAME group received L-NAME (100 mg x kg(-1) x day(-1)) for 4 wk, the 1wL + 3wNT group received L-NAME for 1 wk and no treatment for the subsequent 3 wk, and the 1wL + 3wACEI group received L-NAME for 1 wk and the ACE inhibitor imidapril (20 mg x kg(-1) x day(-1)) for the subsequent 3 wk. After 4 wk, we observed significant arteriosclerosis of the coronary artery (medial thickening and fibrosis) and increased cardiac ACE activity in the 1wL + 3wNT group as well as in the 4wL-NAME group, but not in the 1wL + 3wACEI group. In a separate study, we examined apoptosis formation and found that posttreatment with imidapril (20 mg x kg(-1) x day(-1)) or an ANG II AT1-receptor antagonist, CS-866 (5 mg x kg(-1) x day(-1)), induced apoptosis (TdT-mediated nick end-labeling) in monocytes and myofibroblasts appearing in the inflammatory lesions associated with a clear degradation in the heart (DNA electrophoresis). In conclusion, treatment with the ACE inhibitor after 1 wk of L-NAME administration inhibited arteriosclerosis by inducing apoptosis in the cells with inflammatory lesions in this study, suggesting that increased ANG II activity inhibited apoptosis of the cells with inflammatory lesions and thus contributed to the development of arteriosclerosis.     

Daily expression of clock genes in whole blood cells in healthy subjects and a patient with circadian rhythm sleep disorder

In recent years, circadian rhythm sleep disorders in humans have been increasing. Clinical features characteristic of this disorder are well known, but the specific causes remain unknown. However, various derangements of circadian expression of the clock gene are a probable cause of this disease. We have attempted to elucidate the relationship between the expression of the clock genes in whole blood cells and the clinical features characteristic of this disorder. In this study, we indicate the daily expression of clock genes period (Per) 1, 2, 3, Bmal1, and Clock in whole blood cells in 12 healthy male subjects. The peak phase of Per1, Per2, and Per3 appeared in the early morning, whereas that of Bmal1 and Clock appeared in the midnight hours. Furthermore, in one patient case with circadian rhythm sleep disorder, we observed variations of the peak phase in clock genes by treatments such as light therapy, exercise therapy, and medicinal therapy. This study suggested that the monitoring of human clock genes in whole blood cells, which may be functionally important for the molecular control of the circadian pacemaker as well as in suprachiasmatic nucleus, might be useful to evaluate internal synchronization.     

Relative contribution of CD2 and LFA-1 to murine T and natural killer cell functions.

We have examined the functional property of murine CD2 as an intercellular adhesion molecule by using five anti-murine CD2 mAb which were classified into two groups according to their mutual competition in binding to cell surface CD2. Hamster fibroblasts transfected with murine CD2 cDNA exhibited increased conjugate formation with a murine mastocytoma P815 which expresses the putative murine LFA-3 mRNA detected by cross-hybridization with human LFA-3 cDNA under conditions of low stringency. This increase in conjugate formation was abrogated by both groups of anti-CD2 mAb, although some differences in the extent of inhibition were observed at lower concentrations of the mAb. We then examined the involvement of CD2 in several murine T cell responses by using these mAb to abrogate CD2-mediated cellular interactions. Anti-CD2 mAb significantly inhibited mitogenic T cell responses induced by suboptimal doses of Con A and PHA. In the allogenic MLR response and in the Ag response of two KLH/I-Ak-specific Th cell clones, the inhibitory effect of anti-CD2 mAb was also greatest under suboptimal conditions, i.e., with lesser doses of the Ag. These results indicate that the contribution of CD2 as an accessory molecule is variable, depending on the Ag dose used for stimulation, and they suggest that CD2 is involved in the Ag response of murine T cells under the physiologic conditions where only a limited amount of Ag is available. We next examined the contribution of CD2 to MHC-restricted cytotoxicity by CTL and to MHC-unrestricted cytotoxicity by NK and lymphokine-activated killer cells. Only a marginal inhibition by anti-CD2 mAb alone was observed. Anti-lymphocyte function-associated Ag (LFA)-1 mAb alone exhibited greater inhibitory effects than anti-CD2 mAb in all of the cases tested. In most cases, however, substantial levels of cytotoxicity remained, even in the presence of both anti-CD2 and anti-LFA-1 mAb. These results indicate a minor contribution of CD2, as compared with LFA-1, to cytotoxicity by murine CTL, NK cells, and lymphokine-activated killer cells, and they reveal the presence of undefined cellular interaction pathways other than those mediated by CD2 and LFA-1.     

A murine very late activation antigen-like extracellular matrix receptor involved in CD2- and lymphocyte function-associated antigen-1-independent killer-target cell interaction

CD2 and lymphocyte function-associated antigen (LFA)-1 are well known as T cell adhesion molecules involved in killer-target cell interactions. However, our recent study revealed that molecule(s) other than CD2 and LFA-1 might be involved in the lymphokine-activated killer (LAK) cell cytotoxicity against certain target cells. In order to characterize such unknown molecules, we established a mAb (RMV-7) which could inhibit CD2/LFA-1-independent LAK cell cytotoxicity and binding to target cells at the effector site. The Ag identified by RMV-7 appeared on splenic T cells late after mitogenic stimulation and was a noncovalently linked heterodimer composed of a 140-kDa alpha-chain and a 95-kDa beta-chain. RMV-7 blocked LAK cell binding to fibronectin (FN), fibrinogen, and vitronectin but not that to laminin or type IV collagen, indicating that the RMV-7-defined molecule is a unique extracellular matrix receptor for FN, fibrinogen, and vitronectin. One of its ligand, FN, was found on the surface of several target cells, and LAK cell cytotoxicity against them was blocked by anti-FN antibody at the target site. Similarly, cytotoxicity of a H-2d-specific CTL clone was inhibited by RMV-7 and anti-FN antibody as well. These results indicate that a unique very late activation Ag-like extracellular matrix receptor on murine CTL and LAK cells contributes to target cell binding and cytotoxicity.