A new approach to understanding the initial step in visual transduction.

Data from picosecond spectroscopic studies of the formation kinetics of bathorhodopsin upon photolysis of rhodopsin and isorhodopsin was analyzed in terms of the Englman-Jortner theory of radiationless transitions. It was found that low frequency vibrations of the protein and/or chromophore are important in coupling bathorhodopsin to its precursor. The results were consistent with a mechanism for bathorhodopsin formation involving only a simple chromophore isomerization. A similar analysis of the formation kinetics of the K state of bacteriorhodopsin showed that different low frequency vibrations than those calculated for rhodopsin couple it to its precursor. The frequency of these vibrations increases upon deuteration for rhodopsin, while it decreases upon deuteration for bacteriorhodopsin. This points out the importance the specific protein matrix has on the primary photolysis reaction.     

Femtosecond formation dynamics of primary photoproducts of visual pigment rhodopsin

The coherent 11-cis-retinal photoisomerization dynamics in bovine rhodopsin was studied by femtosecond time-resolved laser absorption spectroscopy at 30-fs resolution. Femtosecond pulses of 500, 535, and 560 nm wavelength were used for rhodopsin excitation to produce different initial Franck-Condon states and relevant distinct values of the vibrational energy of the molecule in its electron excited state. Time evolution of the photoinduced rhodopsin absorption spectra was monitored after femtosecond excitation in the spectral range of 400–720 nm. Oscillations of the time-resolved absorption signals of rhodopsin photoproducts represented by photorhodopsin₅₇₀ with vibrationally-excited all-trans-retinal and rhodopsin₄₉₈ in its initial state with vibrationally-excited 11-cis-retinal were studied. These oscillations reflect the dynamics of coherent vibrational wave-packets in the ground state of photoproducts. Fourier analysis of these oscillatory components has revealed frequencies, amplitudes, and initial phases of different vibrational modes, along which the motion of wave-packets of both photoproducts occurs. The main vibrational modes established are 62, 160 cm⁻¹ and 44, 142 cm⁻¹ for photorhodopsin₅₇₀ and for rhodopsin₄₉₈, respectively. These vibrational modes are directly involved in the coherent reaction under the study, and their amplitudes in the power spectrum obtained through the Fourier transform of the kinetic curves depend on the excitation wavelength of rhodopsin.     

The influence of arrestin (48K protein) and rhodopsin kinase on visual transduction.

The shutoff of the phototransduction cascade in retinal rods requires the inactivation of light-activated rhodopsin. The underlying mechanisms were studied in functionally intact detached rod outer segments by testing the effect of either sangivamycin, an inhibitor of rhodopsin kinase, or phytic acid, an inhibitor of 48K protein binding to phosphorylated rhodopsin, on light responses recorded in whole-cell voltage clamp. The results suggest that isomerized rhodopsin is inactivated fully by multiple phosphorylation and that the binding of 48K protein accelerates recovery by quenching partially phosphorylated rhodopsin. Higher concentrations of sangivamycin cause changes in the light response that cannot be explained by selective inhibition of rhodopsin kinase and suggest that other protein kinases are needed for normal rod function.     

Calcium and signal transduction in plants

Environmental and hormonal signals control diverse physiological processes in plants. The mechanisms by which plant cells perceive and transduce these signals are poorly understood. Understanding biochemical and molecular events involved in signal transduction pathways has become one of the most active areas of plant research. Research during the last 15 years has established that Ca2+ acts as a messenger in transducing external signals. The evidence in support of Ca2+ as a messenger is unequivocal and fulfills all the requirements of a messenger. The role of Ca2+ becomes even more important because it is the only messenger known so far in plants. Since our last review on the Ca2+ messenger system in 1987, there has been tremendous progress in elucidating various aspects of Ca(2+) -signaling pathways in plants. These include demonstration of signal-induced changes in cytosolic Ca2+, calmodulin and calmodulin-like proteins, identification of different Ca2+ channels, characterization of Ca(2+) -dependent protein kinases (CDPKs) both at the biochemical and molecular levels, evidence for the presence of calmodulin-dependent protein kinases, and increased evidence in support of the role of inositol phospholipids in the Ca(2+) -signaling system. Despite the progress in Ca2+ research in plants, it is still in its infancy and much more needs to be done to understand the precise mechanisms by which Ca2+ regulates a wide variety of physiological processes. The purpose of this review is to summarize some of these recent developments in Ca2+ research as it relates to signal transduction in plants.     

Sensitivity and specificity amplification in signal transduction

Intracellular signal transduction pathways transmit signals from the cell surface to various intracellular destinations, such as cytoskeleton and nucleus through a cascade of protein-protein interactions and activation events, leading to phenotypic changes such as cell proliferation, differentiation, and death. Over the past two decades, numerous signaling proteins and signal transduction pathways have been discovered and characterized. There are two major classes of signaling proteins: phosphoproteins (e.g., mitogen-activated protein kinases) and guanosine triphosphatases (GTPases; e.g., Ras and G proteins). They both function as molecular switches by addition and removal of one or more high-energy phosphate groups. This review discusses developments that seek to quantify the signal transduction processes with kinetic analysis and mathematical modeling of the signaling phosphoproteins and GTPases. These studies have provided insights into the sensitivity and specificity amplification of biological signals in integrated systems.     

Molecular physiology of visual pigment rhodopsin

The key physiological functions of the rhodopsin molecule are reviewed. Molecular mechanisms of visual pigments spectral tuning, photoisomerization of the 11-cis-retinal chromophore that triggers the phototransduction process, formation of physiologically active state of rhodopsin as a G-protein-coupled receptor, rhodopsin visual cycle, and consequences of its impairment are evaluated. Visual pigment rhodopsin performs several functions, providing spectral sensitivity of photoreceptor cells, phototransduction processes and light and dark adaptation. Genetically determined defects of visual pigment molecule and proteins involved into mechanisms of phototransduction and adaptation or into mechanism of visual cycle are directly linked to pathogenesis of different forms of degenerative retina diseases. Understanding the molecular mechanisms of these physiological processes uncovers the way to direct investigation of pathogenesis of these severe eye diseases.     

Electrogenic Na(+)/Ca(2+) exchange. A novel amplification step in squid olfactory transduction

Olfactory receptor neurons (ORNs) from the squid, Lolliguncula brevis, respond to the odors l-glutamate or dopamine with increases in internal Ca(2+) concentrations ([Ca(2+)](i)). To directly asses the effects of increasing [Ca(2+)](i) in perforated-patched squid ORNs, we applied 10 mM caffeine to release Ca(2+) from internal stores. We observed an inward current response to caffeine. Monovalent cation replacement of Na(+) from the external bath solution completely and selectively inhibited the caffeine-induced response, and ruled out the possibility of a Ca(2+)-dependent nonselective cation current. The strict dependence on internal Ca(2+) and external Na(+) indicated that the inward current was due to an electrogenic Na(+)/Ca(2+) exchanger. Block of the caffeine-induced current by an inhibitor of Na(+)/Ca(2+) exchange (50-100 microM 2',4'-dichlorobenzamil) and reversibility of the exchanger current, further confirmed its presence. We tested whether Na(+)/Ca(2+) exchange contributed to odor responses by applying the aquatic odor l-glutamate in the presence and absence of 2', 4'-dichlorobenzamil. We found that electrogenic Na(+)/Ca(2+) exchange was responsible for approximately 26% of the total current associated with glutamate-induced odor responses. Although Na(+)/Ca(2+) exchangers are known to be present in ORNs from numerous species, this is the first work to demonstrate amplifying contributions of the exchanger current to odor transduction.     

Magnetic anisotropy of the visual pigment rhodopsin.

A new estimate of diamagnetic anisotropy of the frog rhodopsin is reported. The estimate is obtained by combining the data of magnetic field induced orientation of isolated frog rod outer segments as measured by Chagneux and Chalazonitis (1972) and the data of diamagnetic anisotropy of lecithin membranes as recently reported by Boroske and Helfrich (1978). The anisotropy of the volume susceptibilities of frog rhodopsin is calculated to be 4.4 X 10(-8) cgs unit/cm3, which corresponds to 1.5 X 10(-27) cgs unit/molecule, or 9.0 X 10(-4) cgs unit/mol.     

Characteristics of Drosophila rhodopsin in wild-type and norpA vision transduction mutants

The properties of the major visual pigment of Drosophila melanogaster were evaluated. The visual pigment was isolated from other protein components using acrylamide gel electrophoresis and spectral identification. Sodium dodecyl sulfate (SDS) acrylamide gels of the isolated visual pigment gave a single protein subunit with a mol wt of 37,000 daltons. The rhodopsin480 molar extinction coefficient was 35,000 liter/mol-cm (+/- 2,700 SE). The metarhodopsin580 molar extinction coefficient was approximately 56,000 liter/mol-cm. Microspectrophotometry was used to compare the rhodopsin concentrations in wild-type flies and norpA vision transduction mutants. At 2 days of age (12 h dark-12 h light cycle, 19 degrees C) all of the norpA flies exhibited a similar rhodopsin concentration (75% of the wild-type strain). By 21 days of age some of the norpA alleles showed substantially reduced rhodopsin concentrations (16-43% of normal), whereas others showed no major age-dependent decreases (68-77%). Temperature and light-dark cycle affected the reduction. Alleles with no receptor potential exhibited the largest decreases in rhodopsin concentration. The data indicate that the norpA phototransduction mutant has a defect in the system responsible for maintaining the rhodopsin480 concentration. This defect in the rhodopsin maintenance system does not appear to be the cause of the reduced electroretinogram (ERG) amplitude observed in some of these mutants, but instead is a consequence of the decrease in ERG amplitude, or the flaw(s) responsible for the decrease in ERG amplitude.     

Role of the HAMP domain region of sensory rhodopsin transducers in signal transduction

Archaea are able to sense light via the complexes of sensory rhodopsins I and II and their corresponding chemoreceptor-like transducers HtrI and HtrII. Though generation of the signal has been studied in detail, the mechanism of its propagation to the cytoplasm remains obscured. The cytoplasmic part of the transducer consists of adaptation and kinase activity modulating regions, connected to transmembrane helices via two HAMP (histidine kinases, adenylyl cyclases, methyl-accepting chemotaxis proteins, phosphatases) domains. The inter-HAMP region of Natronomonas pharaonis HtrII (NpHtrII) was found to be α-helical [Hayashi, K., et al. (2007) Biochemistry 46, 14380-14390]. We studied the inter-HAMP regions of NpHtrII and other phototactic signal transducers by means of molecular dynamics. Their structure is found to be a bistable asymmetric coiled coil, in which the protomers are longitudinally shifted by ~1.3 ?. The free energy penalty for the symmetric structure is estimated to be 1.2-1.5 kcal/mol depending on the molarity of the solvent. Both flanking HAMP domains are mechanistically coupled to the inter-HAMP region and are asymmetric. The longitudinal shift in the inter-HAMP region is coupled with the in-plane displacement of the cytoplasmic part by 8.6 ? relative to the transmembrane part. The established properties suggest that (1) the signal may be transduced through the inter-HAMP domain switching and (2) the inter-HAMP region may allow cytoplasmic parts of the transducers to come sufficiently close to each other to form oligomers.     

G-protein-coupled receptor rhodopsin regulates the phosphorylation of retinal insulin receptor

We have shown previously that phosphoinositide 3-kinase in the retina is activated in vivo through light-induced tyrosine phosphorylation of the insulin receptor (IR). The light effect is localized to photoreceptor neurons and is independent of insulin secretion (Rajala, R. V., McClellan, M. E., Ash, J. D., and Anderson, R. E. (2002) J. Biol. Chem. 277, 43319-43326). These results suggest that there exists a cross-talk between phototransduction and other signal transduction pathways. In this study, we examined the stage of phototransduction that is coupled to the activation of the IR. We studied IR phosphorylation in mice lacking the rod-specific alpha-subunit of transducin to determine if phototransduction events are required for IR activation. To confirm that light-induced tyrosine phosphorylation of the IR is signaled through bleachable rhodopsin, we examined IR activation in retinas from RPE65(-/-) mice that are deficient in opsin chromophore. We observed that IR phosphorylation requires the photobleaching of rhodopsin but not transducin signaling. To determine whether the light-dependent activation of IR is mediated through the rod or cone transduction pathway, we studied the IR activation in mice lacking opsin, a mouse model of pure cone function. No light-dependent activation of the IR was found in the retinas of these mice. We provide evidence for the existence of a light-mediated IR pathway in the retina that is different from the known insulin-mediated pathway in nonneuronal tissues. These results suggest that IR phosphorylation in rod photoreceptors is signaled through the G-protein-coupled receptor rhodopsin. This is the first study demonstrating that rhodopsin can initiate signaling pathway(s) in addition to its classical phototransduction.     

Insights into the activation mechanism of the visual receptor rhodopsin

Recent advances in the structural biology of GPCRs (G-protein-coupled receptors) have provided insights into their structure and function. Comparisons of the visual and ligand-activated receptors highlight the unique elements of rhodopsin that allow it to function as a highly sensitive dim-light photoreceptor in vertebrates, as well as the common elements that it shares with the large class A GPCR family. However, despite progress, a number of questions remain unanswered about how these receptors are activated.     

Characterization of the primary photointermediates of Drosophila rhodopsin

Invertebrate opsins are unique among the visual pigments because the light-activated conformation, metarhodopsin, is stable following exposure to light in vivo. Recovery of the light-activated pigment to the dark conformation (or resting state) occurs either thermally or photochemically. There is no evidence to suggest that the chromophore becomes detached from the protein during any stage in the formation or recovery processes. Biochemical and structural studies of invertebrate opsins have been limited by the inability to express and purify rhodopsins for structure-function studies. In this study, we used Drosophila to produce an epitope-tagged opsin, Rh1-1D4, in quantities suitable for spectroscopic and photochemical characterization. When expressed in Drosophila, Rh1-1D4 is localized to the rhabdomere membranes, has the same spectral properties in vivo as wild-type Rh1, and activates the phototransduction cascade in a normal manner. Purified Rh1-1D4 visual pigment has an absorption maximum of the dark-adapted state of 474 nm, while the metarhodopsin absorption maximum is 572 nm. However, the metarhodopsin state is not stable as purified in dodecyl maltoside but decays with kinetics that require a double-exponential fit having lifetimes of 280 and 2700 s. We investigated the primary properties of the pigment at low temperature. At 70 K, the pigment undergoes a temperature-induced red shift to 486 nm. Upon illumination with 435 nm light, a photostationary state mixture is formed consisting of bathorhodopsin (lambda(max) = 545 nm) and isorhodopsin (lambda(max) = 462 nm). We also compared the spectroscopic and photochemical properties of this pigment with other vertebrate pigments. We conclude that the binding site of Drosophila rhodopsin is similar to that of bovine rhodopsin and is characterized by a protonated Schiff base chromophore stabilized via a single negatively charged counterion.     

Energy storage in the primary photochemical events of rhodopsin and isorhodopsin

The energetics associated with the photoequilibrium (Formula: see text) are measured at 77 K by using pulsed-laser photocalorimetry and a range of excitation wavelengths and relative starting concentrations. Enthalpies for the photochemical transformations R hv----B and I hv----B are measured to be delta HRB = 32.2 +/- 0.9 kcal mol-1 and delta HIB = 27.1 +/- 3.2 kcal mol-1, respectively. Although the value of delta HRB is slightly lower than that reported previously by Cooper of 34.7 +/- 2.2 kcal mol-1 [Cooper, A. (1979) Nature (London) 282, 531-533], the two values are in agreement within experimental error. The energy difference delta HRB - delta HIB = 5.1 +/- 3.3 kcal mol-1 is identical within experimental error with the difference in enthalpies of isorhodopsin and rhodopsin [5.2 +/- 2.3; Cooper, A. (1979) FEBS Lett. 100, 382-384]. We suggest that this result is consistent with the theory that bathorhodopsin is a single, common photochemical intermediate connecting rhodopsin and isorhodopsin.     

Calcium channels and signal transduction in plant cells

An increasing number of studies indicate that changes in cytosolic free Ca²⁺ ([Ca²⁺]_c) mediate specific types of signal transduction in plant cells. Modulation of [Ca²⁺]_c is likely to be achieved through changes in the activity of Ca²⁺ channels, which catalyse passive influx of Ca²⁺ to the cytosol from extracellular and intracellular compartments. Voltage-sensitive Ca²⁺ channels have been detected in the plasma membranes of algae, where they control membrane electrical properties and cell turgor. These channels are sensitive to 1,4-dihydropyridines, which in animal cells specifically affect one class of voltage-regulated plasma membrane Ca²⁺ channel. Ca²⁺-permeable channels with different pharmacological properties have been found in the plasma membrane of higher plants. Recent evidence suggests the existence of two discrete classes of Ca²⁺ channel co-resident in the vacuolar membrane (tonoplast) of higher plants. The first is gated by inositol 1,4,5-trisphosphate, and bears a number of similarities to its animal counterpart which is located in the endoplasmic reticulum (ER). The second tonoplast Ca²⁺ channel is voltage-operated. However, the specific roles of these tonoplast channels in signal transduction have yet to be elucidated.     

Temporal cooperativity and sensitivity amplification in biological signal transduction

Sensitivity amplification in signal transduction modules regulated by phosphorylation-dephosphorylation cycles and GTPases results from a new type of cooperativity which is fundamentally different from that of allosterism. This type of cooperativity, termed temporal cooperativity [Qian, H. (2003) Biophys. Chem. 105, 585-593], is analyzed in this paper through stochastic models for molecular interactions. Mathematical analysis is developed through a series of models with different levels of complexity, from which a simple conceptual model based on linear cooperativity is derived. The following are shown: (i) When both kinase and phosphatase are nonsaturating, the distribution of the number of activated substrate molecules is binomial. With increasing kinase activity, the peak of the distribution continuously moves toward 100% activation. (ii) When the kinase is saturated, i.e., zeroth order, the distribution is Poisson. (iii) When both enzymes are saturated, the distribution is geometric. Ultrasensitivity corresponds to an abrupt switching of the peak position of the distribution from 0 to 100%. The theory is applicable to a wide range of processes in cell signaling including the specificity and sensitivity of T-cell activation.     

Purification of visual arrestin from squid photoreceptors and characterization of arrestin interaction with rhodopsin and rhodopsin kinase

Invertebrate visual signal transduction involves photoisomerization of rhodopsin, activating a guanine nucleotide binding protein (G protein) of the G(q) class, iG(q), which stimulates a phospholipase C, increasing intracellular Ca2+. Arrestin binding to photoactivated rhodopsin is a key mechanism of desensitization. We have previously reported the cloning of a retina-specific arrestin cDNA from Loligo pealei displaying 56-64% sequence similarity to other reported arrestin sequences. Here, we report the purification of the 55-kDa squid visual arrestin. Purified squid visual arrestin is able to inhibit light-activated GTPase activity dose-dependently in arrestin-depleted rhabdomeric membranes and associate with the membrane in a light-dependent manner. Membrane association can be partially inhibited by inositol 1,2,3,4,5,6-hexakisphosphate (IP6), a soluble analog of the membrane lipid phosphatidylinositol 3,4,5-triphosphate. In reconstitution assays, we demonstrate arrestin phosphorylation by squid rhodopsin kinase, a novel function among the G protein-coupled receptor kinase family. Phosphorylation of purified arrestin requires squid rhodopsin kinase, membranes, light-activation, and the presence of Ca2+. This is the first large-scale purification of an invertebrate arrestin and biochemical demonstration of arrestin function in the invertebrate visual system.