G protein-coupled receptors (GPCR) are seven transmembrane helix proteins that couple binding of extracellular ligands to conformational changes and activation of intracellular G proteins, GPCR ...kinases, and arrestins. Constitutively active mutants are ubiquitously found among GPCRs and increase the inherent basal activity of the receptor, which often correlates with a pathological outcome. Here, we have used the M257Y6.40 constitutively active mutant of the photoreceptor rhodopsin in combination with the specific binding of a C-terminal fragment from the G protein alpha subunit (GαCT) to trap a light activated state for crystallization. The structure of the M257Y/GαCT complex contains the agonist all-trans-retinal covalently bound to the native binding pocket and resembles the G protein binding metarhodopsin-II conformation obtained by the natural activation mechanism; i.e., illumination of the prebound chromophore 11-cis-retinal. The structure further suggests a molecular basis for the constitutive activity of 6.40 substitutions and the strong effect of the introduced tyrosine based on specific interactions with Y2235.58 in helix 5, Y3067.53 of the NPxxY motif and R1353.50 of the E(D)RY motif, highly conserved residues of the G protein binding site.
Animals have evolved specialized photoreceptors in the retina and in extraocular tissues that allow them to measure light changes in their environment. In mammals, the retina is the only structure ...that detects light and relays this information to the brain. The classical photoreceptors, rods and cones, are responsible for vision through activation of rhodopsin and cone opsins. Melanopsin, another photopigment first discovered in Xenopus melanophores (Opn4x), is expressed in a small subset of retinal ganglion cells (RGCs) in the mammalian retina, where it mediates non-image forming functions such as circadian photoentrainment and sleep. While mammals have a single melanopsin gene (opn4), zebrafish show remarkable diversity with two opn4x-related and three opn4-related genes expressed in distinct patterns in multiple neuronal cell types of the developing retina, including bipolar interneurons. The intronless opn4.1 gene is transcribed in photoreceptors as well as in horizontal cells and produces functional photopigment. Four genes are also expressed in the zebrafish embryonic brain, but not in the photoreceptive pineal gland. We discovered that photoperiod length influences expression of two of the opn4-related genes in retinal layers involved in signaling light information to RGCs. Moreover, both genes are expressed in a robust diurnal rhythm but with different phases in relation to the light-dark cycle. The results suggest that melanopsin has an expanded role in modulating the retinal circuitry of fish.
Our current understanding of the spectral sensitivities of the mysticete whale rod-based visual pigments is based on two species, the gray whale (Eschrichtius robustus) and the humpback whale ...(Megaptera novaeangliae) possessing absorbance maxima determined from difference spectra to be 492 and 497 nm, respectively. These absorbance maxima values are blueshifted relative to those from typical terrestrial mammals (≈500 nm) but are redshifted when compared to those identified in the odontocetes (479-484 nm). Although these mysticete species represent two of the four mysticete families, they do not fully represent the mysticete whales in terms of foraging strategy and underwater photic environments where foraging occurs. In order to better understand the spectral sensitivities of the mysticete whale rod visual pigments, we have examined the rod opsin genes from 11 mysticete species and their associated amino acid substitutions. Based on the amino acids occurring at positions 83, 292, and 299 along with the directly determined dark spectra from expressed odontocete and mysticete rod visual pigments, we have determined that the majority of mysticete whales possess deep-sea and pelagic like rod visual pigments with absorbance maxima between 479 and 484 nm. Finally, we have defined the five amino acid substitution events that determine the resulting absorbance spectra and associated absorbance maxima for the mysticete whale rod visual pigments examined here.
To assess the role that vision plays in the ability of the North Atlantic right whale (Eubalaena glacialis) to detect its primary prey species, the calanoid copepod Calanus finmarchicus, we have ...compared the absorbance spectrum of the E. glacialis rod visual pigment, the transmittance spectra of C. finmarchicus carotenoid pigments, as well as the downwelling irradiance and horizontal radiance spectra collected during springtime at three locations in the western Gulf of Maine. The E. glacialis rod visual pigment absorbs light maximally at 493 nm, while microspectrophotometric measurements of the C. finmarchicus carotenoid pigments reveal transmission spectra with minima matching very well with the E. glacialis rod visual pigment absorbance spectra maximum. Springtime spectral downwelling irradiance and horizontal radiance values from the surface waters of Cape Cod Bay and at all depths in Great South Channel overlap the E. glacialis rod absorbance spectrum, allowing C. finmarchicus to appear as a high‐contrast dark silhouette against a bright background spacelight, thus facilitating visually guided contrast foraging. In contrast, spectral downwelling irradiance and horizontal radiance at depth in Cape Cod Bay, and all depths in Wilkinson Basin, do not overlap the E. glacialis rod absorbance spectrum, providing little if any useful light for contrast vision.
We report experiments designed to test the hypothesis that the aqueous solubility of 11-cis-retinoids plays a significant role in the rate of visual pigment regeneration. Therefore, we have compared ...the aqueous solubility and the partition coefficients in photoreceptor membranes of native 11-cis-retinal and an analogue retinoid, 11-cis 4-OH retinal, which has a significantly higher solubility in aqueous medium. We have then correlated these parameters with the rates of pigment regeneration and sensitivity recovery that are observed when bleached intact salamander rod photoreceptors are treated with physiological solutions containing these retinoids. We report the following results: (a) 11-cis 4-OH retinal is more soluble in aqueous buffer than 11-cis-retinal. (b) Both 11-cis-retinal and 11-cis 4-OH retinal have extremely high partition coefficients in photoreceptor membranes, though the partition coefficient of 11-cis-retinal is roughly 50-fold greater than that of 11-cis 4-OH retinal. (c) Intact bleached isolated rods treated with solutions containing equimolar amounts of 11-cis-retinal or 11-cis 4-OH retinal form functional visual pigments that promote full recovery of dark current, sensitivity, and response kinetics. However, rods treated with 11-cis 4-OH retinal regenerated on average fivefold faster than rods treated with 11-cis-retinal. (d) Pigment regeneration from recombinant and wild-type opsin in solution is slower when treated with 11-cis 4-OH retinal than with 11-cis-retinal. Based on these observations, we propose a model in which aqueous solubility of cis-retinoids within the photoreceptor cytosol can place a limit on the rate of visual pigment regeneration in vertebrate photoreceptors. We conclude that the cytosolic gap between the plasma membrane and the disk membranes presents a bottleneck for retinoid flux that results in slowed pigment regeneration and dark adaptation in rod photoreceptors.
Opsins comprise a family of proteins belonging to the superfamily of G-protein coupled receptors (GPCRs). The opsins combined with vitamin-A derived chromophores make up the photopigments responsible ...for such diverse functions as image- and nonimage-forming vision, entrainment of circadian cycles, and the pupillary light response. The elucidation of the structural and biochemical properties of photopigments is of extreme importance to understanding the biological processes that they control. Currently the crystal structure of two photopigments have been solved; bovine rhodopsin from the vertebrate visual opsins, and squid rhodopsin from the invertebrate visual opsins. In the vertebrate visual opsins, five main subclasses exist; the RH1 class containing bovine rhodopsin makes up the rod based opsins, and the SWS1, SWS2, RH2, and M/LWS classes make up the cone based opsins. I question how similar structurally and biochemically the cone opsin classes are to the RH1 class. Using in vitro G-protein activation assays it is shown that mutations targeting key structural bonds within the cone opsin classes, compared to the RH1 class, result in different profiles of biochemical behavior reflective of structural variation surrounding the chromophore-binding pocket. This structural variation is further explored through the use of spin-labeling techniques aimed at analyzing the helical arrangement within the M/LWS cone opsin class compared to the RH1 class. In addition, a sixth class of opsins, the melanopsins, which maintains greater sequence identity to the invertebrate visual opsins than to the vertebrate visual opsins, is analyzed to identify the location of the counterion to the protonated Schiff base. The counterion is a key functional residue within the opsin family responsible for stabilizing the inactive dark state pigment as well as tuning the wavelength absorbance into the visible spectrum. Through spectroscopic analysis of various melanopsin mutants, evidence is presented that shows the counterion residue is not conserved across opsin groups. Through the evolution of the opsins, the location of the counterion has changed at least twice. This work highlights the large level of complexity that exists in the structure of the opsin family emphasizing the need for further structural analysis.
Vertebrate opsins are classified into one of five classes on the basis of amino acid similarity. These classes are short wavelength sensitive 1 and 2 (SWS1, SWS2), medium/long wavelength sensitive ...(M/LWS), and rod opsin like 1 and 2 (RH1, RH2). In bovine rod opsin (RH1), two critical amino acids form a salt bridge in the apoprotein that maintains the opsin in an inactive state. These residues are K296, which functions as the chromophore binding site, and E113, which functions as the counterion to the protonated Schiff base. Corresponding residues in each of the other vertebrate opsin classes are believed to play similar roles. Previous reports have demonstrated that mutations in these critical residues result in constitutive activation of transducin by RH1 class opsins in the absence of chromophore. Additionally, recent reports have shown that an E113Q mutation in SWS1 opsin is constitutively active. Here we ask if the other classes of vertebrate opsins maintain activation characteristics similar to that of bovine RH1 opsin. We approach this question by making the corresponding substitutions which disrupt the K296/E113 salt bridge in opsins belonging to the other vertebrate opsin classes. The mutant opsins are tested for their ability to constitutively activate bovine transducin. We demonstrate that mutations disrupting this key salt bridge produce constitutive activation in all classes. However, the mutant opsins differ in their ability to be quenched in the dark state by the addition of chromophore as well as in their level of constitutive activation. The differences in constitutive activation profiles suggest that structural differences exist among the opsin classes that may translate into a difference in activation properties.
Emphysema, a component of chronic obstructive pulmonary disease (COPD), is characterized by irreversible alveolar destruction that results in a progressive decline in lung function. This alveolar ...destruction is caused by cigarette smoke, the most important risk factor for COPD. Only 15%‐20% of smokers develop COPD, suggesting that unknown factors contribute to disease pathogenesis. We postulate that the aryl hydrocarbon receptor (AHR), a receptor/transcription factor highly expressed in the lungs, may be a new susceptibility factor whose expression protects against COPD. Here, we report that Ahr‐deficient mice chronically exposed to cigarette smoke develop airspace enlargement concomitant with a decline in lung function. Chronic cigarette smoke exposure also increased cleaved caspase‐3, lowered SOD2 expression, and altered MMP9 and TIMP‐1 levels in Ahr‐deficient mice. We also show that people with COPD have reduced expression of pulmonary and systemic AHR, with systemic AHR mRNA levels positively correlating with lung function. Systemic AHR was also lower in never‐smokers with COPD. Thus, AHR expression protects against the development of COPD by controlling interrelated mechanisms involved in the pathogenesis of this disease. This study identifies the AHR as a new, central player in the homeostatic maintenance of lung health, providing a foundation for the AHR as a novel therapeutic target and/or predictive biomarker in chronic lung disease.
Understanding the characteristics of human immunodeficiency virus (HIV) necessary for infection in a new host is a critical goal for acquired immunodeficiency syndrome (AIDS) research. We studied the ...characteristics of HIV-1 envelope genes in 38 men in the Multicenter AIDS Cohort Study cohort before seroconversion. We found a range of diversity (0.2%–5.6% median, 0.86%), V1-V2 loop length (58–93 aa), and potential N-linked glycosylation sites (n=2–9). However, at least 46% of the men had replicating virus that appeared to have been derived from a single viral variant. Nearly all variants were predicted to be CCR5 tropic. We found no correlation between these viral characteristics and the HIV outcomes of time to clinical AIDS or death and/or a CD4 cell count <200 cells/μL
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