Expression of functional delta opioid receptors in vascular smooth muscle.

We evaluated smooth muscle from human internal mammary artery and rat aorta for the presence of delta opioid receptors. Radioligand receptor competition studies using the delta-receptor selective agonist, [3H]-[D-Ala2, Met5] enkephalinamide (DAMA) suggested the expression of a high affinity binding site in rat and human blood vessels that was consistent with the delta-2 opioid receptor subtype. Using RT-PCR with primers to the cloned delta opioid receptor (DOR), a cDNA fragment identical to the known DOR sequence was obtained from the smooth muscle cell line, A-10. Stimulation of A-10 cells with DAMA resulted in a significant mobilization of intracellular calcium and membrane depolarization. Exposure of aortic rings denuded of endothelium to DAMA induced a naltrindole-senstive increase in contractile tone. These data demonstrate the presence of a functional DOR in vascular smooth muscle and a direct impact of opioids on vascular contractile tone.

Overexpression of Arabidopsis phytochrome B inhibits phytochrome A function in the presence of sucrose.

Overexpression of phytochrome B (phyB) in Arabidopsis has previously been demonstrated to result in dominant negative interference of phytochrome A (phyA)-mediated hypocotyl growth inhibition in far-red (FR) light. This phenomenon has been examined further in this study and has been found to be dependent on the FR fluence rate and on the availability of metabolizable sugars in the growth medium. Poorly metabolized sugars capable of activating the putative hexokinase sensory function were not effective in eliciting the phytochrome interference response. Overexpressed phyB lacking the chromophore-binding site was also effective at inhibiting the phyA response, especially at higher fluence rates of FR. Overexpressed phyB produces the dominant negative phenotype without any apparent effect on phyA abundance or degradation. It is possible that phyA and phyB interact with a common reaction partner but that either the energy state of the cell or a separate sugar-signaling mechanism modulates the phytochrome-signaling interactions.

Missense mutations define a restricted segment in the C-terminal domain of phytochrome A critical to its regulatory activity.

The phytochrome family of photoreceptors has dual molecular functions: photosensory, involving light signal perception, and regulatory, involving signal transfer to downstream transduction components. To define residues necessary specifically for the regulatory activity of phytochrome A (phyA), we undertook a genetic screen to identify Arabidopsis mutants producing wild-type levels of biologically defective but photochemically active and dimeric phyA molecules. Of eight such mutants identified, six contain missense mutations (including three in the same residue, glycine 727) clustered within a restricted segment in the C-terminal domain of the polypeptide. Quantitative photobiological analysis revealed retention of varying degrees of partial activity among the different alleles--a result consistent with the extent of conservation at the position mutated. Together with additional data, these results indicate that the photoreceptor subdomain identified here is critical to the regulatory activity of both phyA and phyB.

Phytochromes: photosensory perception and signal transduction.

The phytochrome family of photoreceptors monitors the light environment and dictates patterns of gene expression that enable the plant to optimize growth and development in accordance with prevailing conditions. The enduring challenge is to define the biochemical mechanism of phytochrome action and to dissect the signaling circuitry by which the photoreceptor molecules relay sensory information to the genes they regulate. Evidence indicates that individual phytochromes have specialized photosensory functions. The amino-terminal domain of the molecule determines this photosensory specificity, whereas a short segment in the carboxyl-terminal domain is critical for signal transfer to downstream components. Heterotrimeric GTP-binding proteins, calcium-calmodulin, cyclic guanosine 5'-phosphate, and the COP-DET-FUS class of master regulators are implicated as signaling intermediates in phototransduction.

Blue Light Induces Phosphorylation at Seryl Residues on a Pea (Pisum sativum L.) Plasma Membrane Protein.

We have partially characterized the blue-light-stimulated in vitro phosphorylation of a membrane protein from etiolated Pisum sativum L. stems. Properties of the response have implicated its involvement in signal transduction of phototropic stimuli (T.W. Short, W.R. Briggs [1990] Plant Physiol 92: 179-185; P. Reymond, T.W. Short, W.R. Briggs [1992] Proc Natl Acad Sci USA 89: 4718- 4721). Analysis of proteolysis products and phosphoamino acidanalysis indicate that the substrate protein is phosphorylated on multiple seryl residues. Kinetics of the in vitro reaction show phosphorylation to be complete within 2 to 5 min at 30[deg]C in either light-exposed or dark-control plasma membrane preparations, regardless of whether the membranes were first solubilized in Triton X-100. Nucleotide competition assays show the kinase to be ATP specific. The pH optimum covers a broad range with a maximum near 7.5. A wide array of salts inhibits the phosphorylation at high concentrations, but millimolar concentrations of Mg2+ are required to form Mg.ATP complexes for maximal activity, whereas excess free Mg2+ or Ca2+ are not required for the reaction.

Arabidopsis HY8 locus encodes phytochrome A.

hy8 long hypocotyl mutants of Arabidopsis defective in responsiveness to prolonged far-red light (the so-called "far-red high-irradiance response") are selectively deficient in functional phytochrome A. To define the molecular lesion in these mutants, we sequenced the phytochrome A gene (phyA) in lines carrying one or other of two classes of hy8 alleles. The hy8-1 and hy8-2 mutants that express no detectable phytochrome A each have a single nucleotide change that inserts a translational stop codon in the protein coding sequence. These results establish that phyA resides at the HY8 locus. The hy8-3 mutant that expresses wild-type levels of photochemically active phytochrome A has a glycine-to-glutamate missense mutation at residue 727 in the C-terminal domain of the phyA sequence. Quantitative fluence rate response analysis showed that the mutant phytochrome A molecule produced by hy8-3 exhibited no detectable regulatory activity above that of the phyA-protein-deficient hy8-2 mutant. This result indicates that glycine-727, which is invariant in all sequenced phytochromes, has a function important to the regulatory activity of phytochrome A but not to photoperception.

Blue Light-Induced Phosphorylation of a Plasma Membrane-Associated Protein in Zea mays L.

Blue light induces a variety of photomorphogenic responses in higher plants, among them phototropic curvature, the bending of seedlings toward a unidirectional light source. In dark-grown coleoptiles of maize (Zea mays L.) seedlings, blue light induces rapid phosphorylation of a 114-kD protein at fluence levels that are sufficient to stimulate phototropic curvature. Phosphorylation in response to blue light can be detected in vivo in coleoptile tips preincubated in 32Pi or in vitro in isolated membranes supplemented with [[gamma]-32P]ATP. Phosphorylation reaches a maximum level in vitro within 2 min following an inductive light pulse, but substantial labeling occurs within the first 15 s. Isolated membranes remain activated for several minutes following an in vitro blue light stimulus, even in the absence of exogenous ATP. Phosphoamino acid analysis of the 114-kD protein detected phosphoserine and a trace of phosphothreonine. The kinase involved in phosphorylating the protein in vitro is not dependent on calcium. The 114-kD protein itself has an apparent binding site for ATP, detected by incubating with the nonhydrolyzable analog, 5[prime]-p-fluorosulfonyl-benzoyladenosine. This result suggests that the 114-kD protein, which becomes phosphorylated in response to blue light, may also be capable of kinase activity.

Correlation of Blue Light-Induced Phosphorylation to Phototropism in Zea mays L.

The physiology of light-induced phototropic curvature has been studied extensively in coleoptiles of grasses, particularly in Avena and Zea mays L. In Z. mays L., we have found that, in addition to curvature, blue light also induces rapid phosphorylation of a 114-kD protein in the tips of coleoptiles, and, in a previous report, we reported several characteristics of the phosphorylated substrate protein and kinase (J.M. Palmer, T.W. Short, S. Gallagher, W.R. Briggs [1993] Plant Physiol 102: 1211-1218). Here, we compare the phosphorylation response to several known aspects of phototropism physiology. Blue light-induced phosphorylation occurs only in the upper portion of the coleoptile and is absent from the node and mesocotyl. The specific activity of phosphorylation is highest in the extreme apical portion of the tip, which is also the site of maximal sensitivity to phototropic stimuli (A. W. Galston [1959] In Physiology of Movements, Encyclopedia of Plant Physiology, Springer, Berlin). Fluence-response determinations indicate that light dosage levels that stimulate curvature also stimulate phosphorylation. However, the threshold for inducing detectable phosphorylation in maize cannot be matched to the threshold for curvature induction. The recovery of sensitivity to phototropic stimuli after exposure to high fluences of light occurs with kinetics that are very similar to those for recovery of the phosphorylation response after a previous high-fluence light exposure. In addition, wavelengths of light in the blue and near-ultraviolet regions of the spectrum that maximally stimulate phototropic curvature also maximally stimulate in vitro phosphorylation in maize. The pattern of stimulation matches the absorption spectra of flavoproteins, which have been proposed as candidates for blue light photoreceptors.

A Pea Plasma Membrane Protein Exhibiting Blue Light-Induced Phosphorylation Retains Photosensitivity following Triton Solubilization.

Phosphorylation of a polypeptide of approximately 120 kD in pea (Pisum sativum L.) plasma membranes in response to blue light has been shown to be involved in phototropic curvature, but the relationship of this protein to the kinase and photoreceptor acting upon it is uncertain. Using two-phase aqueous partitioning to isolate right-side-out plasma membrane vesicles, we have obtained evidence suggesting that the photoreceptor, kinase, and substrate are localized to the plasma membrane fraction. Latent phosphorylation accessible through Triton X-100 or freeze/thaw treatments of purified plasma membrane vesicles indicates that at least the kinase moiety is present on the internal face of the plasma membrane. Effects of solubilization of vesicles on fluence-response characteristics and on phosphorylation levels provide evidence that the receptor, kinase, and protein substrate are present together in individual mixed detergent micelles, either as a stable complex or as domains of a single polypeptide. In vivo blue-light irradiation results in a small but significant decrease in mobility of the 120-kD phosphorylated protein on sodium dodecylsulfate gel electrophoresis. This mobility shift is evident on Coomassie-stained gels and on western blots probed with polyclonal antibodies raised against the 120-kD protein. Among the plasma membrane proteins bound to the reactive nucleotide analog fluorosulfonylbenzoyladenine (FSBA), a distinct protein band at 120 kD can be detected on blots probed with anti-FSBA antibodies. This band exhibits an in vivo light-dependent mobility shift identical to that observed for the protein band and antibodies specific for the 120-kD protein, implying that the 120-kD protein has an integral nucleotide binding site and consistent with the possibility that the substrate protein is also a kinase.

Blue light activates a specific protein kinase in higher plants.

Blue light mediates the phosphorylation of a membrane protein in seedlings from several plant species. When crude microsomal membrane proteins from dark-grown pea (Pisum sativum L.), sunflower (Helianthus annuus L.), zucchini (Cucurbita pepo L.), Arabidopsis (Arabidopsis thaliana L.), or tomato (Lycopersicon esculentum L.) stem segments, or from maize (Zea mays L.), barley (Hordeum vulgare L.), oat (Avena sativa L.), wheat (Triticum aestivum L.), or sorghum (Sorghum bicolor L.) coleoptiles are illuminated and incubated in vitro with [gamma-(32)P]ATP, a protein of apparent molecular mass from 114 to 130 kD is rapidly phosphorylated. Hence, this system is probably ubiquitous in higher plants. Solubilized maize membranes exposed to blue light and added to unirradiated solubilized maize membranes show a higher level of phosphorylation of the light-affected protein than irradiated membrane proteins alone, suggesting that an unirradiated substrate is phosphorylated by a light-activated kinase. This finding is further demonstrated with membrane proteins from two different species, where the phosphorylated proteins are of different sizes and, hence, unambiguously distinguishable on gel electrophoresis. When solubilized membrane proteins from one species are irradiated and added to unirradiated membrane proteins from another species, the unirradiated protein becomes phosphorylated. These experiments indicate that the irradiated fraction can store the light signal for subsequent phosphorylation in the dark. They also support the hypothesis that light activates a specific kinase and that the systems share a close functional homology among different higher plants.

Light-induced phosphorylation of a membrane protein plays an early role in signal transduction for phototropism in Arabidopsis thaliana.

Blue light is known to cause rapid phosphorylation of a membrane protein in etiolated seedlings of several plant species, a protein that, at least in etiolated pea seedlings and maize coleoptiles, has been shown to be associated with the plasma membrane. The light-driven phosphorylation has been proposed on the basis of correlative evidence to be an early step in the signal transduction chain for phototropism. In the Arabidopsis thaliana mutant JK224, the sensitivity to blue light for induction of first positive phototropism is known to be 20- to 30-fold lower than in wild type, whereas second positive curvature appears to be normal. While light-induced phosphorylation can be demonstrated in crude membrane preparations from shoots of the mutant, the level of phosphorylation is dramatically lower than in wild type, as is the sensitivity to blue light. Another A. thaliana mutant, JK218, that completely lacks any phototropic responses to up to 2 h of irradiation, shows a normal level of light-induced phosphorylation at saturation. Since its gravitropic sensitivity is normal, it is presumably blocked in some step between photoreception and the confluence of the signal transduction pathways for phototropism and gravitropism. We conclude from mutant JK224 that light-induced phosphorylation plays an early role in the signal transduction chain for phototropism in higher plants.

Characterization of a Rapid, Blue Light-Mediated Change in Detectable Phosphorylation of a Plasma Membrane Protein from Etiolated Pea (Pisum sativum L.) Seedlings.

When crude microsomal membranes from apical stem segments of etiolated Pisum sativum L. cv Alaska are mixed in vitro with gamma-[(32)P]ATP, a phosphorylated band of apparent molecular mass 120 kilodaltons can be detected on autoradiographs of sodium dodecyl sulfate electrophoresis gels. If the stem sections are exposed to blue light immediately prior to membrane isolation, this band is not evident. The response is observed most strongly in membranes from the growing region of the stem, but no 120 kilodalton radiolabeled band is detected in membranes from the developing buds. Fluence-response curves for the reaction show that the system responds to blue light above about 0.3 micromole per square meter, and the visible phosphorylation completely disappears above 200 micromoles per square meter. Reciprocity is valid for the system, because varying illumination time or fluence rate give similar results. If the stem segments are left in the dark following a saturating blue irradiation, the radio-labeled band begins to return after about 10 minutes and is as intense as that from the dark controls within 45 to 60 minutes. A protein that comigrates with the phosphorylated protein on polyacrylamide gels is also undetectable after saturating blue light irradiations. The fluence range in which the protein band disappears is the same as that for the disappearance of the phosphorylation band. Its dark recovery kinetics and tissue distribution also parallel those for the phosphorylation. In vitro irradiation of the isolated membranes also results in a phosphorylation change at that molecular mass, but in the opposite direction. Comparisons of the kinetics, tissue distribution, and dark recovery of the phosphorylation response with those published for blue light-mediated phototropism or rapid growth inhibition indicate that the phosphorylation could be linked to one or both of those reactions. However, the fluence-response relationships for the change in detectable phosphorylation match quite closely those reported for phototropism but not those for growth inhibition. Blue light has also been found to regulate the capacity for in vitro phosphorylation of a second protein. It has an apparent molecular mass of 84 kilodaltons and is localized primarily in basal stem sections.

Light-mediated changes in two proteins found associated with plasma membrane fractions from pea stem sections.

Irradiation of etiolated pea (Pisum sativum L.) seedlings with white light affects two proteins, both of monomer molecular mass near 120 kDa. Both proteins have been detected in association with plasma membrane fractions. The first is identifiable in that it becomes heavily phosphorylated when the membranes are incubated with exogenous ATP. The second of these proteins is phytochrome, as determined by electrophoretic transfer (Western) blot analysis. Measurable phosphorylation and phytochrome (the latter detected by antigenicity) decline when the tissue is irradiated with white light prior to membrane isolation and in vitro phosphorylation. The phosphorylated protein is probably not phytochrome for three reasons. (i) It shows a slightly different distribution in sucrose gradients. (ii) Red light causes a gradual decline in the phytochrome that is associated with membrane fractions but has a negligible effect on the phosphorylatable protein; blue light, on the other hand, causes significantly slower loss of phytochrome than does red light but brings about a rapid decline in the phosphorylation signal. (iii) The molecular masses are not identical. The association of both proteins with membrane fractions is probably neither ionic nor, at least for the phosphorylatable protein, the consequence of entrapment of soluble proteins in vesicles formed during tissue extraction. Phytochrome is lost from the membrane fractions during irradiation, as judged by loss of antigenicity. Whether the phosphorylatable protein is lost, a specific kinase is lost, phosphatase activity increases, or phosphorylatable sites are blocked as a consequence of blue light treatment is not known.