Modulation of CRX transactivation activity by phosducin isoforms.

Phosducin (Phd) and Phd-like proteins (PhLPs) selectively bind guanine nucleotide protein (G protein) betagamma subunits (Gbetagamma), while Phd-like orphan proteins (PhLOPs) lack the major functional domain for the binding of Gbetagamma. A retina- and pineal gland-specific transcription factor, cone-rod homeobox (CRX), was identified by a yeast two-hybrid screen using PhLOP1 as the bait. Direct protein-protein interactions between Phd or PhLOP1 and CRX were demonstrated using a beta-galactosidase quantitative assay in the yeast two-hybrid system and were confirmed by an in vitro binding assay and a glutathione S-transferase (GST) pull-down assay. To determine if the interaction with Phd or PhLOP1 affected CRX transactivation, a 120-bp interphotoreceptor retinoid binding protein (IRBP) promoter-luciferase reporter construct containing a CRX consensus element (GATTAA) was cotransfected into either COS-7 or retinoblastoma Weri-Rb-1 cells with expression constructs for CRX and either Phd or PhLOP1. Phd and PhLOP1 inhibited the transcriptional activation activity of CRX by 50% during transient cotransfection in COS-7 cells and by 70% in Weri-Rb-1 cells and COS-7 cells stably transfected with CRX. Phd inhibited CRX transactivation in a dose-dependent manner. Whereas Phd is a cytoplasmic phosphoprotein, coexpression of Phd with CRX results in Phd being localized both in the cytoplasm and nucleus. By contrast, PhLOP1 is found in the nucleus even without CRX coexpression. To address the physiological relevance of these potential protein interacting partners, we identified immunoreactive proteins for Phd and CRX in retinal cytosolic and nuclear fractions. Immunohistochemical analysis of bovine retinas reveals colocalization of Phd isoforms with CRX predominantly in the inner segment of cone cells, with additional costaining in the outer nuclear layer and the synaptic region. Our findings demonstrate that both Phd and PhLOP1 interact directly with CRX and that each diminishes the transactivation activity of CRX on the IRBP promoter. A domain that interacts with CRX is found in the carboxyl terminus of the Phd isoforms. Phd antibody-immunoreactive peptides are seen in light-adapted mouse retinal cytosolic and nuclear extracts. Neither Phd nor PhLOP1 affected CRX binding to its consensus DNA element in electrophoretic mobility shift assays. A model that illustrates separate functional roles for interactions between Phd and either SUG1 or CRX is proposed. The model suggests further a mechanism by which Phd isoforms could inhibit CRX transcriptional activation.

NonO, a non-POU-domain-containing, octamer-binding protein, is the mammalian homolog of Drosophila nonAdiss.

We have cloned the ubiquitous form of an octamer-binding, 60-kDa protein (NonO) that appears to be the mammalian equivalent of the Drosophila visual and courtship song behavior protein, no-on-transient A/dissonance (nonAdiss). A region unprecedently rich in aromatic amino acids containing two ribonuclear protein binding motifs is highly conserved between the two proteins. A ubiquitous form of NonO is present in all adult tissues, whereas lymphocytes and retina express unique forms of NonO mRNA. The ubiquitous form contains a potential helix-turn-helix motif followed by a highly charged region but differs from prototypic octamer-binding factors by lacking the POU DNA-binding domain. In addition to its conventional octamer duplex-binding, NonO binds single-stranded DNA and RNA at a site independent of the duplex site.

Species-specific differences in expression of G-protein-coupled receptor kinase (GRK) 7 and GRK1 in mammalian cone photoreceptor cells: implications for cone cell phototransduction.

Desensitization plays an important role in the rapid termination of G-protein signaling pathways. This process, which involves phosphorylation by a G-protein-coupled receptor kinase (GRK) followed by arrestin binding, has been studied extensively in the rod photoreceptor cell of the mammalian retina. In contrast, less is known regarding desensitization in cone photoreceptor cells, which occurs more rapidly than in rod cells. Recently, our laboratory has cloned a novel GRK family member, GRK7, from the retina of a cone-dominant mammal, the 13-lined ground squirrel. Here we report the cloning of GRK7 from rod-dominant pig and human retinas, suggesting that this kinase plays a role in human visual signaling. Because GRK1 (rhodopsin kinase), the GRK that mediates rhodopsin desensitization in the rod cell, is reportedly expressed in both rods and cones, a detailed comparison of the localization of the two kinases is a necessary step toward determining their potential roles in cone visual signaling. Immunocytochemical analysis using antibodies selective for these two GRKs unexpectedly demonstrated species-specific differences in GRK7 and GRK1 expression in cones. In pigs and dogs, cones express only GRK7, whereas in mice and rats, we detected only GRK1 in cones. These results suggest that either GRK7 or GRK1 may participate in cone opsin desensitization, depending on the expression pattern of the kinases in different species. In contrast, GRK7 and GRK1 are coexpressed in monkey and human cones, suggesting that coordinate regulation of desensitization by both kinases may occur in primates.

Age-associated reduction in nocturnal pineal melatonin levels in female rats.

Pineal N-acetyltransferase (NAT) activity and radioimmunoassayable levels of melatonin were compared in 2-month-old (young), 12-month-old (middle-aged), and 29-month-old (old) female rats killed at 1600 h (during the light) and at 2300 h (4 h after darkness onset) and 0100 h (6 h after darkness onset). During the light period, NAT levels were equivalent in pineals from each age group. With the onset of darkness NAT levels rose sharply and were again equivalent in all groups at 2300 h. At 0100 h pineal NAT values in the old rats were lower than in the other two groups. Melatonin values were low in pineal glands of all animals killed at 1600 h in light. By 4 h after darkness onset pineal melatonin content in the young rats had increased 12-fold compared to a 6-fold rise in the old animals. Melatonin levels in the middle aged rats were intermediate between the other two groups. Similar relationships were observed in the rats killed at 0100 h. By this time the young rats had melatonin levels 17 times higher than during the day while the increase in the old rats was only 7-fold; 12-month-old rats again had intermediate levels. The finding show a marked reduction in pineal melatonin with aging in female rats.

The arrestin superfamily: cone arrestins are a fourth family.

Arrestins constitute a superfamily of regulatory proteins that down-regulate phosphorylated G-protein membrane receptors, including rod and cone photoreceptors and adrenergic receptors. The potential role of arrestin in color visual processes led us to identify a cDNA encoding a cone-like arrestin in Xenopus laevis, the principle amphibian biological model system. Alignment of 18 deduced amino acid sequences of all known arrestins from both invertebrate and vertebrate species reveals five arrestin families. Further analysis identifies 7 variable and 4 conservative arrestin structural motifs that may identify potential functional domains. The adaptive evolutionary relationship of Xenopus cone arrestin to the arrestin gene tree suggests high intrafamily homology and early gene duplication events.

Linkage of photoreceptor degeneration by apoptosis with inherited defect in phototransduction.

PURPOSE: To investigate the developing retina of normal and rd/rd mice to establish if the inherited defect in the retinal degeneration (rd) gene, encoding the beta subunit of the cascade phosphodiesterase, is associated with rd photoreceptor degeneration by apoptosis. METHODS: DNA content of developing normal and rd/rd retinas was measured spectrophotometrically and analyzed for differential loss during the course of photoreceptor degeneration. Degenerating rd photoreceptors were evaluated by electron microscopy for cytoplasmic features and chromosomal condensation. DNA fragmentation was analyzed by agarose gel electrophoresis at daily intervals during the developmental period in which rd/rd cell death occurs. RESULTS: DNA loss from developing rd/rd retinas is maximal between 10 and 15 postnatal days. Photoreceptor cells die individually throughout the postnatal period of degeneration, with pycnotic nuclei dispersed among morphologically normal rd photoreceptors. DNA fragmentation into 200 base pair multiples occurs maximally in rd/rd retinas between 10 and 15 postnatal days. CONCLUSION: Photoreceptor cell death in developing rd/rd retinas occurs by a mechanism that links a defect in the phototransduction cascade with a program for cell death, called apoptosis.

Interaction of phosducin and phosducin isoforms with a 26S proteasomal subunit, SUG1.

PURPOSE: Retinal phosducin (Phd) and phosducin-like protein 1 (PhLP1) selectively bind G-protein beta/gamma subunits (Gbetagamma). Our laboratory has recently identified two phosducin-like orphan proteins (PhLOP1 and PhLOP2) that lack the ability to interact with Gbetagamma. In search of potential functional protein partner(s) for these phosducin orphans, we examined their protein-protein interactions using a yeast two-hybrid screen. METHODS: A bovine retina yeast expression cDNA library was screened with the GAL4 DNA binding domain (BD) fusion of PhLOP1. Quantitative analysis of the selected positives with PhLOP1 and other Phd isoforms was assessed by growth and beta-galactosidase activity. Further molecular, biochemical, and immunological detection methods utilizing glutathione S-transferase (GST)-Phd isoform fusion proteins and the potential partner were also performed. RESULTS: A member of the superfamily of putative ATPases was selected in the yeast two hybrid screen. Further characterization identified a direct interaction of this putative ATPase with PhLOP1, as well as Phd and PhLP1, but not with PhLOP2. A database search verified this ATPase as a bovine orthologue of the yeast SUG1 (ySUG1), a putative transcriptional mediator and a subunit of the 26S proteasome complex. Our experiments reveal that the carboxy-terminus of PhLOP1 is essential for the protein-protein interaction with SUG1, but it alone is not sufficient to mediate SUG1 interaction. CONCLUSIONS: Based on these experimental results, Phd, PhLP1 and PhLOP1 have protein-protein interaction with SUG1. PhLOP1, a truncated amino-terminal splice variant of Phd, is the best candidate for the interaction with SUG1 among the four Phd isoforms studied, which suggests a potential function for PhLOP1.

Identification of specific histidine residues and the carboxyl terminus are essential for serotonin N-acetyltransferase enzymatic activity.

Melatonin is synthesized in pinealocytes of the pineal gland and in photoreceptors of the retina. Synthesis rate from serotonin to melatonin is controlled by the rapid and dramatic enzymatic increase in darkness of serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AA-NAT, EC 2.3.1.87) and hydroxyindole-O-methyltransferase (HIOMT, EC 2.1.1.4). The primary structure of these critical indoleamine enzymes is now known and the regulation of the enzyme catalysis can be examined. As a first step, the conserved cysteine (C) and histidine (H) residues were targeted for site-directed mutagenesis as potential amino acid residues involved in the N-acetylation reaction of AA-NAT. Our studies concluded that among 6 histidine (H) to alanine (A) mutations, three residues (H110A, H118A, H120A) within the AA-NAT protein showed little or no enzymatic activity, whereas the others (H28A, H70A, H125A) retained enzymatic activity, compared to the unaltered AA-NAT protein. Cysteine to alanine mutations, C37A and C177A, had no significant effect on the AA-NAT enzymatic activity; however, C61A had a four-fold increase in K(m) for acetyl CoA and an altered sensitivity to the thiol modification chemical, N-ethylmaleimide (NEM), implying that C61 may participate in the acetyl CoA binding. Further studies examined the AA-NAT enzyme regulation of the highly conserved carboxyl terminus. When 12 terminal amino acid residues were deleted systematically from the carboxyl terminus of the 205 amino acid residue AA-NAT protein, enzyme activity was retained. However, further residue deletion resulted in enzyme activity plummeting, implicating that the essential information either for the correct structural folding into an active enzyme form or for enzyme stability is in the 193 residues. To test the relative importance of the AA-NAT carboxyl terminal region, a single leucine (L) was altered to alanine (A) or proline (P). Both mutants, either L193A or L193P, had a marked decrease in AA-NAT enzymatic activity and a decrease in thermal stability, suggesting the leucine, in addition to the cysteine and histidine residues, is involved in either enzyme catalysis or stability. In light of the recently reported three-dimensional structure of AA-NAT (17,18), the site-directed mutagenesis data demonstrate experimentally the importance of essential amino acid residues for acetyl CoA binding and AA-NAT activation.

Bovine arylalkylamine N-acetyltransferase activity correlated with mRNA expression in pineal and retina.

Arylalkylamine N-acetyltransferase (AA-NAT, E. C. 2.3.1.87) is the enzyme that catalyzes the transfer of an acetyl group from acetyl-CoA to serotonin to form N-acetylserotonin (NAS) in the indoleamine biosynthetic pathway. Bovine pineal AA-NAT, partially purified on an anion exchange column, displayed an 8-fold higher enzymatic activity in pineals from animals killed in early morning (0800) compared to an afternoon group (1430). Poly A(+) mRNA was isolated from early morning bovine pineals, used to construct a mammalian expression cDNA library (lambdaZAP Express), and then screened with a rat AA-NAT cDNA to isolate a 924 basepair cDNA that encodes the bovine pineal AA-NAT. The amino acid sequence alignment reveals that bovine AA-NAT shares 94.20%, 78.54%, 76.33% and 56.3% identity to ovine, rat, human and chicken sequences, respectively. Northern blot analysis demonstrates a 0.7-fold higher mRNA level in pineal glands taken from animals from the 0800 time-point compared with mRNA from the 1430 time-point. AA-NAT mRNA was expressed at high levels in pineal and retina, but the message was undetectable in adrenal, cerebellum, cortex, small intestine, testis and thyroid. Based on the significant identity of amino acid sequence and the similar mRNA expression pattern, these data suggest that the bovine AA-NAT is more analogous to the ovine rather than either the rat, human or chicken AA-NAT.

Circadian regulation of hydroxyindole-O-methyltransferase mRNA levels in rat pineal and retina.

Hydroxyindole-O-methyltransferase (HIOMT, EC 2.1.1.4) catalyzes the methylation of acetylserotonin to complete the synthesis of melatonin in the pineal and retina. A complete 1728 nucleotide cDNA encoding rat pineal HIOMT was isolated, characterized, and used to evaluate day/night levels of HIOMT mRNA. As previously reported for HIOMT enzyme activity, HIOMT mRNA levels were also greater in the pineal than in the retina. Northern blot analysis and in situ hybridization were useful for detection of HIOMT mRNA in the pineal but not the retina, whereas the reverse transcriptase-polymerase chain reaction or RNase protection assay revealed transcripts for HIOMT both in the pineal and retina. Investigating HIOMT mRNA levels in rat pineal and retina at 6 time-points throughout a 24 h period revealed higher levels of HIOMT message during darkness. The daily fluctuation in HIOMT mRNA persisted in constant darkness, verifying an endogenous circadian rhythm both in the pineal and retina. In mammalian pineals, sympathetic innervation, synthesizing norepinephrine that activates beta (beta) adrenergic receptors, entrain several circadian bodily functions through the synthesis and release of melatonin. A single injection of the beta-adrenergic agonist, isoproterenol, induced a dramatic increase of HIOMT mRNA levels in the light-adapted pineal, in vivo. Moreover, a single injection of the beta-adrenergic antagonist, propranolol, prevented the nocturnal increase of pineal HIOMT mRNA. Using a combination of methods, it has been shown that the level of HIOMT mRNA fluctuates daily in both the pineal gland and retina. This day/night rhythm can be modulated either by beta receptor agonists or antagonists when applied appropriately during the circadian cycle, suggesting that the mRNA changes in HIOMT may be controlled at the transcriptional level.

Hydroxyindole-O-methyltransferase is another target for L-glutamate-evoked inhibition of melatonin synthesis in rat pinealocytes.

Rat pinealocytes use L-glutamate as a modulator for melatonin synthesis. Upon binding of L-glutamate to the class II metabotropic glutamate receptor, norepinephrine (NE)-dependent formation of cAMP was inhibited, resulting in decreased serotonin-N-acetyltransferase (NAT) activity and melatonin output. Although L-glutamate at 1 mM caused 90% inhibition of melatonin synthesis, about 30% of the NAT activity remained, suggesting the presence of another target for L-glutamate. In this study, we found that L-glutamate also inhibits hydroxyindole-O-methyltransferase (HIOMT). The inhibition is reversible and dose-dependent: the maximal inhibition was obtained with more than 0.4 mM L-glutamate. Contrary to L-glutamate-evoked inhibition of NAT, agonists for class II metabotropic receptors such as (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (DCG IV) had no effect on HIOMT. Neither (2S,3S,4S)-2-methyl-2-(carboxycyclopropyl)glycine (MCCG), an specific antagonist for class II mGluRs, nor dibutyryl cAMP restored the L-glutamate-evoked inhibition of HIOMT. Northern blot analyses revealed that L-glutamate significantly inhibits the expression of mRNA of NAT, but not that of HIOMT. These results indicated that HIOMT is an another target for L-glutamate due to its inhibition of melatonin synthesis, and the signaling pathway toward the inhibition is distinct from that of NAT.

Localization of mRNA encoding the indolamine synthesizing enzyme, hydroxyindole-O-methyltransferase, in chicken pineal gland and retina by in situ hybridization.

Hydroxyindole-O-methyltransferase (HIOMT) is the enzyme that catalyzes the final step in the synthesis of the hormone melatonin. We have examined the localization of expression of the mRNA encoding HIOMT by in situ hybridization in the 3-day-old and adult chicken retina and pineal gland. The riboprobe utilized for this study was transcribed from the complete coding region of HIOMT cDNA synthesized from retina RNA and amplified by polymerase chain reaction (PCR). High levels of HIOMT mRNA were present in the pinealocytes of the pineal gland. In the retina, the hybridization signal was localized to the photoreceptors. The retinal photoreceptors of the 3-day-old chick displayed a much lower level of hybridization than did the photoreceptors of the adult chicken. This study strongly suggests that the photoreceptors are the sites of melatonin synthesis in the retina.

Identification of circadian gene expression in the rat pineal gland and retina by mRNA differential display.

To identify unique gene products that could be crucial for circadian and light-dark regulations, using the technology of differential display, we have investigated mRNAs that were isolated from rat pineals and retinas collected either during subjective day (D) or subjective night (N). A subpopulation of about 50 mRNAs were amplified from pineal using designed primers and reverse transcriptase-polymerase chain reaction. Thirty five of the mRNAs were expressed equivalently in both D and N samples. From the 15 mRNAs showing differential expression, four amplified products were selected, based on higher expression during the subjective night (N310, N320, N383, N420) and each was subcloned and sequenced. Of the four cDNAs studied, only N310 was determined to have significant identity to a known cell adhesion protein, called F3. This protein is highly expressed in developing mouse neurons and is related to chicken contactin. Each differentially displayed product identified is being examined further in order to understand their potential significance in the circadian regulation of pineal and retinal activities.

Derivatives of [3H]serotonin in organ cultures of hamster pineal gland.

The purpose of this study was to determine the viability of the hamster pineal gland in organ culture and to test the effect of norepinephrine (NE) on [3H]serotonin derivatives. In this study, elevated levels of melatonin (7-fold, p less than .05), 5- hydroxytrytophol (5-fold, p less than .001), 5-methoxytryptophol (1.78-fold, p less than .05), and depressed levels of 5-hydroxyindoleacetic acid (3.8-fold, p less than .02) and methoxyindoleacetic acid (1.78-fold, p less than .05) were detected in the glands following the addition of NE to the medium. In a separate experiment, melatonin concentration in the media was also periodically measured by radioimmunoassay to determine the viability of the organ culture over a four-day period. The melatonin level on day 2 (2321 +/- 106 pg/gland) was significantly higher (p less than 0.01) than on day 3 (1542 +/- 86 pg/gland) or day 4 (805 +/- 39 pg/gland). The results of these experiments verify the viability of the hamster pineal organ culture and show that the gland responds to NE in vitro.

Decreases in pineal melatonin content during the hibernation bout in the golden-mantled group squirrel, Spermophilus lateralis.

The role of the pineal gland in modulating the rhythmic bouts of hibernation in the golden-mantled ground squirrel (S. lateralis) was explored by comparing pineal melatonin content in hibernating animals with that of euthermic animals at the same time of year. Significant decreases in pineal melatonin content were found in hibernating versus euthermic animals. In addition, significantly lower values for pineal melatonin were observed in hibernating animals that were sacrificed in the late bout period, just prior to expected spontaneous arousal, as compared to hibernating animals that were sacrificed on the first day of their respective bouts. A strong correlation was evident between pineal melatonin content and the duration of the individual hibernation bout. These data suggest that pineal melatonin may be important in determining the duration of individual bouts of hibernation in this species.

Microbial Properties of Composts That Suppress Damping-Off and Root Rot of Creeping Bentgrass Caused by Pythium graminicola.

Composts prepared from a variety of feedstocks were tested for their ability to suppress seedling and root diseases of creeping bentgrass caused by Pythium graminicola. Among the most suppressive materials in laboratory experiments were different batches of a brewery sludge compost and a biosolids compost from Endicott, N.Y. Batches of these composts that were initially not suppressive to Pythium damping-off became more suppressive with increasing compost age. Leaf, yard waste, food, and spent mushroom composts as well as certain biosolids, cow manure, chicken-cow manure, and leaf-chicken manure composts were not suppressive to Pythium damping-off. In some cases, turkey litter, chicken manure, chicken-leaf, and food waste composts were inhibitory to creeping bentgrass seed germination in laboratory experiments. Microbial populations varied among all of the composts tested. Bacterial populations were high in all composts except the turkey litter compost, in which populations were 1,000- to 10,000-fold lower than in the other composts tested. Among the highest populations of heterotrophic fungi and antibiotic-producing actinomycetes were those found in all batches of the brewery sludge compost, whereas the lowest populations were found in turkey litter, chicken manure, and food waste composts. Heat treatment of suppressive composts reduced populations of bacteria, fungi, and actinomycetes in all composts tested. Disease suppressiveness was also reduced or eliminated in heated composts. Amending heated composts with small amounts of nonheated compost restored suppressive properties and partially restored microbial populations to wild-type levels. A strong negative relationship between compost microbial activity (as measured by the hydrolysis of fluorescein diacetate) and Pythium damping-off severity was observed. When composts were applied to creeping bentgrass in field experiments, a significant level of suppressiveness was evident with some composts when disease pressure was high (i.e., disease ratings high in uninoculated plots). A 1991 batch of turkey litter compost and the 1990 batch of Endicott biosolids were consistently suppressive to foliar symptoms of Pythium root rot on creeping bentgrass. This study indicates that suppression of Pythium diseases of creeping bentgrass in batches of brewery sludge and Endicott biosolids composts, and possibly in other suppressive composts examined in less detail in this study, is related directly to the microbial activities in the composts. On the other hand, the mechanisms of Pythium suppression in turkey litter and perhaps other poultry-based composts is not related directly to the compost microbial activity. Although turkey litter showed a lack of suppressiveness in laboratory bioassays and low microbial populations and activity, it resulted in a significant and consistent level of suppressiveness in field experiments. Therefore, the microbiological properties of Pythium-suppressive composts may differ substantially, and measurements of microbial populations and activity may not be predictive of the level of disease suppression in all composts.

The carboxyl terminal domain of phosducin functions as a transcriptional activator.

In previous work, we identified a set of phosducin (Phd) isoforms with unknown function including the phosducin (Phd)-like orphan protein 1 (PhLOP1), an amino terminal truncated isoform of the retinal Phd lacking the Gbetagamma binding domain. To investigate the potential biological function of PhLOP1, PhLOP1 was fused at its amino terminus with the DNA binding domain (BD) of the yeast transcriptional factor, GAL4, and used as bait in a yeast two-hybrid screen. Two potential functional protein partners were identified during the screen: SUG1, a subunit of the 26S proteasome and a putative transcriptional mediator, and CRX, a retina- and pineal-specific transcription factor. Upon localizing the interacting domain of PhLOP1 with one of the new partners, SUG1, we found that a domain of 40 amino acids at the carboxyl terminus of Phd and PhLOP1 had intrinsic transcriptional activation activity in yeast. The transactivation activity was further confirmed in mammalian cells. This region contains an acidic domain that has been shown to be involved in the function of several transcriptional activators. In addition, we showed that Phd is cytoplasmic while PhLOP1 is localized predominantly to the nucleus when fused to an enhanced green fluorescent protein (EGFP) and transiently expressed in transfected cells, suggesting that PhLOP1 may play a distinct functional role in transcriptional regulation independent of the known Phd interaction/regulation of Gbetagamma transduction.

Biochemical characterization of recombinant serotonin N-acetyltransferase.

Pineal and retinal melatonin synthesis is controlled by the enzymatic activity of arylalkylamine N-acetyltransferase (AA-NAT, EC 2.3.1.87), which is regulated by light/dark signals and circadian factors. This enzyme converts serotonin to N-acetylserotonin by the transfer of an acetyl group from acetyl coenzyme A. Endogenous AA-NAT instability during routine purification has made enzyme characterization difficult, but now a stable recombinant protein for AA-NAT has been synthesized to investigate the intrinsic biochemical properties of AA-NAT from a rat pineal cDNA encoding a 205 amino acid, 23 kilodalton protein, by using a glutathione-S-transferase (GST) fusion protein system. Recombinant GST-AA-NAT showed substrate specificity for arylalkylamines and stability at 4 degrees C; however, the enzyme activity was reduced by 40% upon preincubation at 37 degrees C for 2 hr. GST-AA-NAT is preferentially phosphorylated by either cyclic AMP- or cyclic GMP-dependent kinases in vitro, but no detrimental effect was observed on AA-NAT enzymatic activity. Among the metal cations tested in this study, Ca2+, Mg2+, Mn2+, Fe2+, and Co2 showed little or no inhibitory potency, while either 1 mM Zn2+ or 0.1 mM Cu2+ nearly abolished the enzymatic activity. GST-AA-NAT enzyme activity is also inhibited by reagents that are known biochemically to modify thiol groups (N-ethylmaleimide, NEM) and histidine residues (p-chloromercuribenzoate, NBS and diethyl pyrocarbonate, DEPC), suggesting the presence of essential cysteine and histidine moieties. Moreover, preincubation of acetyl CoA completely protects the recombinant AA-NAT from inactivation by NEM and DEPC, indicating that specific cysteine and histidine residues may be at the acetylation site. The conclusion is that the biochemical properties of rat recombinant AA-NAT is similar to the endogenous pineal and retinal AA-NAT with respect to the sensitivity to temperature, metal cations, as well as the thiol modification reagents. These data also suggest that the phosphorylation status of the AA-NAT does not affect enzymatic activity directly, and histidine residues are potentially important residues required for high catalytic activity.

Differential expression of mRNA and protein encoding retinal and pineal S-antigen during the light/dark cycle.

S-Antigen is a soluble cell protein unique to the retina and pineal gland. In the former, it is a well-characterized molecule that participates in light-induced signal transduction in photoreceptor cells. In the latter, the functional role is presently not known. The expression of S-antigen and its mRNA was examined in the rat retina and pineal gland throughout the diurnal cycle and with light interruption of the dark cycle. A cDNA for rat S-antigen was isolated from a pineal gland library to examine the mRNAs. A 1.7-kb mRNA for S-antigen was observed in both the pineal gland and the retina. Retinal S-antigen mRNA was expressed throughout the diurnal cycle and increased with light interruption of the dark cycle. In contrast, pineal gland S-antigen mRNA levels were detectable only during the dark and were absent preceding and during light. The phenotypic expression of immunoreactive S-antigen, identified with two S-antigen monoclonal antibodies (MAbs), MAb A9C6 and MAb C10C10, was analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel (PAGE) and isoelectric focusing (IEF) electrophoresis. Immunoblot analysis of gels after SDS-PAGE revealed a single 46-kDa protein in retina. In contrast, two bands of approximately 43 and 46 kDa were identified in the pineal gland. Immunoblots of the retinal extracts separated by IEF electrophoresis revealed five S-antigen isomers, which vary quantitatively throughout the diurnal cycle and when light interrupted the dark cycle. Immunoblots of the pineal gland samples separated by IEF electrophoresis indicated that the pineal gland possesses four pineal gland-specific forms of S-antigen in addition to the five forms present in the retina. The differences observed in the mRNA and protein analyses suggest tissue-specific structural components for S-antigen in the retina and pineal gland that are not regulated in the same manner.

Photoreceptors of the retina and pinealocytes of the pineal gland share common components of signal transduction.

Light absorbed by retinal photoreceptors triggers a cascade of reactions that initiate cGMP hydrolysis, cation channel closure and membrane hyperpolarization. Down-regulation of the cascade involves additional proteins that interfere with amplification along the cascade. Pinealocytes are activated by norepinephrine during the dark phase of the day/night cycle. Mature pinealocytes of the mammalian pineal express the known photoreceptor proteins that are implicated in down-regulation of the visual cascade, but the cascade components that produce cGMP hydrolysis and membrane hyperpolarization are absent. Pinealocytes accumulate cyclic AMP minimally when norepinephrine activates their beta adrenergic receptors alone, but the response is potentiated by the simultaneous activation of their alpha-1 adrenergic receptors. A model is proposed whereby phosducin, a phosphoprotein that binds the beta,gamma subunit of G-proteins, could modulate the synthesis of cyclic AMP by buffering the amount of beta,gamma G-protein subunits that are available for activating adenylate cyclase.