Signal transduction in the pineal gland is studied. The primary focus is on the biochemistry and molecular biology of first enzyme in the serotonin -> melatonin pathway, serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AANAT, EC 2.3.1.87). Recent microarray efforts have identified other gene products of interest which are also being investigated because of dynamic night/day changes in expression they exhibit - similar to the large changes seen in AANAT. REGULATION OF AANAT: AANAT is important because it regulates large changes in the production of melatonin, which occur in all vertebrates at night. As such, AANAT plays an essential role in vertebrate physiology in converting night into a circulating signal - melatonin. High levels of melatonin = darkness at night. Melatonin is also synthesized in the retinae of some, but not all vertebrates. The formation of melatonin in the retina is also thought to enhance dark adaptation. However, primate retinae do not express the second enzyme in melatonin synthesis, hydroxyindole-O-methyltransferase. In these tissues, the high levels of AANAT appear to play a different role, which may also occur in all vertebrates - protection by detoxification of arylalkylamines, thereby preventing the accumulation of the adduct product formed by reaction of serotonin and other arylalkylamines with retinaldehyde, the essential molecule of the visual cycle. The products - described as bis-retinyl arylalkylamines (A2-AAs) are likely to be detrimental for two reasons - depletion of retinaldehyde and reactivity A2-AAs with macromolecules in the retina, leading to cell death. The genetic code of AANAT in several vertebrate classes has been determined and is being used to study the regulation of mRNA encoding the enzyme and to generate proteins and peptides to use as antigens to raise antisera against the protein. Using this information, it has been found that transcriptional mechanisms play a regulatory role in controlling melatonin production in some but not all species. However, in all species, it is becoming clear that a common mechanism regulating AANAT activity is second messenger control of proteasomal proteolysis. This mechanism appears to play a central role in producing the rapid light induced decrease in AANAT activity and melatonin production. X-Ray crystallographic analysis has established the 3-D structure of an active form of AANAT. Studies on the rat, chicken and zebra fish promoter has made it possible to identify elements involved in the tissue-specific expression of AANAT. With this information, it has been possible to construct a rat and zebra fish transgenic animals that express reporters selectively in the pineal gland under the control of the AANAT promoter A major step forward has been the development of a new cell line expressing human AANAT. This has had a major impact on research, with the most important advance being evidence indicating a new level of regulation of AANAT activity - through an activation/inactivation mechanism. Recent work has resulted in the development of AANAT cell lines, which have provided a new tool to study regulation of the enzyme. Using this, it has been discovered that cAMP can modulate the activity of AANAT, without changing AANAT protein levels. Perhaps the most important recent advance in understanding the role AANAT plays in the regulation of melatonin synthesis is the discovery that AANAT exists in the cell in a complex composed of at least one phosphorylated AANAT molecule and two molecules of 14-3-3 protein. The complex has been characterized and crystallized. Analysis reveals the complex is held together by many interactions, but the key one - the switch - is a set of interactions involving phosphorylation of two residues in AANAT T31 and S205. Binding via both sites is required for activation of the enzyme, apparently by restricting the mobility of a highly mobile protein loop that completes the serotonin binding pocket. GLOBAL ANALYSIS OF GENE EXPRESSION: Recent work on global analysis of gene expression in the rat, human and monkey pineal glands, using microarray analysis has reveal global similarities in genes expressed in these tissues, most notably those dedicated specifically to melatonin synthesis (i.e. tryptophan hydroxylase, AANAT and hydroxyindole-O-methyltransferase), those required for synthesis of cofactors AcCoA and S-adenosylmethionine, and signal transduction genes linked to phototransduction (Arrestin, phosducin, retinaldehyde binding proteins) reflecting the primitive photodetector as the common origin of the retinal photoreceptor and the pineal gland. In addition to these similarities, there are species specific differences in the genes expressed and in rhythmic gene expression: the rat exhibits large changes in expression of many genes whereas primates do not. This effort has stimulated in depth analyses of the expression of several genes in the pineal gland, which have not been studied. These include two which are involved in synthesis of cofactors (methionine adenosyltransferase and ATP dependent citrate lyase/AcCoA synthetase) and one which is involved in the MAP signaling pathway (MAP kinase phosphatase). In the case of most genes, it appears they are regulated in a manner generally similar to that of AANAT, involving adrenergic control of cAMP and cAMP control of gene expression. There is an overarching effort to determine the nature of the signaling system through which adrenergic activation and second messengers control gene expression. This is thought to involve a cascade of transcription factors, some of which are known to be controlled by cAMP. In addition, there is a dedication to determining the specific transcription factor code which control pineal specific expression. This work will draw on microarray analysis of gene expression in mammals and in zebra fish; it will be dedicated to the identification of highly conserved expression mechanisms, i.e. regulatory elements and transcription factors. Initial analysis of the rat and zebra fish AANAT promoters, as described above, have identified common elements which are found in the promoters of other pineal-specific genes. These are E-boxes, photoreceptor conserved elements and cAMP response elements. The transcription factors conferring pineal specificity include members of the CRX/OTX family; in addition, other candidates are being investigated. Many similarities exist between gene expression in the pinealocyte and retinal photoreceptor and attention is being directed to the basis of differences. EVOLUTION OF THE PINEAL GLAND: The PI has developed a revolutionary theory to explain the evolution of the pineal gland, based on evidence that both the pinealocyte and the retinal photoreceptor evolved from a common cell. It is proposed that melatonin synthesis was 'acquired' early in chordate evolution by lateral gene transfer from bacteria. The initial selective advantage of this was detoxification. A night/day rhythm in detoxification evolved so that retinaldehyde would be protected when it was most needed - at night. This led to the association of melatonin with darkness. The evolution of robust melatonin synthesis conflicted with the evolution of enhanced photosensitivity, because high levels of serotonin increased A2AA formation. This was resolved by the evolution of the common ancestral photoreceptor to a melatonin factory - the pinealocyte ? and to the retinal photoreceptor. The high levels of AANAT in the primate retina ? in the absence of melatonin synthesis - may reflect a detoxification function of the enzyme ? and a role of AANAT in preventing blindness due to A2-AA dependent macular degeneration. This issue is being strongly pursued by the Section.
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