Extracellular signal-regulated kinases (ERK1 and 2) are synaptic signaling components necessary for several forms of learning. In mice lacking ERK1, we observe a dramatic enhancement of striatum-dependent long-term memory, which correlates with a facilitation of long-term potentiation in the nucleus accumbens. At the cellular level, we find that ablation of ERK1 results in a stimulus-dependent increase of ERK2 signaling, likely due to its enhanced interaction with the upstream kinase MEK. Consistently, such activity change is responsible for the hypersensitivity of ERK1 mutant mice to the rewarding properties of morphine. Our results reveal an unexpected complexity of ERK-dependent signaling in the brain and a critical regulatory role for ERK1 in the long-term adaptive changes underlying striatum-dependent behavioral plasticity and drug addiction.

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1 Mouse ®broblasts (NIH3T3) transfected with the full-length coding region of the Mel 1a melatonin receptor stably expressed the receptor, coupled to a pertussis toxin-sensitive G-protein(s) and exhibiting high a nity and adequate pharmacological pro®le. 2 The receptor protein had the tendency of a strong coupling to the G-protein and therefore lowa nity state was induced by uncoupling the receptor from its G-protein in presence of high concentrations of NaCl (500 ± 700 mM) and/or GTPgS (100 mM). Thereafter, the a nity of a series of melatonin analogues was determined to both, high-and low-a nity receptor states, thus providing a basis for the prediction of their e cacy, according to the ternary complex model. 3 The cells were subsequently used to study the agonist-induced G-protein activation, determined by calculating the rate of GDP-GTP exchange measured in presence of 35 S-labelled GTPgS. The natural ligand melatonin induced a signi®cant increase in the GDP-GTP exchange rate, the presence of GDP and NaCl being necessary to observe this e ect. 4 The full agonists 2-phenylmelatonin, 2-bromomelatonin and 6-chloromelatonin equally induced an increase of the GDP-GTP exchange. 5-Hydroxy-N-acetyltryptamine activated the GTP-GDP exchange to a much lesser extent (53%) than melatonin, thus behaving as a partial agonist. As predicted by the model, the melatonin antagonist (N-[(2-phenyl-1H-indol-3-yl)ethyl]cyclobutanecarboxamide) was without e ect on basal G protein activation. Coincubation of this compound with melatonin induced a dosedependent rightward shift in the melatonin concentration-e ect curve, thus exhibiting the behaviour of a competitive and surmountable antagonist. 5 Using the equation proposed by Venter (1997) we were able to determine that there were no`spare' receptors in the system. Therefore, the approach proposed in the present work can be successfully used for the determination of`drug action' at the level of the human Mel 1a melatonin receptor and evaluation of the e cacy of new selective melatonin analogues.

Ras-related guanosine triphosphatases (GTPases) couple receptor activity to a number of intracellular signalling events culminating in the control of cell morphology and gene transcription. In culture cells, the best-understood Ras-dependent signalling pathway involves the mitogen-activated protein kinase/extracellular-regulated kinase (MAPK/ERK) cascade. A growing body of evidence has recently been accumulating to suggest a crucial role of Ras and MAPK signalling in neuronal functions connected to synaptic plasticity. In the present review article we discuss the experimental basis supporting the notion that the Ras/MAPK pathway interacts with other synaptic mechanisms to regulate invertebrate and vertebrate behavioural responses such as those implicated in learning and memory processes.

Naturally occurring nondeletional mutations affecting the distal CCAAT box of the human ␥-globin gene promoter result in hereditary persistence of fetal hemoglobin in adult life. Although the distal CCAAT box is the target of several factors, including CP1/NFY, CDP, GATA-1 and NFE3, only NFE3 binding activity is consistently sensitive to well characterized mutations in this region such as G ؊117 3 A, C ؊114 3 T, and ⌬13 hereditary persistence of fetal hemoglobin. We extensively characterized the binding specificities of NFE3 and demonstrated that NFE3 has unique properties with respect to other CCAAT box-binding proteins. Affinity-purified NFE3 from erythroid K562 cells binds the distal but not the proximal human ␥-globin CCAAT box, the single CCAAT box of the human ⑀-globin promoter, and the proximal CCAAT box of the evolutionarily related Galago crassicaudatus ␥-globin gene. Within the ⑀-globin CCAAT box, NFE3 represents the major and almost exclusive binding activity. Disruption of such a binding site essentially inactivates the ⑀-globin promoter, suggesting that NFE3 plays an important role in the embryonic expression of this gene.In humans, different types of hemoglobins are produced during the embryonic, fetal, and adult stages (1). The first hemoglobin switch occurs early in gestation and involves the substitution of ␣-and ␥-globin for -and ⑀-globin, respectively. At birth, ␥-globin synthesis is almost completely switched off and is replaced by the synthesis of the major ␤-globin and the minor ␦-globin chains. Functional studies of the ␤-like globin cluster highlighted the role of the upstream locus control region in promoting high level erythroid-specific expression of the globin genes (2). However, temporal regulation of the expression of various globin genes, in particular ⑀-and ␥-globin, appears to be largely dependent on sequences comprised within the genes themselves or in their immediate vicinity (3, 4).Several different approaches have been taken to analyze functional elements in the promoters of the human globin genes. Evolutionarily conserved DNA motifs were identified and provided clues to the discovery of DNA binding motifs (5-7). Deletion and site-directed mutagenesis also identified potentially important motifs (8 -10).Finally, the existence of natural mutants within the human population provided in vivo evidence for the role of specific sequences (reviewed in Refs. 11 and 12). In particular, ␤-globin promoter mutations (␤-thalassemia) demonstrated an important role of two conserved motifs, the TATA and the CACC box, in promoter function; surprisingly, no mutations have so far been identified in another highly conserved element, the CCAAT box. However, although these mutations significantly decrease ␤-globin expression, they do not appear to modify its temporal regulation.On the other hand, mutations within the promoter of the fetal ␥-globin gene do increase, often substantially, its postnatal and adult expression. Some of these mutations have been suggested to create better binding sites fo...

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The CREM gene encodes both repressors and activators of cAMP-dependent transcription in a tissue and developmentally regulated manner. In addition, multiple and cooperative phosphorylation events regulate the function of the CREM proteins. CREM plays a key physiological and developmental role within the hypothalamic-pituitary axis. There is a functional switch in CREM expression during the development of male germ cells which is directed by the pituitary hormone FSH. The CREM protein in germ cells is a powerful activator which appears to function as a master-switch in the regulation of postmeiotic genes. CREM is inducible by activation of the cAMP signalling pathway with the kinetics of an early response gene. The induction is transient, cell-specific, does not involve increased transcript stability and does not require protein synthesis. The subsequent decline in CREM expression requires de novo protein synthesis. The induced transcript encodes ICER and is generated from an alternative, intronic promoter. ICER functions as a powerful repressor of cAMP-induced transcription, and represses the activity of its own promoter, thus constituting a negative autoregulatory loop.

Reverse transcription-polymerase chain reaction (RT-PCR) analysis performed on total RNA from different tissues of Xenopus laevis showed that the melatonin receptor gene cloned from dermal melanophores is expressed in the whole brain, skin, and retina, and that apart from the ovary, there is no expression in tissues having origin outside the central nervous system. Comparative studies using in vitro autoradiography and in situ hybridization demonstrated that the melatonin receptor is expressed with discrete allocation in Xenopus brain. Though the distribution pattern of the specific messenger RNA conforms well with that of the corresponding receptor protein, it is not always coincident.