Adenosine A2A receptor gene disruption provokes marked changes in melanocortin content and pro-opiomelanocortin gene expression.

A2A receptor knockout (A2AR-/-) mice are more anxious and aggressive, and exhibit reduced exploratory activity than their wild-type littermates (A2AR+/+). Because alpha-melanocyte-stimulating hormone (alpha-MSH) influences anxiety, aggressiveness and motor activity, we investigated the effect of A2AR gene disruption on alpha-MSH content in discrete brain regions and pro-opiomelanocortin (POMC) expression in the hypothalamus and pituitary. No modification in alpha-MSH content was observed in the hypothalamus and medulla oblongata where POMC-expressing perikarya are located. In the arcuate nucleus of the hypothalamus, POMC mRNA levels were not affected by A2AR disruption. Conversely, in A2AR-/- mice, a significant increase in alpha-MSH content was observed in the amygdala and cerebral cortex, two regions that are innervated by POMC terminals. In the pars intermedia of the pituitary, A2AR disruption provoked a significant reduction of POMC mRNA expression associated with a decrease in alpha-MSH content. By contrast, in the anterior lobe of the pituitary, a substantial increase in POMC mRNA and adrenocorticotropin hormone concentrations was observed, and plasma corticosterone concentration was significantly higher in A2AR-/- mice, revealing hyperactivity of their pituitary-adrenocortical axis. Together, these results suggest that adenosine, acting through A2A receptors, may modulate the release of alpha-MSH in the cerebral cortex and amygdala. The data also indicate that A2A receptors are involved in the control of POMC gene expression and biosynthesis of POMC-derived peptides in pituitary melanotrophs and corticotrophs.

Constitutively active mutants of 5-HT4 receptors are they in unique active states?

Somatic mutations leading to constitutively active G-protein coupled receptors (GPCRs) are responsible for certain human diseases. A consistent structural description of the molecular change underlying the conversion of GPCRs from an inactive R state to an active R* state is lacking. Here, we show that a series of constitutively active 5-HT4 receptors (mutated or truncated in the C-terminal and the third intracellular loop) were characterized by an increase in their denaturation rate at 55 degrees C. The thermal denaturation kinetics were monophasic, suggesting that we were measuring mainly the denaturation rate of R*. Analysis of these kinetics revealed that constitutively active C-terminal domain mutants, were due to a change in the J constant governing the R/R* equilibrium. However, the constitutive activity of the receptor mutated within the third intracellular loop was the result of both a change in the allosteric J constant and a change in the R* conformation.

Cloning of a human purinergic P2Y receptor coupled to phospholipase C and adenylyl cyclase.

Clones encoding a new human P2Y receptor, provisionally called P2Y11, have been isolated from human placenta complementary DNA and genomic DNA libraries. The 1113-base pair open reading frame is interrupted by one intron. The P2Y11 receptor is characterized by considerably larger second and third extracellular loops than the subtypes described so far. The deduced amino acid sequence exhibits 33% amino acid identity with the P2Y1 receptor, its closest homolog. Northern blot analysis detected human P2Y11 receptor messenger RNA in spleen and HL-60 cells. The level of P2Y11 transcripts was strongly increased in these cells after granulocyte differentiation induced by retinoic acid or dimethyl sulfoxide. The new receptor was stably expressed in 1321N1 astrocytoma and CHO-K1 cells, where it couples to the stimulation of both the phosphoinositide and adenylyl cyclase pathways, a unique feature among the P2Y family. The rank order of agonists potency was: ATP > 2-methylthio-ATP >>> ADP, whereas UTP and UDP were inactive, indicating that it behaves as a selective purinoceptor.

Coupling of metabotropic glutamate receptors 2 and 4 to G alpha 15, G alpha 16, and chimeric G alpha q/i proteins: characterization of new antagonists.

Together with the calcium-sensing receptor, the metabotropic glutamate receptors (mGluRs) share no sequence homology with the other G protein-coupled receptors (GPCRs) and therefore constitute a new family of receptors. Recently, it was reported that G alpha 15 and G alpha 16 subunits allow many GPCRs to activate phospholipase C (PLC). Furthermore, the exchange of a few carboxyl-terminal residues of G alpha q by those of G alpha 12 or G alpha o allows the resulting chimeric G alpha subunits (G alpha ql and G alpha qol respectively) to couple Gi-coupled receptors to PLC. We report that mGluR2 and mGluR4, two receptors negatively coupled to adenylyl cyclase, activate PLC when coexpressed with G alpha 15, G alpha ql or G alpha qo. This indicates that the carboxyl-terminal end of the G alpha subunit also plays an important role in the specific interaction between mGluRs and the G proteins. In addition, the measurement of PLC activation by Gi-coupled mGluRs coexpressed with these G alpha subunits constitutes an easy functional assay for the pharmacological characterization of these receptors. The rank order of potency of antagonists was found to be (2S,3S,4S)-2-methyl-2-(carboxycyclopropyl)glycine approximately (R,S)- alpha-methyl-4-phosphonophenylglycine > (R,S)-alpha-methyl-4-sulfonophenylglycine > (R,S)-alpha-methyl-4-tetrazolylphenylglycine = (S)-2-amino-2-methyl-4-phosphonobutyrate for mGluR2 and to be (R,S)-alpha-methyl-4-phosphonophenylglycine > or = (S)-2-amino-2-methyl-4-phosphonobutyrate > > (R,S)-alpha-methyl-4-sulfonophenylglycine [(R,S)-alpha-methyl-4-tetrazolylphenylglycine and (2S,3S,4S)-2-methyl-2-(carboxycyclopropyl)glycine being inactive at 1 mM] for mGluR4. Using this functional assay, (R,S)-alpha-methyl-4-phosphonophenylglycine was found to have a similar KB value for mGluR2 and mGluR4.

Cloning, functional expression and tissue distribution of the human P2Y6 receptor.

In order to isolate new subtypes of P2Y purinoceptors, a human placenta cDNA library was screened at middle stringency with a P2Y4 probe. The purification and the sequencing of several clones led us to identify a 984 base pair open reading frame encoding a new human P2Y receptor. It appeared later that this sequence corresponds to the human ortholog (88% amino acid identity) of the rat receptor recently cloned by Chang et al (J. Biol. Chem. 270, 26152-26158, 1995) and called P2Y6. Northern blot analysis detected human P2Y6 receptor messenger RNA in human spleen, placenta, thymus, intestine, and blood leukocytes. In 1321N1 cells stably expressing the human P2Y6 receptor, the formation of IP3 was stimulated by nucleotides with the following order of potency: UDP > 5-bromo-UPT > UTP > ADP > 2-methylthio-ATP >> ATP. The P2Y6 receptor, together with the previously cloned P2Y4 subtype (Communi et al., J. Biol. Chem., 270, 30849-30852, 1995), belongs thus to a subfamily of pyrimidinoceptors inside the P2Y family.

Human 27-kDa calbindin complementary DNA sequence. Evolutionary and functional implications.

Human 27-kDa calbindin cDNA clones were selected by antibody screening from lambda gt11 brain libraries. The sequence revealed an open reading frame coding for a protein of 261 amino acids, containing four active calcium-binding domains, and two modified domains that had presumably lost their calcium-binding capability. Comparison with chick and bovine calbindins showed that the protein was highly conserved in evolution (evolutionary rate: 0.3 x 10(-9) amino acid-1 year-1) and that active and inactive domains were equally conserved. From the data we postulate that calbindin has an important physiological function involving protein--protein interactions. Comparison of calcium-binding domains from various proteins suggested that all members of the troponin C superfamily derive from a common two-domained ancestor, but that duplications leading to calbindin and to the four-domained calcium-binding proteins took place independently on different branches of the evolutionary tree. Preliminary data showed that another calcium-binding protein, homologous to calbindin, is present in the brain and encoded by a different gene.

Compared localizations of prolactin-like and adrenocorticotropin immunoreactivities within the brain of the rat.

By an immunoperoxidase (PAP) technique, ACTH- and prolactin-like substances were detected in neurons of several hypothalamic nuclei in young and adult rats. Neurons in the arcuate and ventromedial nuclei were simultaneously both ACTH-like and PRL-like positive. Labelled fibres were followed to their fields of projections. Intensity of labelling was not influenced by experimental procedures known to change PRL and ACTH content of the pituitary. Colchicine enhanced labelling of perikarya while lowering that of fibres. The results support neuronal synthesis of hormonal ACTH and PRL related molecules and their involvement in the control of autonomic nerve functions.