G proteins are involved in cellular signalling and regulate a variety of biological processes including differentiation and development. We have generated mice deficient for the G protein subunit alpha i2 (G alpha i2) by homologous recombination in embryonic stem cells. G alpha i2-deficient mice display growth retardation and develop a lethal diffuse colitis with clinical and histopathological features closely resembling ulcerative colitis in humans, including the development of adenocarcinoma of the colon. Prior to clinical symptoms, the mice show profound alterations in thymocyte maturation and function. The study of these animals should provide important insights into the pathogenesis of ulcerative colitis as well as carcinogenesis.

Antidiuresis, the recovery of water from the lumen of the renal collecting tubule, is regulated by the hypothalamic release of antidiuretic hormone (ADH), which binds to specific receptors on renal collecting tubule cells, stimulates adenylyl cyclase and promotes the cyclic AMP-mediated incorporation of water pores into the luminal surface of these cells. We report here the isolation of the human ADH receptor gene using a genomic expression cloning approach. The gene was used to clone the complementary DNA from a human renal library. The deduced amino-acid sequence of the receptor yields a hydropathy profile characteristic of receptors with seven putative transmembrane regions. This and the comparison with other cloned receptors indicates that the ADH receptor is a member of the superfamily of G-protein-coupled receptors.

The G protein G o is highly expressed in neurons and mediates effects of a group of rhodopsin-like receptors that includes the opioid, ␣ 2 -adrenergic, M2 muscarinic, and somatostatin receptors. In vitro, G o is also activated by growth cone-associated protein of M r 43,000 (GAP43) and the Alzheimer amyloid precursor protein, but it is not known whether this occurs in intact cells. G o is an ␣␤␥ heterotrimeric G protein discovered in 1984 in brain by Neer and collaborators (1, 2) and by Sternweis and Robishaw (3), who all characterized it as a substrate for the ADP-ribosyltransferase activity of pertussis toxin. G o has received special attention for the following reasons: (i) It is the most abundant G protein in neurons, where it can constitute up to 2% of membrane protein (3). (ii) In addition to neurons, it appears to be expressed only on endocrine cells and heart, albeit at much lower levels comparable to those of the other heterotrimeric G proteins. (iii) G o is activated not only by the same class of seven-transmembrane receptors that activate the inhibitory G proteins G i1 -G i3 (4-9) but also by at least two proteins that do not belong to the rhodopsin-like family of G protein-coupled receptors, GAP43, an intracellular growth cone-associated protein active in neurite outgrowth (10), and the Alzheimer amyloid protein precursor protein responsible for familial forms of this disease (11). (iv) Except for inhibition of neuronal Ca 2ϩ channels, for which the mechanism of action of G o has been elucidated at the molecular level and shown to be due to the interaction of its ␤␥ moiety with the ␣ 1 subunit of the channel (6, 12-16), the mode of action of G o is essentially unknown. Tests for an ␣ i -like function for ␣ o have failed (17,18). Activated ␣ o has transformed NIH-3T3 cells (19) and activated mitogen-activated protein kinase activity in Chinese hamster ovary cells (20), phospholipase C in Xenopus oocytes, and K ϩ channels in neurons (21, 22), but how these effects come about has not been established. In fact, there is scant knowledge of the gamut of effector systems that may be the target(s) of activated G o .Gene ablation in mice is a powerful yet technically complex approach to identify as yet unknown functions of proteins that become manifest in mutated animals and͞or in cell lines derived from them. It has been applied to several G proteins with the following interesting results. G i2 -deficient mice were found to develop ulcerative colitis and adenocarcinomas, revealing an unexpected and as yet unexplained role of G i2 in the development of a chronic inflammatory response and very likely in lymphocyte homing to enteric epithelia (23, 24). Ablation of G q revealed an essential role for this G protein in platelet activation, because G q -deficient mice bleed and their platelets fail to be activated by physiologic activators such as collagen, thrombin, thromboxane, and ADP (25). Also the ablation of G o has been reported (26). Live mice, homozygous for loss of ␣ o , were obtained showing that...

Pituitary adenylate cyclase-activating polypeptide (PACAP)-27 and PACAP-38 are neuropeptides of the vasoactive intestinal peptide/secretin/glucagon family. We previously described alternative splicing of the region encoding the third intracellular loop of the PACAP receptor generating six isoforms with differential signal transduction properties (Spengler, D., Waeber, C., Pantaloni, C., Holsboer, F., Bockaert, J., Seeburg, P. H., and Journot, L. (1993) Nature 365, 170 -175). In addition, we demonstrated that the potencies of the two forms of PACAP are similar for adenylate cyclase stimulation, whereas PACAP-38 is more potent than PACAP-27 in phospholipase C activation. In the present work, we document the existence of a new splice variant of the PACAP receptor that was characterized by a 21-aminoacid deletion in the N-terminal extracellular domain. We demonstrate that this domain modulates receptor selectivity with respect to PACAP-27 and -38 binding and controls the relative potencies of the two agonists in phospholipase C stimulation.Pituitary adenylate cyclase-activating polypeptides (PACAP) 1 are recently purified neuropeptides (1, 2) that are named according to their amino acid number, PACAP-27 and PACAP-38. The 27-amino acid form corresponds to the 27 N-terminal amino acids of PACAP-38 and shares 68% identity with vasoactive intestinal peptide (VIP). Two classes of PACAP binding sites were pharmacologically defined. Type I PACAP receptors bind PACAP-27 and -38 about two orders of magnitude more efficiently than VIP, whereas type II PACAP receptors do not discriminate between PACAP-27, -38, and VIP. At present, three PACAP/VIP receptor genes have been identified: PACAP 1 -R corresponds to PACAP type I receptors whereas PACAP/VIP 1 -R and PACAP/VIP 2 -R correspond to type II receptors. No VIP-specific receptor has so far been cloned.PACAP 1 -R is abundantly expressed in the brain, the pituitary and pineal glands, and the adrenal medulla. It mediates the neurotrophic action of PACAP-38 on PC12 cells (3) and sympathetic neuroblasts (4, 5). In the pituitary gland, PACAP-38 modulates the release of several hormones (1) and of interleukin-6 (6) through activation of PACAP 1 -R but also of PACAP/VIP 1 -R and PACAP/VIP 2 -R (7-11). PACAP 1 -R activation also controls proliferation of chromaffin cells (12) as well as catecholamine release by the adrenal medulla (13, 14).We and others (15-19) recently cloned the rat PACAP 1 -R cDNA. In addition, we demonstrated that PACAP 1 -R hnRNA is alternatively spliced and gives rise to six variants (18,20). Two cassettes named "hip" and "hop" are possibly inserted at the end of the third intracellular loop of the receptor. The resulting variants were named PACAP 1 -R s (the shortest form, without cassette), PACAP 1 -R hip, PACAP 1 -R hop1, PACAP 1 -R hop2, PACAP 1 -R hip-hop1, and PACAP 1 -R hip-hop2. They display differential signal transduction properties upon expression into LLC PK1 cells and Xenopus oocytes. The short as well as the hop variants potently activate both AC an...

No abstract

No abstract

The circadian clock located in the suprachiasmatic nucleus (SCN) organizes autonomic and behavioral rhythms into a near 24 hr time that is adjusted daily to the solar cycle via a direct projection from the retina, the retinohypothalamic tract (RHT). This neuronal pathway costores the neurotransmitters PACAP and glutamate, which seem to be important for light-induced resetting of the clock. At the molecular level the clock genes mPer1 and mPer2 are believed to be target for the light signaling to the clock. In this study, we investigated the possible role of PACAP-type 1 receptor signaling in light-induced resetting of the behavioral rhythm and light-induced clock gene expression in the SCN. Light stimulation at early night resulted in larger phase delays in PACAP-type 1 receptor-deficient mice (PAC1 Ϫ /Ϫ ) compared with wild-type mice accompanied by a marked reduction in light-induced mPer1, mPer2, and c-fos gene expression. Light stimulation at late night induced mPer1 and c-fos gene expression in the SCN to the same levels in both wild type and PAC1 Ϫ /Ϫ mice. However, in contrast to the phase advance seen in wild-type mice, PAC1Ϫ /Ϫ mice responded with phase delays after photic stimulation. These data indicate that PAC1 receptor signaling participates in the gating control of photic sensitivity of the clock and suggest that mPer1, mPer2, and c-fos are of less importance for light-induced phase shifts at night.

Pituitary adenylate cyclase-activating polypeptide (PACAP) and the proopiomelanocortin (POMC)-derived peptide, a-melanocytestimulating hormone (a-MSH), exert anorexigenic activities. While a-MSH is known to inhibit food intake and stimulate catabolism via activation of the central melanocortin-receptor MC4-R, little is known regarding the mechanism by which PACAP inhibits food consumption. We have recently found that, in the arcuate nucleus of the hypothalamus, a high proportion of POMC neurons express PACAP receptors. This observation led us to investigate whether PACAP may inhibit food intake through a POMC-dependent mechanism. In mice deprived of food for 18 h, intracerebroventricular administration of PACAP significantly reduced food intake after 30 min, and this effect was reversed by the PACAP antagonist PACAP6-38. In contrast, vasoactive intestinal polypeptide did not affect feeding behavior. Pretreatment with the MC3-R/MC4-R antagonist SHU9119 significantly reduced the effect of PACAP on food consumption. Central administration of PACAP induced c-Fos mRNA expression and increased the proportion of POMC neuronexpressing c-Fos mRNA in the arcuate nucleus. Furthermore, PACAP provoked an increase in POMC and MC4-R mRNA expression in the hypothalamus, while MC3-R mRNA level was not affected. POMC mRNA level in the arcuate nucleus of PACAP-specific receptor (PAC1-R) knock-out mice was reduced as compared with wild-type animals. Finally, i.c.v. injection of PACAP provoked a significant increase in plasma glucose level. Altogether, these results indicate that PACAP, acting through PAC1-R, may inhibit food intake via a melanocortin-dependent pathway. These data also suggest a central action of PACAP in the control of glucose metabolism.