The two forms of pituitary adenylyl cyclase-activating polypeptide (PACAP-27 and -38) are neuropeptides of the secretin/glucagon/vasoactive intestinal polypeptide/growth-hormone-releasing hormone family and regulate hormone release from the pituitary and adrenal gland. They may also be involved in spermatogenesis, and PACAP-38 potently stimulates neuritogenesis and survival of cultured rat sympathetic neuroblast and promotes neurite outgrowth of PC-12 cells. The PACAP type-I receptor (found in hypothalamus, brain stem, pituitary, adrenal gland and testes), specific for PACAP, is positively coupled to adenylyl cyclase and phospholipase C. The recently cloned type II receptor does not discriminate between PACAP and vasoactive intestinal polypeptide and is coupled to only adenylyl cyclase. Here we have used a new expression cloning strategy, based on the induction of a reporter gene by cyclic AMP, to isolate a complementary DNA encoding the type-I PACAP receptor. On transfection of this cDNA, both PACAP-27 and -38 stimulate adenylyl cyclase with similar EC50 values (50% effective concentration, 0.1-0.4 nM), whereas only PACAP-38 stimulates phospholipase C with high potency (EC50 = 15 nM). Four other splice variants were isolated with insertions at the C-terminal end of the third intracellular loop. Expression of these cDNAs revealed altered patterns of adenylyl cyclase and phospholipase C stimulation, suggesting a novel mechanism for fine tuning of signal transduction.

The proliferation rate of a cell population reflects a balance between cell division, cell cycle arrest, differentiation and apoptosis. The regulation of these processes is central to development and tissue homeostasis, whereas dysregulation may lead to overt pathological outcomes, notably cancer and neurodegenerative disorders. We report here the cloning of a novel zinc finger protein which regulates apoptosis and cell cycle arrest and was accordingly named Zac1. In vitro Zac1 inhibited proliferation of tumor cells, as evidenced by measuring colony formation, growth rate and cloning in soft agar. In vivo Zac1 abrogated tumor formation in nude mice. The antiproliferative activity of Zac1 was due to induction of extensive apoptosis and of G1 arrest, which proceeded independently of retinoblastoma protein and of regulation of p21(WAF1/Cip1), p27Kip1, p57Kip2 and p16INK4a expression. Zac1-mediated apoptosis was unrelated to cell cycle phase and G1 arrest was independent of apoptosis, indicating separate control of apoptosis and cell cycle arrest. Zac1 is thus the first gene besides p53 which concurrently induces apoptosis and cell cycle arrest.

We previously reported the identification of mZac, a novel mouse zinc finger protein that shared with p53 the ability to regulate concomitantly apoptosis and cell cycle progression. We describe here the isolation, chromosomal localization, and functional in vitro characterization of its human homolog. hZAC is a widely expressed zinc finger protein that reveals transactivation and DNA-binding activity. hZAC inhibits tumor cell growth through induction of apoptotic cell death and G 1 arrest. Thus hZAC, like its mouse counterpart, displays antiproliferative properties through pathways known to be central to the activity of p53. We mapped hZAC on chromosome 6q24-q25, a region frequently deleted in many solid tumors. Indeed, allelic loss at 6q24-q25 has been shown in breast and ovary cancers, melanomas, astrocytomas, and renal cell carcinomas. Furthermore, Abdollahi et al.

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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...

Loss of chromosome 6q21-qter is the second most frequent loss of chromosomal material in sporadic breast neoplasms suggesting the presence of at least one tumor suppressor gene on 6q. We recently isolated a cDNA encoding a new zinc ®nger protein which we named ZAC according to its functional properties, namely induction of apoptosis and control of cell cycle progression. ZAC is expressed in normal mammary gland and maps to 6q24-q25, a recognized breast cancer hot spot on 6q. In the present report, we investigated the possible inactivation of ZAC in breast cancer cell lines and primary tumors. We detected no mutation in ZAC coding region in a panel of 45 breast tumors with allelic imbalance of 6q24-q25. However, a survey of eight breast cancer cell lines showed a deeply reduced (three cell lines) or complete loss of (®ve cell lines) ZAC expression. Treatment of three of these cell lines with the methylation-interfering agent 5-azacytidine induced ZAC re-expression. In addition, Northern blot and RNase protection assay analysis of ZAC expression in 23 unselected primary breast tumors showed a reduced expression in several samples. Together with its functional properties and chromosomal localization, these ®ndings substantiate ZAC as a good candidate for the tumor suppressor gene on 6q24-q25.

Myosin polymorphism in muscles has been studied by a variety of electrophoretic techniques, in non-dissociating and in dissociating conditions. The analysis of myosin isozymes in the native state was achieved in pyrophosphate buffer and required only minute amounts of protein ; identical results were obtained with purified or crudely extracted myosin. The determination of the subunit content of each isozyme was done in the presence of sodium dodecyl sulphate or urea for light chain, and in a phenol, acetic acid and urea system for heavy chain screening.Electrophoresis in non-dissociating conditions has led to the separation of up to a dozen of myosin isozymes, differing in mobilities by as much as 30 %. Muscle specificity of myosin was clearly established. Apart from a few exceptions, all the muscles tested were shown to contain more than one myosin species ; fast-twitch muscles for instance all contained the same three isozymes, but in variable ratios. Class specificity of myosin appeared related to the relative proportions of isozymes in a given muscle. A second electrophoresis in dissociating solvents of the myosin bands first resolved in pyrophosphate buffer has then allowed a further characterization of the various isozymes. The differences in mobilities observed in the native state were shown to come either from the light chains, or from the heavy chains, or from both. The first case was illustrated by the three species present in fast muscles, which were shown to correspond to three alkali light-chain isozymes, the heterodimer representing in some instances up to 40 % of the total. Next to light-chain muscle type specificity, electrophoresis in the phenol, acetic acid, urea system has led to the detection of differences in the heavy chains of fast, slow and cardiac myosins.The application of these various electrophoretic techniques to the analysis of the modification of myosin isozymes during development or in pathology studies can be considered.

ZAC encodes a zinc ®nger protein with antiproliferative activity, is maternally imprinted and is a candidate for the tumor suppressor gene on 6q24. ZAC expression is frequently lost in breast and ovary tumor-derived cell lines and down-regulated in breast primary tumors. In this report, we describe ZACD2, an alternatively spliced variant of ZAC lacking the sequence encoding the two N-terminal zinc ®ngers. Messenger RNAs encoding ZAC or ZACD2 were equally abundant and both proteins were nuclear. ZACD2 displayed an improved transactivation activity and an enhanced anity for a ZAC binding site, suggesting that the two N-terminal zinc ®ngers negatively regulated ZAC binding to its target DNA sequences. Both proteins were equally ecient in preventing colony formation, indicating similar overall antiproliferative activities. However, these activities resulted from a dierential regulation of apoptosis vs cell cycle progression since ZACD2 was more ecient at induction of cell cycle arrest than ZAC, whereas it was the reverse for apoptosis induction. Hence, these data further underline that ZAC gene is critically controlled, both at the transcriptional level through imprinting and at the functional level through alternative splicing.