Synthesis and binding characteristics of two sulfhydryl-reactive probes for vasopressin receptors

AbstractThe present study describes the synthesis and receptor binding affinities of the sulfhydryl-reactive vasopressin analogs deamino[Dab(N8-N-maleoyl-β-alanin e)4]AVP (1a) and deamino[Lys(N8-N-maleoyl-β-alanine)8VP (2a). The analogs were obtained by introducing the sulfhydryl-reactive maleoyl-β-alanyl group at the δ-amino group of Dab4 in deamino[Dab4]IAVP (1) and at the ε-amino group of Lys8 in deamino[Lys8]VP (2), which were synthesized by the solid-phase method. Furthermore, the analog modified at Dab4 was prepared as tritium labeled compound (1b) after catalytic iodine tritium exchange at Tyr2 in deamino[Dab4]AVP. The sulfhydryl-reactive vasopressin analogs retained high binding affinity for the V2 vasopressin receptor in membranes derived from bovine kidney inner medulla. Apparent dissociation constants Kd of 45 nM (compound 1a) and 15 nM (compound 2a) were determined. Incubation of the ligand receptor complexes at pH 5.5 resulted in dissociation of the sulfhydryl-reactive vasopressin analogs from the V2 receptor. No indications of a covalent reaction between analogs 1a, 2a and 1b and sulfhydryl groups in or close to the hormone binding site of the V2 receptor were found

Differential gene expression in ADAM10 and mutant ADAM10 transgenic mice

<p>Abstract</p> <p>Background</p> <p>In a transgenic mouse model of Alzheimer disease (AD), cleavage of the amyloid precursor protein (APP) by the α-secretase ADAM10 prevented amyloid plaque formation, and alleviated cognitive deficits. Furthermore, ADAM10 overexpression increased the cortical synaptogenesis. These results suggest that upregulation of ADAM10 in the brain has beneficial effects on AD pathology.</p> <p>Results</p> <p>To assess the influence of ADAM10 on the gene expression profile in the brain, we performed a microarray analysis using RNA isolated from brains of five months old mice overexpressing either the α-secretase ADAM10, or a dominant-negative mutant (dn) of this enzyme. As compared to non-transgenic wild-type mice, in ADAM10 transgenic mice 355 genes, and in dnADAM10 mice 143 genes were found to be differentially expressed. A higher number of genes was differentially regulated in double-transgenic mouse strains additionally expressing the human APP_[V717I]mutant.</p> <p>Overexpression of proteolytically active ADAM10 affected several physiological pathways, such as cell communication, nervous system development, neuron projection as well as synaptic transmission. Although ADAM10 has been implicated in Notch and β-catenin signaling, no significant changes in the respective target genes were observed in adult ADAM10 transgenic mice.</p> <p>Real-time RT-PCR confirmed a downregulation of genes coding for the inflammation-associated proteins S100a8 and S100a9 induced by moderate ADAM10 overexpression. Overexpression of the dominant-negative form dnADAM10 led to a significant increase in the expression of the fatty acid-binding protein Fabp7, which also has been found in higher amounts in brains of Down syndrome patients.</p> <p>Conclusion</p> <p>In general, there was only a moderate alteration of gene expression in ADAM10 overexpressing mice. Genes coding for pro-inflammatory or pro-apoptotic proteins were not over-represented among differentially regulated genes. Even a decrease of inflammation markers was observed. These results are further supportive for the strategy to treat AD by increasing the α-secretase activity.</p

Ectodomain shedding of L1 adhesion molecule promotes cell migration by autocrine binding to integrins

The L1 adhesion molecule plays an important role in axon guidance and cell migration in the nervous system. L1 is also expressed by many human carcinomas. In addition to cell surface expression, the L1 ectodomain can be released by a metalloproteinase, but the biological function of this process is unknown. Here we demonstrate that membrane-proximal cleavage of L1 can be detected in tumors and in the developing mouse brain. The shedding of L1 involved a disintegrin and metalloproteinase (ADAM)10, as transfection with dominant-negative ADAM10 completely abolishes L1 release. L1-transfected CHO cells (L1-CHO) showed enhanced haptotactic migration on fibronectin and laminin, which was blocked by antibodies to αvβ5 and L1. Migration of L1-CHO cells, but not the basal migration of CHO cells, was blocked by a metalloproteinase inhibitor, indicating a role for L1 shedding in the migration process. CHO and metalloproteinase-inhibited L1-CHO cells were stimulated to migrate by soluble L1-Fc protein. The induction of migration was blocked by αvβ5-specific antibodies and required Arg-Gly-Asp sites in L1. A 150-kD L1 fragment released by plasmin could also stimulate CHO cell migration. We propose that ectodomain-released L1 promotes migration by autocrine/paracrine stimulation via αvβ5. This regulatory loop could be relevant for migratory processes under physiological and pathophysiological conditions

Molecular cloning of a new bombesin receptor subtype expressed in uterus during pregnancy

The homology screening approach has been used to clone a new member of the guanine-nucleotidebinding-protein-coupled receptor superfamily from guinea pig uterus. The cloned cDNA encodes a 399-amino-acid protein and shows the highest amino acid similarity to members of the bombesin receptor family; 52% and 47% similarity to the gastrin-releasing-peptide (GRP) receptor and the neuromedin-B receptor, respectively. Bindingexperiments with the stably transfected LLC-PK1 cell line expressing the new receptor protein confmned the bombesin-like nature of the cloned receptor. The relative order ofligand affinity, GRP = neuromedin C >> neuromedin B, suggests that the cloned cDNA represents the GRP subtype rather than the neuromedin-B subtype of bombesin receptors. Northern-blot analysis of mRNA species from several guinea-pig tissues showed that the mRNA for the new bombesin receptor subtype is expressed mainly in uteri of pregnant animals

Molecular cloning of substance P receptor cDNA from guinea-pig uterus

A cDNA encoding guinea-pig uterine substance P (SP) receptor has been isolated using the homology screening approach. Northern blot analysis reveals that the corresponding mRNA, of approx. 4.8 kb, is expressed in all tissues tested, but predominantly in the uteri of non-pregnant animals; during pregnancy its expression is reduced. The guinea-pig SP receptor was expressed in COS-7 cells and demonstrated relative Iigand affinity in the order: SP >> neurokinin A > neurokinin B

Differential inactivation of vasopressin receptor subtypes in isolated membranes and intact cells by N-ethylmaleimide

AbstractVasopressin receptors in plasma membranes and on cell monolayers were treated with sulfhydryl reagents. Specific binding of [3H]AVP to renal V2 receptors in membranes from bovine and porcine kidney and on LLC-PK1 cells was markedly (80–90%) reduced after treatment with NEM but that to V1 receptors on rat liver membranes and A7r5 smooth muscle cells only slightly (10–30%). Inactivation of receptors by NEM reduced the number of binding sites without altering the affinity of unmodified receptor molecules. High affinity ligands (agonists and antagonists), in complex with the V2 receptor, protected against its inactivation. The results suggest that one or more cysteine residues are located in the ligand-binding site of the V2 receptor, and are essential for hormone binding. Furthermore, it is possible to use NEM to differentiate between vasopressin isoreceptors

Alpha-secretase activation : an approach to Alzheimer's disease therapy

The nonamyloidogenic pathway of processing the amyloid precursor protein (APP) involves the cleavage within the amyloid-beta peptide sequence, and thus precludes amyloid-beta formation. The identification of a member of the disintegrin and metalloproteinase family, ADAM10, as an alpha-secretase that prevents plaque formation and hippocampal deficits in vivo gave us the possibility to examine the alpha-secretase as a potential target for the therapy of Alzheimer&#039;s disease. Within the priority program Cellular Mechanisms of Alzheimer&#039;s Disease,we investigated several approaches to stimulate the alpha-secretase pathway. Two protein convertases were found to be responsible for the removal of the prodomain, and for the formation of the mature enzyme with alpha-secretase activity. The cloning and characterization of the human ADAM10 promoter provided the basis to examine ADAM10 gene expression. We found a common upregulation of ADAM10, APP, and APP-like protein 2 during differentiation of neuronal cells by retinoic acid, and increased alpha-secretase cleavage of the two substrates. Other approaches for enhancing alpha-secretase activity are the reduction of cellular cholesterol and the stimulation of G protein-coupled neuropeptide receptors. Our results suggest medications and dietary regiments which enhance the nonamyloidogenic pathway of APP processing to be a valuable approach to Alzheimer&#039;s disease therapy