Heinz Lother

Increased AT(1) receptor heterodimers in preeclampsia mediate enhanced angiotensin II responsiveness.

Several examples of functional G-protein–coupled receptor heterodimers have been identified. However, it is not known whether receptor heterodimerization is involved in the pathogenesis of human disorders. Here we show that in preeclamptic hypertensive women, a significant increase in heterodimerization occurs between the AT1-receptor for the vasopressor angiotensin II and the B2-receptor for the vasodepressor bradykinin. AT1–B2-receptor heterodimerization in preeclampsia correlated with a 4–5-fold increase in B2-receptor protein levels. Expression of the AT1–B2 heterodimer increased the responsiveness to angiotensin II and conferred resistance in AT1-receptors to inactivation by reactive oxygen species raised in normotensive and preeclamptic pregnancies. We suggest that AT1–B2 heterodimers contribute to angiotensin II hypersensitivity in preeclampsia. Moreover, we identify preeclampsia as the first disorder associated with altered G-protein–coupled receptor heterodimerization.

Factor XIIIA Transglutaminase Crosslinks AT1 Receptor Dimers of Monocytes at the Onset of Atherosclerosis

Many G protein-coupled receptors form dimers in cells. However, underlying mechanisms are barely understood. We report here that intracellular factor XIIIA transglutaminase crosslinks agonist-induced AT1 receptor homodimers via glutamine315 in the carboxyl-terminal tail of the AT1 receptor. The crosslinked dimers displayed enhanced signaling and desensitization in vitro and in vivo. Inhibition of angiotensin II release or of factor XIIIA activity prevented formation of crosslinked AT1 receptor dimers. In agreement with this finding, factor XIIIA-deficient individuals lacked crosslinked AT1 dimers. Elevated levels of crosslinked AT1 dimers were present on monocytes of patients with the common atherogenic risk factor hypertension and correlated with an enhanced angiotensin II-dependent monocyte adhesion to endothelial cells. Elevated levels of crosslinked AT1 receptor dimers on monocytes could sustain the process of atherogenesis, because inhibition of angiotensin II generation or of intracellular factor XIIIA activity suppressed the appearance of crosslinked AT1 receptors and symptoms of atherosclerosis in ApoE-deficient mice.

Regulation of transcription of the chromosomaldnaA gene ofEscherichia coli

SummaryBy comparative S1 analysis we investigated the in vivo regulation of transcription of the chromosomaldnaA gene coding for a protein essential for the initiation of replication at the chromosomal origin. Inactivation of the protein indnaA mutants results in derepression, whereas excess DnaA protein (presence of a DnaA overproducing plasmid) leads to repression ofdnaA transcription. BothdnaA promoters are subject to autoregulation allowing modulation of transcriptional efficiency by at least 20-fold. Increasing the number oforiC sequences (number of DnaA binding sites) in the cell by introducingoriC plasmids leads to a derepression of transcription. Autoregulation and binding tooriC suggest that the DnaA protein exerts a major role in the regulation of the frequency of initiation atoriC. The efficiency of transcription of thednaA2 promoter is reduced in the absence ofdam methylation, which is involved in the regulation oforiC replication.

Mesangial AT1/B2 receptor heterodimers contribute to angiotensin II hyperresponsiveness in experimental hypertension.

Angiotensin II plays a central role in the pathogenesis of hypertension and of related cardiovascular disorders by binding to and activating angiotensin II receptors (AT1 receptors). Sensitization to the vasopressor response of angiotensin II is a key feature in many cardiovascular disorders. However, underlying mechanisms responsible for angiotensin II hypersensitivity are barely understood. Because angiotensin II responsiveness of AT1 receptors can be specifically modified by AT1/B2 receptor dimerization, we determined the AT1 receptor dimerization status in an experimental model of hypertension. AT1/B2 receptor heterodimers were abundant on renal mesangial cells isolated from spontaneously hypertensive rats compared with that on cells from normotensive controls. Heterodimerization of AT1 with B2 receptors was correlated with high levels of B2 receptor protein on kidneys and on mesangial cells of hypertensive rats, as determined in immunoblot with receptor-specific antibodies. Specific inhibition of AT1/B2 receptor heterodimers revealed that these receptor heterodimers mediated an enhanced angiotensin II-stimulated Gαq/11 activation and an increased endothelin-1 secretion of mesangial cells from hypertensive rats. Thus, AT1/B2 receptor heterodimerization contributes to angiotensin II hyperresponsiveness of mesangial cells in experimental hypertension.

Angiotensin II AT2 Receptor Oligomers Mediate G-protein Dysfunction in an Animal Model of Alzheimer Disease

Progressive neurodegeneration and decline of cognitive functions are major hallmarks of Alzheimer disease (AD). Neurodegeneration in AD correlates with dysfunction of diverse signal transduction mechanisms, such as the G-protein-stimulated phosphoinositide hydrolysis mediated by Gαq/11. We report here that impaired Gαq/11-stimulated signaling in brains of AD patients and mice correlated with the appearance of cross-linked oligomeric angiotensin II AT2 receptors sequestering Gαq/11. Amyloid β (Aβ) was causal to AT2 oligomerization, because cerebral microinjection of Aβ triggered AT2 oligomerization in the hippocampus of mice in a dose-dependent manner. Aβ induced AT2 oligomerization by a two-step process of oxidative and transglutaminase-dependent cross-linking. The induction of AT2 oligomers in a transgenic mouse model with AD-like symptoms was associated with Gαq/11 dysfunction and enhanced neurodegeneration. Vice versa, stereotactic inhibition of AT2 oligomers by RNA interference prevented the impairment of Gαq/11 and delayed Tau phosphorylation. Thus, Aβ induces the formation of cross-linked AT2 oligomers that contribute to the dysfunction of Gαq/11 in an animal model of Alzheimer disease.

Dominant-negative AT2 receptor oligomers induce G-protein arrest and symptoms of neurodegeneration

Neurodegeneration in Alzheimers disease (AD) correlates with dysfunction of signaling mediated by Gαq/11. Nondissociable angiotensin II AT2 receptor oligomers are linked to the impaired Gαq/11-stimulated signaling of AD patients and transgenic mice with AD-like symptoms. To further analyze the role of AT2 receptor oligomers, we induced the formation of AT2 oligomers in an in vitro cell system. Similarly as in vivo, sequential oxidative and transglutaminase-dependent cross-linking steps triggered the formation of AT2 oligomers in vitro. Elevated reactive oxygen species mediated oxidative cross-linking of AT2 monomers to dimers involving tyrosine residues located at putative interreceptor contact sites of the cytoplasmic loop connecting transmembrane helices III/IV. Cross-linked AT2 dimers were subsequently a substrate of activated transglutaminase-2, which targeted the carboxyl terminus of AT2 dimers, as assessed by truncated and chimeric AT2 receptors, respectively. AT2 oligomers acted as dominant negative receptors in vitro by mediating Gαq/11 protein sequestration and Gαq/11 protein arrest. The formation of AT2 oligomers and G-protein dysfunction could be suppressed in vitro and in vivo by an AT2 receptor mutant. Inhibition of AT2 oligomerization upon stereotactic expression of the AT2 receptor mutant revealed that Gαq/11-sequestering AT2 oligomers enhanced the development of neurodegenerative symptoms in the hippocampus of transgenic mice with AD-like pathology. Thus, AT2 oligomers inducing Gαq/11 arrest are causally involved in inducing symptoms of neurodegeneration.

Promoters in the region of the E. coli replication origin

SummaryIn vitro binding of RNA polymerase to DNA of the minichromosome pCM959 revealed 5 binding sites, one within the origin of replication, oriC. A comparison of the location of the binding sites with the nucleotide sequence allowed the definition of 4 promoters, the transcripts of which allow the synthesis of 4 polypeptides.