The corticotropin-releasing factor (CRF) 3 receptor family is involved in the regulation of the hypothalamic-pituitary-adrenal stress axis in mammals (1-3). A large body of evidence points to a major role of the receptors in mediating CRF effects in anxiety and depressive disorders and in stress-associated pathologies. Two types of CRF receptors are known, the CRF 1 and the CRF 2 receptors. The CRF 1 receptor is expressed mainly in the pituitary and central nervous system and binds CRF with high affinity. It mediates adrenocorticotrophic hormone release from the anterior pituitary and is involved in the endocrine, autonomic, and cognitive responses to stress stimuli. The CRF 2 receptors are expressed in the central nervous system but also in the periphery including skeletal muscle cells, cardiac myocytes, and cells of the gastrointestinal tract. Three splice variants of CRF 2 receptors have been described: CRF 2(a) , CRF 2(b) , and CRF 2(c) receptors. They bind CRF with low and the urocortins 1-3 with high affinity. The CRF 2 receptors are involved in the regulation of feeding behavior (4) and in recovery from a stress response (5). It is likely that they are also involved in modulating anxiety-related behavior.The CRF receptors belong to the small subgroup of GPCRs (5-10%) possessing putative N-terminal signal peptides. These peptides are believed to be cleaved-off after mediating the ER targeting/insertion process (6, 7). The majority (90 -

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Botulism is a severe neurological disease caused by the complex family of botulinum neurotoxins (BoNT). Based on the different serotypes known today, a classification of serotype variants termed subtypes has been proposed according to sequence diversity and immunological properties. However, the relevance of BoNT subtypes is currently not well understood. Here we describe the isolation of a novel Clostridium botulinum strain from a food-borne botulism outbreak near Chemnitz, Germany. Comparison of its botulinum neurotoxin gene sequence with published sequences identified it to be a novel subtype within the BoNT/A serotype designated BoNT/A8. The neurotoxin gene is located within an ha-orfX+ cluster and showed highest homology to BoNT/A1, A2, A5, and A6. Unexpectedly, we found an arginine insertion located in the HC domain of the heavy chain, which is unique compared to all other BoNT/A subtypes known so far. Functional characterization revealed that the binding characteristics to its main neuronal protein receptor SV2C seemed unaffected, whereas binding to membrane-incorporated gangliosides was reduced in comparison to BoNT/A1. Moreover, we found significantly lower enzymatic activity of the natural, full-length neurotoxin and the recombinant light chain of BoNT/A8 compared to BoNT/A1 in different endopeptidase assays. Both reduced ganglioside binding and enzymatic activity may contribute to the considerably lower biological activity of BoNT/A8 as measured in a mouse phrenic nerve hemidiaphragm assay. Despite its reduced activity the novel BoNT/A8 subtype caused severe botulism in a 63-year-old male. To our knowledge, this is the first description and a comprehensive characterization of a novel BoNT/A subtype which combines genetic information on the neurotoxin gene cluster with an in-depth functional analysis using different technical approaches. Our results show that subtyping of BoNT is highly relevant and that understanding of the detailed toxin function might pave the way for the development of novel therapeutics and tailor-made antitoxins.

The corticotropin-releasing factor receptor type 2a (CRF 2(a) R) belongs to the family of G protein-coupled receptors. The receptor possesses an N-terminal pseudo signal peptide that is unable to mediate targeting of the nascent chain to the endoplasmic reticulum membrane during early receptor biogenesis. The pseudo signal peptide remains uncleaved and consequently forms an additional hydrophobic receptor domain with unknown function that is unique within the large G protein-coupled receptor protein family. Here, we have analyzed the functional significance of this domain in comparison with the conventional signal peptide of the homologous corticotropin-releasing factor receptor type 1 (CRF 1 R). We show that the presence of the pseudo signal peptide leads to a very low cell surface receptor expression of the CRF 2(a) R in comparison with the CRF 1 R. Moreover, whereas the presence of the pseudo signal peptide did not affect coupling to the G s protein, G i -mediated inhibition of adenylyl cyclase activity was abolished. The properties mediated by the pseudo signal peptide were entirely transferable to the CRF 1 R in signal peptide exchange experiments. Taken together, our results show that signal peptides do not only influence early protein biogenesis. In the case of the corticotropin-releasing factor receptor subtypes, the use of conventional and pseudo signal peptides have an unexpected influence on signal transduction.The family of corticotropin-releasing factor (CRF) 3 receptors encompasses two subtypes, the CRF 1 R and CRF 2 R (1, 2). The CRF 1 R is expressed mainly in the anterior pituitary and plays a central role in the regulation of the hypothalamic-pituitaryadrenal stress axis in mammals (3). It binds CRF with high affinity and mediates ACTH release from the pituitary leading to cortisol biosynthesis in the adrenal cortex. A large body of evidence points to a major role of the receptor in mediating CRF effects in anxiety and depressive disorders (4 -6).In the case of the CRF 2 R, three splice variants have been described as follows: the CRF 2(a) R, CRF 2(b) R, and CRF 2(c) R. All splice variants bind CRF with low affinity and the urocortins 1-3 with high affinity. They are involved in the regulation of feeding behavior (7) and in recovery from a stress response (8).It is likely that they are also involved in modulating anxietyrelated behavior.Both the CRF 1 R and the CRF 2(a) R usually couple to the G s / adenylyl cyclase system and consequently increase cytosolic cAMP as a second messenger. However, a promiscuous coupling behavior was described previously in particular for the CRF

The detection of botulinum neurotoxins (BoNT) is extremely challenging due to their high toxicity and the multiple BoNT variants. To date, seven serotypes with more than 30 subtypes have been described, and even more subtypes are expected to be discovered. The fact that the BoNT molecules are released as large complexes of different size and composition adds further complexity to the issue. Currently, in the diagnostics of botulism, the mouse bioassay (MBA) is still considered as gold standard for the detection of BoNT in complex sample materials. Over the years, different functional, immunological, and spectrometric assays or combinations thereof have been developed, supplemented by DNA-based assays for the detection of the organism. In this review, advantages and limitations of the current technologies will be discussed, highlighting some of the intricacies of real sample analysis.

CC2D1A and CC2D1B belong to the evolutionary conserved Lgd protein family with members in all multi-cellular animals. Several functions such as centrosomal cleavage, involvement in signalling pathways, immune response and synapse maturation have been described for CC2D1A. Moreover, the Drosophila melanogaster ortholog Lgd was shown to be involved in the endosomal trafficking of the Notch receptor and other transmembrane receptors and physically interacts with the ESCRT-III component Shrub/CHMP4. To determine if this function is conserved in mammals we generated and characterized Cc2d1a and Cc2d1b conditional knockout mice. While Cc2d1b deficient mice displayed no obvious phenotype, we found that Cc2d1a deficient mice as well as conditional mutants that lack CC2D1A only in the nervous system die shortly after birth due to respiratory distress. This finding confirms the suspicion that the breathing defect is caused by the central nervous system. However, an involvement in centrosomal function could not be confirmed in Cc2d1a deficient MEF cells. To analyse an influence on Notch signalling, we generated intestine specific Cc2d1a mutant mice. These mice did not display any alterations in goblet cell number, proliferating cell number or expression of the Notch reporter Hes1-emGFP, suggesting that CC2D1A is not required for Notch signalling. However, our EM analysis revealed that the average size of endosomes of Cc2d1a mutant cells, but not Cc2d1b mutant cells, is increased, indicating a defect in endosomal morphogenesis. We could show that CC2D1A and its interaction partner CHMP4B are localised on endosomes in MEF cells, when the activity of the endosomal protein VPS4 is reduced. This indicates that CC2D1A cycles between the cytosol and the endosomal membrane. Additionally, in rescue experiments in D. melanogaster, CC2D1A and CC2D1B were able to functionally replace Lgd. Altogether our data suggest a functional conservation of the Lgd protein family in the ESCRT-III mediated process in metazoans.

Organic anions are taken up from the blood into proximal tubule cells by organic anion transporters 1 and 3 (OAT1 and OAT3) in exchange for dicarboxylates. The released dicarboxylates are recycled by the sodium dicarboxylate cotransporter 3 (NaDC3). In this study, we tested the substrate specificities of human NaDC3, OAT1, and OAT3 to identify those dicarboxylates for which the three cooperating transporters have common high affinities. All transporters were stably expressed in HEK293 cells, and extracellularly added dicarboxylates were used as inhibitors of3 H]estrone-3-sulfate (OAT3) uptake. Human NaDC3 was stably expressed as proven by immunochemical methods and by sodiumdependent uptake of succinate (K 0.5 for sodium activation, 44.6 mM; Hill coefficient, 2.1; K m for succinate, 18 M). NaDC3 was best inhibited by succinate (IC 50 25.5 M) and less by ␣-ketoglutarate (IC 50 69.2 M) and fumarate (IC50 95.2 M). Dicarboxylates with longer carbon backbones (adipate, pimelate, suberate) had low or no affinity for NaDC3. OAT1 exhibited the highest affinity for glutarate, ␣-ketoglutarate, and adipate (IC50 between 3.3 and 6.2 M), followed by pimelate (18.6 M) and suberate (19.3 M). The affinity of OAT1 to succinate and fumarate was low. OAT3 showed the same dicarboxylate selectivity with ϳ13-fold higher IC50 values compared with OAT1. The data 1) reveal ␣-ketoglutarate as a common high-affinity substrate of NaDC3, OAT1, and OAT3 and 2) suggest potentially similar molecular structures of the binding sites in OAT1 and OAT3 for dicarboxylates.EFFICIENT EXCRETION OF ANIONIC endogenous waste products and exogenous compounds including drugs and toxins is an important task of the kidneys. Many organic anions (OA) undergo active trans-cellular secretion in renal proximal tubules, involving OA uptake across the basolateral membrane and OA release across the luminal membrane of proximal tubule cells. The uptake of OA from blood across the basolateral membrane into tubule cells is remarkable in several ways. First, OA uptake is considered the rate-limiting step in secretion; second, the uptake occurs against an opposing intracellular negative membrane potential difference; third, a multitude of chemically different OA is accepted as substrates for secretion (8,30,36). The mechanism by which OA overcome the opposing driving force during basolateral uptake was clarified in studies with basolateral membrane vesicles isolated from rat kidneys. Shimada et al. (29) and Pritchard (25) suggested the cooperation of two transporters: a sodium-driven dicarboxylate cotransporter and an OA/dicarboxylate exchanger.As regards the molecular identity of these cooperating transporters, the sodium-dicarboxylate cotransporter 3 (NaDC3, SLC13A3) takes up dicarboxylates into the cells and thus provides substrates for exchange against extracellular OA through the organic anion transporters 1 and 3 (OAT1, SLC22A6; and OAT3, SLC22A8). NaDC3 was cloned from human, rat, mouse, and winter flounder and found to be expressed in kidneys, liver, placenta, and...

In this study, we introduce a new methodology to study post-endocytotic GPCR trafficking using fusions with the recently cloned Kaede protein. In contrast to the widely used green fluorescent protein, the fluorescence of Kaede can be converted from green to red using ultraviolet irradiation. Our methodology allows to study recycling of GPCRs microscopically in real-time bypassing problems with secretory pathway receptors. Initially, receptors are internalized using an agonist. Fluorescence signals in endosomes are switched, and trafficking of the receptors to the plasma membrane can be easily visualized by monitoring their new fluorescence. Using this methodology, we show that the corticotropin-releasing factor receptor type 1 belongs to the family of recycling GPCRs. Moreover, we demonstrate by fluorescence correlation spectroscopy that Kaede does not oligomerize when fused to membrane proteins, representing an additional advantage of this technique. The Kaede technology may be a powerful tool to study membrane protein trafficking in general.