Anja Schweizer

The endoplasmic reticulum—Golgi intermediate compartment

Abstract The recent identification of an endoplasmic reticulum-Golgi intermediate compartment has added to the complexity of the structural and functional organization of the early secretory pathway. Protein sorting along the endoplasmic reticulum-Golgi pathway depends on different signals and mechanisms, some of which guarantee recycling from various levels of the Golgi apparatus to biosynthetically earlier compartments.

XCE, a new member of the endothelin-converting enzyme and neutral endopeptidase family, is preferentially expressed in the CNS

In the present study, we have isolated a cDNA encoding a novel member of the family of zinc metallopeptidases that includes neutral endopeptidase and endothelin-converting enzyme. The predicted amino-acid sequence of this enzyme, termed XCE, consists of 775 amino-acids with a single putative membrane-spanning region, an N-terminal cytoplasmic domain of 59 residues, and a large luminal domain that contains a characteristic zinc-binding motif. Western blot analysis of cells stably expressing this new metallopeptidase revealed a glycosylated protein of approximately 95 kDa. XCE mRNA was found to be predominantly expressed in the central nervous system, sympathetic ganglia and in uterine subepithelial cells. In the rat and human CNS, a very specific pattern of neuronal labelling (in presumptive cholinergic interneurons of basal ganglia, basal forebrain neurons, as well as brainstem and spinal cord motoneurons) was detected by in situ hybridization histochemistry. The enzyme substrate, as yet unidentified, might be found among the numerous neuropeptide transmitters which are colocalized with acetylcholine in these neurons.

Neonatal lethality in mice deficient in XCE, a novel member of the endothelin-converting enzyme and neutral endopeptidase family

XCE, a new member of the endothelin-converting enzyme and neutral endopeptidase family, is preferentially expressed in specific areas of the central nervous system including spinal chord and medulla. To elucidate the importance and function of XCE, we disrupted its gene in mouse embryonic stem cells by homologous recombination and created mice deficient in XCE. The resulting phenotype is characterized by neonatal lethality. All XCE −/− homozygous mice died of respiratory failure shortly after birth, and in most cases their lungs were never ventilated. Apart from the atelectasis, anatomical and histological examinations of embryonic day 18.5 XCE −/− embryos and newborn homozygotes did not reveal any obvious abnormalities in organs and tissues. Malformations that are related to the knock-out were also not found in the skeletons of XCE −/− mice. In addition, XCE knock-out animals showed no deficiency of pulmonary surfactant proteins and had normal heart beat frequencies. Taken together, our results demonstrate that XCE is an essential gene. The phenotype of the XCE-deficient mice together with the central nervous system-specific expression further suggest that XCE may play a vital role in the control of respiration.

A di-aromatic motif in the cytosolic tail of the mannose receptor mediates endosomal sorting.

The mannose receptor (MR), the prototype of a new family of multilectin receptor proteins important in innate immunity, undergoes rapid internalization and recycling from the endosomal system back to the cell surface. Sorting of the MR in endosomes prevents the receptor from entering lysosomes where it would be degraded. Here, we focused on a diaromatic sequence (Tyr18-Phe19) in the MR cytoplasmic tail as an endosomal sorting signal. The subcellular distribution of chimeric constructs between the MR and the cation-dependent mannose 6-phosphate receptor was assessed by Percoll density gradients and cell surface assays. Unlike the wild type constructs, mutant receptors with alanine substitutions of Tyr18-Phe19 were highly missorted to lysosomes, indicating that the di-aromatic motif of the MR cytoplasmic tail mediates sorting in endosomes. Within this sequence Tyr18is the key residue with Phe19 contributing to this function. Moreover, Tyr18 was also found to be essential for internalization, consistent with the presence of overlapping signals for internalization and endosomal sorting in the cytosolic tail of the MR. A di-aromatic amino acid sequence in the cytosolic tail has now been shown to function in two receptors known to be internalized from the plasma membrane, the MR and the cation-dependent mannose 6-phosphate receptor. This feature therefore appears to be a general determinant for endosomal sorting.

A new family of orphan G protein‐coupled receptors predominantly expressed in the brain1

The cloning of a cDNA encoding a G protein‐coupled receptor homologous to the endothelin type B receptor, but unable to bind endothelin, was recently reported and termed ETBR‐LP. We report here the isolation of a human cDNA encoding a receptor that is highly related to ETBR‐LP and which was therefore termed ETBR‐LP‐2. Comparison of the two amino acid sequences revealed 68% overall homology and 48% identity. As is the case for ETBR‐LP, the new receptor is strongly expressed in the human central nervous system (e.g. in cerebellar Bergmann glia, cerebral cortex, internal capsule fibers). Membranes of HEK‐293 cells stably expressing ETBR‐LP‐2 did not bind endothelin‐1, endothelin‐2, endothelin‐3, bombesin, cholecystokinin‐8 or gastrin‐releasing peptide.