Human endothelin-converting enzyme (ECE-1) has been shown to exist as three isoforms (ECE-1a, ECE-1b and ECE-1c) diverging in their N-terminal sequence and displaying different patterns of subcellular localization. We report here the cloning of ECE-1d, a novel isoform of 767 amino acids, which is generated from the same gene via the existence of an additional promoter located upstream from the third exon of the ECE-1 gene. ECE-1d converting activity is comparable to that of the other three isoenzymes. In contrast to ECE-1b, ECE-1d is expressed at the cell surface, although less strongly than ECE-1a. We have also shown, by identifying ECE-1b and ECE-1d in rat, that the ECE-1 diversity is conserved between human and rodent, suggesting its physiological relevance. The mRNA levels of the four isoforms were assessed in the two species in various cell types, revealing some differences. In particular, the ECE-1a isoform, strongly expressed at the plasma membrane, was found to be highly expressed in primary cultures of endothelial cells but absent from primary cultures of smooth muscle cells.

Endothelin-converting enzyme (ECE) is a membrane metalloprotease that generates endothelin from its direct precursor big endothelin. Four isoforms of ECE-1 are produced from a single gene through the use of alternate promoters. These isoforms share the same extracellular catalytic domain and contain unique cytosolic tails, which results in their specific subcellular targeting. We investigated the distribution of ECE-1 isoforms in transfected AtT-20 neuroendocrine cells. Whereas ECE-1a and 1c were present at the plasma membrane, ECE-1b and ECE-1d were retained inside the cells. We found that both intracellular isoforms were concentrated in the endosomal system: ECE-1d in recycling endosomes, and ECE-1b in late endosomes/multivesicular bodies. Leucine-based motifs were involved in the intracellular retention of these isoforms, and the targeting of ECE-1b to the degradation pathway required an additional signal in the N terminus. The concentration of ECE-1 isoforms in the endosomal system suggested new functions for these enzymes. Potential novel functions include redistribution of other isoforms through direct interaction. We have showed that ECE-1 isoforms could heterodimerize, and that in such heterodimers the ECE-1b targeting signal was dominant. Interaction of a plasma membrane isoform with ECE-1b resulted in its intracellular localization and decreased its extracellular activity. These data demonstrated that the targeting signals specific for ECE-1b constitute a regulatory domain per se that could modulate the localization and the activity of other isoforms.Endothelins (ET 1 -1, ET-2, and ET-3) are 21-residue peptides derived from three distinct genes (1). They are pleiotropic factors that play an important role in the regulation of the cardiovascular and endocrine systems (for review see Refs. 2 and 3). Endothelins are also crucial developmental factors as demonstrated by the targeted disruption of the genes coding for the precursors of ET-1 and ET-3, and of the genes coding for their receptors, ETA and ETB (4 -7). In addition, the expression of endothelin is associated with many pathological processes and with tumor growth (2, 8 -10). In order to fulfill such a wide spectrum of physiological functions, endothelins act through autocrine and paracrine mechanisms. Their biosynthesis thus requires tight local control. Endothelins are synthesized in the endoplasmic reticulum as precursors that undergo a two-step proteolytic maturation. Pro-endothelins are first processed at conserved multibasic sites by furin or a furin-like enzyme, in order to release an intermediate called big endothelin (big-ET) (11, 12), which is devoid of biological activity (13). Big-ET is then processed by endothelin converting enzyme (ECE) at a Trp-Val/Ile bond, which releases the biologically active peptide. This latter proteolytic step can occur in the extracellular medium and in the secretory pathway, so that cells secrete either big-ETs alone or together with endothelins (14, 15, 16). Endothelial cells co-express the precursor, the c...

Three aspects of the secretory process in male rat prolactin (PRL) cells grown in primary cultures for 7--14 days have been investigated by cytochemical methods. The subcellular localization of prolactin has been studied using preembedding or postembedding immunocytochemical methods after various fixatives. With the postembedding method, PRL is localized essentially in secretory granules. The maximum intensity of staining is obtained with PAF fixative. With the preembedding method, subcellular localization of the staining varies depending on the fixative. After PAF-fixation, positive staining is observed on secretory granules, ground cytoplasm, the outer face of some RER cisternae and, in a few cells, on the innermost Golgi cisternae, as well as on masses of condensing secretory material. After Ohtsuki's hypotonic fixative followed by saponin permeabilization, PRL is visualized within the totality of RER cisternae, including the perinuclear cisternae and the peripheral saccules on the cis-Golgi face. Secretory granules are unstained. Membrane traffic was investigated using the Con A-HRP indirect method as a tracer of surface saccharides. Plasma membrane, coated with Con A-HRP at 4 degrees C, is slowly internalized at 37 degrees C. This involves both randomly distributed invaginations and capping. The final step of endocytosis (1--2 hours) is located in the Golgi zone, where very few smooth membranes are stained. In contrast, a conspicuous deposit is found around the dense content of secretory granules. This suggests a recycling of internalized membrane and a transfer of Con A-HRP from the inner face of smooth cisternae to the secretory material. The internalization of Con A-HRP-coated membrane leads to an inhibition of PRL release starting after 30 minutes. This is accompanied by a marked increase of acid phosphatase activity, mostly around forming and mature secretory granules.

(6A0727). trope, thyrotrope, and corticotrepe cells but were very scarce in prolactin cells and absent in somatotrope cells. In features or by immunodetection of its hormone content. These findings suggest that laminin might be synthesized and exported by all the glandular anterior pituitary cells, according to different pathways. Materials and Methods Materials Antibodies. Affinity-purified goat antibodies to laminin (a.laminin)

We have isolated a full-length murine clone corresponding to the rat neuronal p1A75 partial cDNA (Sutcliffe, J. G., Milner, R. J., Shinnick, T. M., and Bloom, F. E. (1983) Cell 33, 671-682). It encodes a 185-residue polypeptide that displays 56% identity with p19, a protein selectively expressed in the Golgi apparatus of neural cells (Sabé ran-Djoneidi, D., Marey-Semper, I., Picart, R., Studler, J.-M., Tougard, C., Glowinski, J., and Lé viStrauss, M. (1995) J. Biol. Chem. 270, 1888 -1893). An antibody directed against the recombinant polypeptide allowed us to demonstrate the existence of the natural 21-kDa protein (p21) in brain and its prominent juxtanuclear Golgi-like localization in cultured neurons. Ultrastructural observation of cultured neurons and analysis of transfected COS cells revealed a specific labeling of the Golgi apparatus, suggesting, as for p19, the presence of a Golgi targeting signal in its primary sequence. Surprisingly, p21, which is much more strongly expressed in the olfactory epithelium than p19, is also present in the Golgi complex of spermatocytes and in the flagellar middle piece of late spermatids.We have previously described a 19-kDa murine protein (p19) selectively expressed in the Golgi apparatus of neural and neuroendocrine cells whose human corresponding gene has been localized in 5q35 (1, 2). The primary sequence of p19 showed a 57% similarity with the translation product of an open reading frame of the rat neuronal p1A75 (3) partial cDNA, which was isolated 14 years ago and whose human corresponding gene is localized in 4p16 (4). Searches in protein data bases for other members of this new family have only revealed that these two proteins share a highly similar short segment with secretogranin III, which is expressed in intracellular vesicles of neural cells (1, 5).The co-localization, in two paralogous chromosomal regions (5q35 and 4p16), of the human p19 and p1A75 genes with other couples of homologous genes such as, for instance, the D1 and D5 dopamine receptors and the FGFR3 and FGFR4 fibroblast growth factor receptors suggested that these genes originated from the same large gene duplication event, which is thought to be the remnant of an ancient round of tetraploidization (6).To characterize this new protein family and to study the functional consequences of a well defined large duplication event, we undertook the thorough analysis of the protein encoded by the p1A75 cDNA.This encoded protein is a 21-kDa protein (p21) that is expressed, like p19, in the Golgi apparatus of neural and neuroendocrine cells. However, unlike p19, which is absent from the testis and faintly expressed in the olfactory epithelium, p21 is very strongly expressed in the olfactory epithelium and in male germ cells. EXPERIMENTAL PROCEDURESRNA Isolation and Northern Blotting-Total cellular RNA was extracted from fresh tissue or cells by the guanidium thiocyanate/phenol chloroform extraction method (7). Timed pregnant OFA rats (IffaCredo) provided a source of fetal and neonatal brains of precise gesta...

The mouse 8.5 mRNA encodes a 171-residue novel protein which displays a highly significant similarity with the product of the previously characterized neuronal p1A75 cDNA (Sutcliffe, J.G., Milner, R.J., Shinnick, T.M., and Bloom, F.E. (1983) Cell 33, 671-682). Northern blot and in situ hybridization experiments indicated that the 8.5 mRNA is specifically expressed in neural and neuroendocrine tissues. An affinity-purified antibody directed against the recombinant 8.5 protein demonstrated the existence of the 19-kDa natural protein in brain and evidenced its prominent juxtanuclear Golgi-like localization in cultured neurons. Ultrastructural analysis of the same preparation revealed a specific labeling of all the Golgi saccules and of some vesicles in the Golgi zone. In transfected COS cells, the exogenous protein was also detected in the Golgi area, indicating, therefore, the presence of a Golgi targeting signal in its primary sequence.

Antibodies against a lysosomal membrane antigen (A-Ly-M) have recently been obtained and characterized (Reggio, H., D. Bainton, E. Harms, E. Coudrier, and D. Louvard, 1984, J. Cell Biol., 99:1511-1526. They recognize a 100,000-mol-wt antigen immunologically related to a purified [H+,K+]ATPase from pig gastric mucosa. In the present study, we have localized this antigen during adsorptive endocytosis in rat prolactin cells in culture using cationized ferritin (CF) as a tracer. CF was rapidly internalized (after 5 min) in coated pits and vesicles that were labeled by antibodies against clathrin. The tracer was then delivered (after 15 rain) to vacuoles and multivesicular bodies. These structures were labeled with A-Ly-M. These organelles were devoid of acid phosphatase activity. At later stages (after 30 min) CF was observed within larger structures that were strongly stained by A-Ly-M and displayed a strong acid phosphatase activity. These findings clearly indicate that A-Ly-M react with prelysosomal and lysosomal compartments involved in the endocytic pathway in cultured prolactin cells. The membrane of these structures therefore contains antigenic determinant(s) related to the 100,000-mol-wt polypeptide. Our results suggest that the prelysosomal structure stained by A-Ly-M may represent in GH3 cells the acidic prelysosomal compartment recently described in the early steps of endocytosis in other cell types (Tycko, B., and F. R. Maxfield, 1982, Cell, 28:643-651).Acidification of endocytic vesicles involved as an early step in adsorptive or receptor-mediated endocytosis has recently been demonstrated using pH-sensitive fluorescent probes (4,12,19,26). This rapid acidification occurs before the fusion of endocytic vesicles with lysosomes and in the absence of lysosomal hydrolases.To explain the molecular mechanisms responsible for the acidification of both the prelysosomal and lysosomal compartments, the existence of an ATP-dependent proton pump has been postulated (for review, see reference 16). Such an activity has recently been demonstrated pharmacologically using isolated brain clathrin-coated vesicles (3), partially isolated endosomes, and lysosomes taken from fibroblasts and macrophages (4), or intact permeabilized fibroblasts (29).A new approach for studying membrane components that are possibly involved in the acidification of the endocytic pathway is provided by the recently characterized antibodies against a lysosomal membrane fraction that recognize a 100,000-mol-wt antigen immunologically related to a purified [H+,K+]ATPase from pig gastric mucosa (17, 18). Immunoelectron microscope localization of this antigen in normal rat kidney cells, rat macrophages, and hepatocytes revealed its presence not only in the membrane of secondary lysosomes but also in the membranes of several intracellular organelles known to be involved in receptor-mediated endocytosis (4,12,19,26). Moreover, some patches on the plasma membrane and some Golgi cisternae were also labeled (18).This prompted us to localize this...

The distribution of laminin was investigated by immunocytochemistry in the rat anterior pituitary in vivo and in primary culture. It was localized by immunofluorescence and by immunoperoxidase in the basement membranes of the pituitary in vivo. In addition it was also found inside glandular cells both in vivo and in culture. The number of immunoreactive cells greatly varied depending on the technical approach used. It was always higher in primary cultures than in vivo. At the electron microscope level, a staining was observed on secretory granules, on rough endoplasmic reticulum cisternae as well as on the membrane of some Golgi saccules and vesicles. Such a localization, at the level of subcellular sites involved in the secretory process, suggests that these cells are able to synthesize and to export in vivo as well as in vitro this component of their basement membranes.