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At least 19 recordsLinked to original sources

PubMed2006

The Drosophila ortholog of the endolysosomal membrane protein, endolyn, regulates cell proliferation.

Endolyn (CD164) is a sialomucin that regulates the proliferation, adhesion, and migration of human haematopoietic stem and progenitor cells. This molecule is predominately localized in endocytotic compartments, where it may contribute to endolysosomal biogenesis and trafficking. In order to more closely define the function of endolyn from an evolutionary view-point, we first analyzed endolyn orthologs in species ranging from insects, fish, and birds to mammals. The predicted molecular structures of the endolyn orthologs from these species are well conserved, particularly with respect to significant O-linked glycosylation of the extracellular domain, and the high degree of amino acid similarities within their transmembrane and cytoplasmic domains, with the latter possessing the lysosomal target signal, YXXphi. Focusing on Drosophila, our studies showed that the subcellular distribution of endolyn in non-polarized Drosophila S2 cells resembles that of its human counterpart in hematopoietic cells, with its predominant localization being within intracellular vesicles, while a small fraction occurs on the cell surface. Both Y --> A and L --> A mutations in the YHTL motif perturbed the normal subcellular distribution of Drosophila endolyn. Interestingly, embryonic and early larval development was often arrested in endolyn-deficient Drosophila mutants. This may partly be due to the role of endolyn in regulating cell proliferation, since knock-down of endolyn expression in S2 cells resulted in up to 50% inhibition of cell growth, with a proportion of cells undergoing apoptosis. Taken together, these results demonstrate that endolyn is an evolutionarily conserved sialomucin fundamentally involved in cell proliferation in both the human and Drosophila melanogaster.

Amino Acid Sequence↗

PubMed2003

Specific N-glycans direct apical delivery of transmembrane, but not soluble or glycosylphosphatidylinositol-anchored forms of endolyn in Madin-Darby canine kidney cells.

The sialomucin endolyn is a transmembrane protein with a unique trafficking pattern in polarized Madin-Darby canine kidney cells. Despite the presence of a cytoplasmic tyrosine motif that, in isolation, is sufficient to mediate basolateral sorting of a reporter protein, endolyn predominantly traverses the apical surface en route to lysosomes. Apical delivery of endolyn is disrupted in tunicamycin-treated cells, implicating a role for N-glycosylation in apical sorting. Site-directed mutagenesis of endolyn's eight N-glycosylation sites was used to identify two N-glycans that seem to be the major determinants for efficient apical sorting of the protein. In addition, apical delivery of endolyn was disrupted when terminal processing of N-glycans was blocked using glycosidase inhibitors. Missorting of endolyn occurred independently of the presence or absence of the basolateral sorting signal, because apical delivery was also inhibited by tunicamycin when the cytoplasmic tyrosine motif was mutated. However, we found that apical secretion of a soluble mutant of endolyn was N-glycan independent, as was delivery of glycosylphosphatidylinositol-anchored endolyn. Thus, specific N-glycans are only essential for the apical sorting of transmembrane endolyn, suggesting fundamental differences in the mechanisms by which soluble, glycosylphosphatidylinositol-anchored, and transmembrane proteins are sorted.

Animals↗

PubMed2001

Competing sorting signals guide endolyn along a novel route to lysosomes in MDCK cells.

We have examined the trafficking of the mucin-like protein endolyn in transfected, polarized MDCK cells using biochemical approaches and immunofluorescence microscopy. Although endolyn contains a lysosomal targeting motif of the type YXXPhi and was localized primarily to lysosomes at steady state, significant amounts of newly synthesized endolyn were delivered to the apical cell surface. Antibodies to endolyn, but not lamp-2, were preferentially internalized from the apical plasma membrane and efficiently transported to lysosomes. Analysis of endolyn-CD8 chimeras showed that the lumenal domain of endolyn contains apical targeting information that predominates over basolateral information in its cytoplasmic tail. Interestingly, surface polarity of endolyn was independent of O-glycosylation processing, but was reversed by disruption of N-glycosylation using tunicamycin. At all times, endolyn was soluble in cold Triton X-100, suggesting that apical sorting was independent of sphingolipid rafts. Our data indicate that a strong, N-glycan-dependent apical targeting signal in the lumenal domain directs endolyn into a novel biosynthetic pathway to lysosomes, which occurs via the apical surface of polarized epithelial cells.

Amino Acid Motifs↗

PubMed2000

Endolyn is a mucin-like type I membrane protein targeted to lysosomes by its cytoplasmic tail.

Endolyn (endolyn-78) is a membrane protein found in lysosomal and endosomal compartments of mammalian cells. Unlike 'classical' lysosomal membrane proteins, such as lysosome-associated membrane protein (lamp)-1, it is also present in a subapical compartment in polarized WIF-B hepatocytes. The structural features that determine sorting of endolyn are unknown. We have identified a rat endolyn cDNA by expression screening. The cDNA encodes a ubiquitously expressed type I membrane protein with a short cytoplasmic tail of 13 amino acids and many putative sites for N- and O-linked glycosylation in the predicted luminal domain. Endolyn is closely related to two human mucin-like proteins, multi-glycosylated core protein (MGC)-24 and CD164 (MGC-24v), expressed in gastric carcinoma cells and bone marrow stromal and haematopoietic precursor cells respectively. The predicted transmembrane and cytoplasmic tail domains of endolyn, as well as parts of its luminal domain, also show some similarities with lamp-1 and lamp-2. Like these and other known lysosomal membrane proteins, endolyn contains a YXXO motif at the C-terminus of its cytoplasmic tail (where O is a bulky hydrophobic amino acid), but with no preceding glycine. Nonetheless, the last ten amino acids of this tail, when transplanted on to human CD8, caused efficient targeting of the chimaeric protein to endosomes and lysosomes in transfected normal rat kidney cells.

Amino Acid Sequence↗

PubMed1989

Endolyn-78, a membrane glycoprotein present in morphologically diverse components of the endosomal and lysosomal compartments: implications for lysosome biogenesis.

A monoclonal antibody (2C5) raised against rat liver lysosomal membranes was used to identify a 78-kD glycoprotein that is present in the membranes of both endosomes and lysosomes and, therefore, is designated endolyn-78. In cultures of rat hepatoma (Fu5C8) and kidney cells (NRK), this glycoprotein could not be labeled with [35S]methionine or with [32P]inorganic phosphate but was easily labeled with [35S]cysteine and [3H]mannose. Pulse-chase experiments and determinations of endoglycosidase H (endo H) sensitivity showed that endolyn-78 is derived from a precursor of Mr 58-62 kD that is processed to the mature form with a t1/2 of 15-30 min. The protein has a 22-kD polypeptide backbone that is detected after a brief pulse in tunicamycin-treated cells. During a chase in the presence of the drug, this is converted into an O-glycosylated product of 46 kD that despite the absence of N-linked oligosaccharides is effectively transferred to lysosomes. This demonstrates that the delivery of endolyn-78 to this organelle is not mediated by the mannose-6-phosphate receptor (MPR). Immunocytochemical experiments showed that endolyn-78 is present in the limiting membranes and the interior membranous structures of morphologically identifiable secondary lysosomes that contain the lysosomal hydrolase beta-glucuronidase, lack the MPR, and could not be labeled with alpha-2-macroglobulin at 18.5 degrees C, a temperature which prevents appearance of endocytosed markers in lysosomes. Endolyn-78 was present at low levels in the plasma membrane and in peripheral tubular endosomes, but was prominent in morphologically diverse components of the endosomal compartment (vacuolar endosomes and various types of multivesicular bodies) which acquired alpha-2-macroglobulin at 18.5 degrees C, and frequently contained substantial levels of the MPR and variable levels of beta-glucuronidase. On the other hand, the MPR was very rarely found in endolyn-containing structures that were not labeled with alpha-2-macroglobulin at the low temperature. Thus, the process of lysosomal maturation appears to involve the progressive delivery of lysosomal enzymes to various types of endosomes that may have already received some of the lysosomal membrane proteins. Although endolyn-78 would be one of the proteins added early to endosomes, other lysosomal membrane proteins may be added only to multivesicular endosomes that represent very advanced stages of maturation.

Animals↗

PubMed2006

N-glycans mediate apical recycling of the sialomucin endolyn in polarized MDCK cells.

Apical and basolateral proteins are maintained within distinct membrane subdomains in polarized epithelial cells by biosynthetic and postendocytic sorting processes. Sorting of basolateral proteins in these processes has been well studied; however, the sorting signals and mechanisms that direct proteins to the apical surface are less well understood. We previously demonstrated that an N-glycan-dependent sorting signal directs the sialomucin endolyn to the apical surface in polarized Madin-Darby canine kidney cells. Terminal processing of a subset of endolyn's N-glycans is key for polarized biosynthetic delivery to the apical membrane. Endolyn is subsequently internalized, and via a cytoplasmic tyrosine-based sorting motif is targeted to lysosomes from where it constitutively cycles to the cell surface. Here, we examine the polarized sorting of endolyn along the postendocytic pathway in polarized cells. Our results suggest that similar N-glycan sorting determinants are required for apical delivery of endolyn along both the biosynthetic and the postendocytic pathways.

Animals↗

PubMed1998

Apical plasma membrane proteins and endolyn-78 travel through a subapical compartment in polarized WIF-B hepatocytes.

We studied basolateral-to-apical transcytosis of three classes of apical plasma membrane (PM) proteins in polarized hepatic WIF-B cells and then compared it to the endocytic trafficking of basolaterally recycling membrane proteins. We used antibodies to label the basolateral cohort of proteins at the surface of living cells and then followed their trafficking at 37 degreesC by indirect immunofluorescence. The apical PM proteins aminopeptidase N, 5'nucleotidase, and the polymeric IgA receptor were efficiently transcytosed. Delivery to the apical PM was confirmed by microinjection of secondary antibodies into the bile canalicular-like space and by EM studies. Before acquiring their apical steady-state distribution, the trafficked antibodies accumulated in a subapical compartment, which had a unique tubulovesicular appearance by EM. In contrast, antibodies to the receptors for asialoglycoproteins and mannose-6-phosphate or to the lysosomal membrane protein, lgp120, distributed to endosomes or lysosomes, respectively, without accumulating in the subapical area. However, the route taken by the endosomal/lysosomal protein endolyn-78 partially resembled the transcytotic pathway, since anti-endolyn-78 antibodies were found in a subapical compartment before delivery to lysosomes. Our results suggest that in WIF-B cells, transcytotic molecules pass through a subapical compartment that functions as a second sorting site for a subset of basolaterally endocytosed membrane proteins reaching this compartment.

5'-Nucleotidase↗

PubMed2005

Epitope recognition of antibodies that define the sialomucin, endolyn (CD164), a negative regulator of haematopoiesis.

Endolyn (CD164) is a sialomucin that functions as an adhesion molecule and a negative regulator of CD34+ CD38- human haematopoietic precursor cell proliferation. The 105A5 and 103B2/9E10 CD164 monoclonal antibodies (mAbs), which act as surrogate ligands, recognize distinct glycosylation-dependent classes I and II epitopes located on domain I of the native and recombinant CD164 proteins. Here, we document five new CD164 mAbs, the 96 series, that rely on conformational integrity, but not glycosylation, of exons 2- and 3-encoded CD164 domains, thereby resembling the class III mAbs, N6B6 and 67D2. Although all the 96 series class III mAbs labelled both the 105A5+ and 103B2/9E10+ cells, cross-competition and immunoblotting studies allow them to be categorized into two distinct class III subgroups, i.e. the N6B6-like subgroup that only recognizes 80-100 kDa proteins and the 67D2-like subgroup that also recognizes a higher molecular weight (>220 kDa) form. To more closely define the reactivity patterns of mAbs to the classes I and II epitopes, the global glycosylation patterns of the soluble human (h) CD164 proteins were determined using lectin binding, high-performance liquid chromatography (HPLC) and mass spectrometry. hCD164 recombinant proteins bound to the lectins, Galanthus nivalis agglutinin, Datura stramonium agglutinin, Sambucus nigra agglutinin, Maackia amurensis agglutinin and peanut agglutinin, indicating the presence of high mannose and complex N-glycans, in addition to core 1 O-glycans (the Tn antigen) and alpha2-3 and alpha2-6 sialic acid moieties. Our HPLC and mass spectrometry results revealed both high mannose and complex N-glycosylation with various numbers of branches increasing the complexity of the glycosylation pattern. Most O-glycans were small, core 1 or 2 based. High levels of sialylation in alpha2-3 and alpha2-6 linkages, without sialyl-Lewis X, indicate that the majority of these hCD164 recombinant proteins are unable to bind to selectins in our assay system, but may interact with Siglec molecules.

Agglutinins↗

PubMed2006

Endolyn (CD164) modulates the CXCL12-mediated migration of umbilical cord blood CD133+ cells.

Hematopoietic stem cell/hematopoietic progenitor cell (HSC/HPC) homing to specific microenvironmental niches involves interactions between multiple receptor ligand pairs. Although CXCL12/CXCR4 plays a central role in these events, CXCR4 regulators that provide the specificity for such cells to lodge and be retained in particular niches are poorly defined. Here, we provide evidence that the sialomucin endolyn (CD164), an adhesion receptor that regulates the adhesion of CD34+ cells to bone marrow stroma and the recruitment of CD34+CD38(lo/-) cells into cycle, associates with CXCR4. The class II 103B2 monoclonal antibody, which binds the CD164 N-linked glycan-dependent epitope or CD164 knockdown by RNA interference, significantly inhibits the migration of CD133+ HPCs toward CXCL12 in vitro. On presentation of CXCL12 on fibronectin, CD164 associates with CXCR4, an interaction that temporally follows the association of CXCR4 with the integrins VLA-4 and VLA-5. This coincides with PKC-zeta and Akt signaling through the CXCR4 receptor, which was disrupted on the loss of CD164 though MAPK signaling was unaffected. We therefore demonstrate a novel association among 3 distinct families of cell-surface receptors that regulate cell migratory responses and identify a new role for CD164. We propose that this lends specificity to the homing and lodgment of these cells within the bone marrow niche.

AC133 Antigen↗

PubMed2006

The role of sialomucin CD164 (MGC-24v or endolyn) in prostate cancer metastasis.

BACKGROUND: The chemokine stromal derived factor-1 (SDF-1 or CXCL12) and its receptor CXCR4 have been demonstrated to be crucial for the homing of stem cells and prostate cancers to the marrow. While screening prostate cancers for CXCL12-responsive adhesion molecules, we identified CD164 (MGC-24) as a potential regulator of homing. CD164 is known to function as a receptor that regulates stem cell localization to the bone marrow. RESULTS: Using prostate cancer cell lines, it was demonstrated that CXCL12 induced both the expression of CD164 mRNA and protein. Functional studies demonstrated that blocking CD164 on prostate cancer cell lines reduced the ability of these cells to adhere to human bone marrow endothelial cells, and invade into extracellular matrices. Human tissue microarrays stained for CD164 demonstrated a positive correlation with prostate-specific antigen levels, while its expression was negatively correlated with the expression of androgen receptor. CONCLUSION: Our findings suggest that CD164 may participate in the localization of prostate cancer cells to the marrow and is further evidence that tumor metastasis and hematopoietic stem cell trafficking may involve similar processes.

Bone Marrow Neoplasms↗

PubMed2004

Differential use of two AP-3-mediated pathways by lysosomal membrane proteins.

The adaptor protein complex AP-3 is involved in the sorting of lysosomal membrane proteins to late endosomes/lysosomes. It is unclear whether AP-3-containing vesicles form at the trans-Golgi network (TGN) or early endosomes. We have compared the trafficking routes of endolyn/CD164 and 'typical' lysosomal membrane glycoproteins (lgp120/lamp-1 and CD63/lamp-3) containing cytosolic YXXPhi-targeting motifs preceded by asparagine and glycine, respectively. Endolyn, which has a NYHTL-motif, is concentrated in lysosomes, but also occurs in endosomes and at the cell surface. We observed predominant interaction of the NYHTL-motif with the mu-subunits of AP-3 in the yeast two-hybrid system. Endolyn was mislocalized to the cell surface in AP-3-deficient pearl cells, confirming a major role of AP-3 in endolyn traffic. However, lysosomal delivery of endolyn (or a NYHTL-reporter), but not GYXXPhi-containing proteins, was practically abolished when AP-2-mediated endocytosis or traffic from early to late endosomes was inhibited in NRK and 3T3 cells. This indicates that endolyn is mostly transported along the indirect lysosomal pathway (via the cell surface), rather than directly from the TGN to late endosomes/lysosomes. Our results suggest that AP-3 mediates lysosomal sorting of some membrane proteins in early endosomes in addition to sorting of proteins with intrinsically strong AP-3-interacting lysosomal targeting motifs at the TGN.

Adaptor Protein Complex 3↗

PubMed2000

Relationship between novel isoforms, functionally important domains, and subcellular distribution of CD164/endolyn.

Functional analyses have indicated that the human CD164 sialomucin may play a key role in hematopoiesis by facilitating the adhesion of human CD34(+) cells to the stroma and by negatively regulating CD34(+)CD38(lo/-) cell proliferation. We have identified three novel human CD164 variants derived by alternative splicing of bona fide exons from a single genomic transcription unit. The predominant CD164(E1-6) isoform, encoded by six exons, is a type I transmembrane protein containing two extracellular mucin domains (I and II) interrupted by a cysteine-rich non-mucin domain. The 103B2/9E10 and 105A5 epitopes, which specify ligand binding characteristics, are located on the exon 1-encoded mucin domain I. Three human CD164(E1-6) mRNA species, exhibiting differential polyadenylation site usage, are differentially expressed in hematopoietic and non-hematopoietic tissues. This study provides additional evidence that human CD164(E1-6) represents the ortholog of murine MGC-24v and rat endolyn. Comparative analysis of murine MGC-24v/CD164(E1-6) with human CD164(E1-6) revealed two potential splice variants and a similar genomic structure. Whereas the human CD164 gene is located on chromosome 6q21, the mouse gene occurs in a syntenic region on chromosome 10B1-B2. By confocal microscopy, human CD164 in CD34(+)CD38(+) hematopoietic progenitor (KG1B) and epithelial cell lines appears to be localized primarily in endosomes and lysosomes, with low concentrations at the cell surface. However, in a minority of KG1B cells, CD164 is more prominently expressed at the plasma membrane and in the recycling endosomes, suggesting that its distribution is regulated in cells of hematopoietic origin.

Amino Acid Sequence↗

PubMed2003

A dominant negative form of the AAA ATPase SKD1/VPS4 impairs membrane trafficking out of endosomal/lysosomal compartments: class E vps phenotype in mammalian cells.

SKD1 is a member of the family of ATPases associated with cellular activities whose yeast homologue Vps4p has been implicated in endosomal/vacuolar membrane transports. When a mutant of SKD1 that lacks ATPase activity [SKD1(E235Q)] was overexpressed in mammalian cells, it induced a dominant negative phenotype characterized by aberrant endosomal structures (denoted as E235Q compartments). Expression of SKD1(E235Q) caused an accumulation of basolateral recycling receptors, such as asialoglycoprotein receptor and low-density lipoprotein in polarized hepatocytes and Madin-Darby canine kidney cells, respectively, in E235Q compartments. In addition, SKD1(E235Q) also abrogated, via endosomes, transport to the trans-Golgi network, as indicated by an accumulation of TGN38 in E235Q compartments. Three lines of evidence further demonstrated that SKD1 participates in the membrane transport from early endosomes to late endosomes/lysosomes: (1) a redistribution of a late endosomal and lysosomal membrane protein endolyn in E235Q compartments; (2) an inhibition of epidermal growth factor receptor degradation, due to an accumulation of the receptors in E235Q compartments; and (3) a mis-sorting of and defect in the proteolytic processing of newly synthesized cathepsin D. An intriguing finding was that the expression of SKD1(E235Q) caused the number of lysosomes to decrease (to one-sixth of control numbers) but their size to increase (2.4-fold larger in diameter than control lysosomes). Indeed, an ultrastructural analysis revealed that the expression of SKD1(E235Q) causes an accumulation of hybrid organelles formed by direct fusion between late endosomes and lysosomes. We conclude that SKD1 regulates multiple steps of membrane transport out of early endosomes and the reformation of lysosomes from a hybrid organelle.

ATPases Associated with Diverse Cellular Activitie↗

PubMed2001

A novel retinoid-response gene set in vascular smooth muscle cells.

A modified suppression subtractive hybridization assay was performed to uncover genes induced by all-trans retinoic acid in cultured smooth muscle cells (SMC). Northern blotting studies confirmed the induction of 14 genes, many of which have heretofore been unrecognized as retinoid-inducible. Temporal expression and cycloheximide studies allowed us to categorize these genes as either immediate-early (LOX-1, endolyn, Stoned B/TFIIA alpha/beta-like factor, Src Suppressed C Kinase Substrate, and tissue transglutaminase) or delayed (cathepsin-L, ceruloplasmin, epithelin, importin alpha, alpha(8)-integrin, lactate dehydrogenase B, retinol dehydrogenase, spermidine/spermine N(1)-acetyltransferase, and VCAM-1) retinoid-response genes. A survey of rat tissues showed two of the genes (tissue transglutaminase and alpha(8)-integrin) to be highly restricted to vascular tissue. In situ hybridization verified expression of both tissue transglutaminase and alpha(8)-integrin to SMC in balloon-injured rat carotid artery. These findings unveil a new retinoid-response gene set that should be exploited to define molecular pathways involved in the antagonistic effects of retinoids on SMC growth and neointimal formation.

Animals↗

PubMed2002

Autophagy in hypothalamic neurones of rats expressing a familial neurohypophysial diabetes insipidus transgene.

We have tested the hypothesis that familial neurohypophysial diabetes insipidus (FNDI) is initiated by a process of autophagy. FNDI is a dominant, progressive inherited disorder characterized by pronounced drinking and urination caused by loss of secretion of antidiuretic hormone (vasopressin). In rats expressing an FNDI mutant transgene (Cys67stop) in vasopressin magnocellular neurones, the mutant protein fails to enter the regulated secretory pathway, and accumulates in a swollen and distended endoplasmic reticulum (ER) that also contains wild-type, endogenous vasopressin. Transmission electron microscopy suggested that these are autophagic vesicles. We have now examined the expression of vesicular markers in our transgenic rats, and demonstrate that activation of autolysosomal processes is a consequence of the expression of Cys67stop. Swollen vesicles containing Cys67stop are immunoreactive for cathepsin D (a lysosomal protease), endolyn (a marker of late endosomes) and lysosomal associated membrane protein 1, suggesting that they may be degradative autolysosomes. In addition, there is an up-regulation of lysosomal markers specifically in cells expressing Cys67stop. The expression of Cys67stop affects neither the trans-Golgi network nor early endosomes. These data support the proposal that Cys67stop mutant protein aggregates within the ER, which is targeted for lysosomal degradation by autophagy.

Animals↗

PubMed2000

Apical endocytosis in rat hepatocytes In situ involves clathrin, traverses a subapical compartment, and leads to lysosomes.

BACKGROUND & AIMS: This study demonstrates and characterizes apical (canalicular) endocytic pathways in hepatocytes in situ. METHODS: Endocytic markers were administered by retrograde infusion through the common bile duct. Colocalization with proteins that are specific for various endocytic compartments was performed on stacks of deconvoluted confocal immunofluorescence images. The subcellular distribution of marker proteins was assessed by electron microscopy (EM). RESULTS: Bulk-phase, as well as membrane-associated markers, were internalized readily at the apical cell pole. At the EM level, marker was found initially in 60-100-nm tubulovesicular structures and 150-200-nm cup-shaped vesicles, whereas multivesicular bodies and lysosomes became labeled after longer time intervals. Apical endocytosis involved clathrin and delivered marker to late endosomes (rab7(+), cathepsin D(+)), as well as lysosomes (rab7(-), cathepsin D(+)). Simultaneous labeling of the basolateral endocytic route resulted in overlap of both pathways in the late endosomal and lysosomal compartments. In addition, apical endocytosis involved a subapical compartment (endolyn-78(+), rab11(+), polymeric IgA receptor [pIgA-R(+)]) that is passed by the transcytotic route, thus constituting a crossroads. pIgA-R immunoreactivity, probably reflecting the cleaved receptor fragment, was associated with apical endocytic marker and colocalized with clathrin and later with cathepsin D. CONCLUSIONS: Apical endocytosis involves coated pits/vesicles, leads to a subapical compartment, and plays a role in the retrieval of canalicular plasma membrane components for lysosomal degradation.

Animals↗

PubMed1993

Incorporation of the pancreatic membrane protein GP-2 into secretory granules in exocrine but not endocrine cells.

The pancreatic zymogen granule membrane protein GP-2 was introduced into cells of exocrine or endocrine origin by transfection of its cDNA in order to investigate the mechanisms by which proteins are specifically incorporated into the membranes of secretory granules. Permanent transformants expressing GP-2 were isolated from exocrine pancreatic-derived AR42J cells as well as AtT20 cells of anterior pituitary origin and insulinoma-derived Rin5F cells. In AR42J cells, GP-2 was localized by immunofluorescence and immunoelectron microscopy to the endogenous zymogen-like granules as well as to the plasma membrane. In experiments supporting the localization data, incubation of the AR42J transformants with the secretagogue cholecystokinin (CCK8) resulted in enhanced release of a shed form of GP-2 into the medium in parallel with amylase, suggesting that the two proteins were secreted from the same compartment. By contrast, when expressed in AtT20 cells, the protein was found by immunofluorescence microscopy on the plasma membrane as well as in intracellular vesicles that differed in size and location from the endogenous secretory vesicles. By electron microscopy, large (approximately 0.5 micron) multivesicular structures were observed. Single- and double-label immunoelectron microscopy demonstrated that these large organelles labeled with anti-GP-2 antibodies, whereas the smaller adrenocorticotropic hormone (ACTH)-containing secretory vesicles did not. In permanent transformants of Rin5F cells, GP-2 was also excluded from the insulin-containing granules and found in multivesicular bodies similar to those in the AtT20 cells and containing the endosomal/lysosomal marker endolyn-78. Despite the apparent accumulation of GP-2 in lysosome-like structures, it turned over slowly and did not undergo rapid endocytosis from the cell surface. We conclude that GP-2 is targeted to secretory granule membranes by cell type-specific mechanisms that likely involve its interaction with other membrane or content proteins expressed only in the exocrine cells.

Amylases↗

PubMed2005

Positive regulation of Fas gene expression by MSSP and abrogation of Fas-mediated apoptosis induction in MSSP-deficient mice.

MSSP has been identified as a transcription factor that regulates the c-myc gene. MSSP was later found to positively or negatively regulate a variety of genes, including alpha-smooth actin, MHC class I, MHC class 2 and the thyrotropin receptor. The knockout mice for the Mssp gene developed by us revealed that these mice became partially embryonic lethal due to a low concentration of progesterone at E2.5. In this study, we further analyzed Mssp-knockout mice and found that the expression of the Fas gene was repressed, resulting in abrogation of Fas-mediated induction of apoptosis both in Mssp-knockout mice and primary thymocytes. MSSP was then found to stimulate promoter activity of the Fas gene by binding to a region spanning -1035 to -635 in chromatin immunoprecipitation assays. Binding of MSSP in the MSSP-binding sequence, TCTAAT, located in this region was confirmed by mobility shift assays, and deletion of this sequence from the Fas promoter was found to result in loss of MSSP-dependent stimulating activity. The results suggest that MSSP is an important mediator for Fas-induced apoptosis in vivo and in vitro.

Animals↗