RESUMEN

Immunological resistance of the chick embryo is dependent upon IgG present in the yolk of the layed egg. Here we show that complement factor 3 (C3), a key component of the humoral complement system, is a yolk component of chicken eggs. C3 is transported into oocytes by LR8-mediated endocytosis. LR8 also binds and transports other major yolk components such as vitellogenin, very-low-density lipoprotein, and alpha(2)-macroglobulin. Expression studies of LR8 during chicken development and oocyte maturation, in combination with studies on the uptake of individual yolk components, suggest the following model for oocyte maturation in the chicken: all oocytes present in the ovary contain high levels of LR8 mRNA and protein long before the onset of oocyte maturation. Selected oocytes gain access to yolk precursors, and LR8 binds, internalizes, and deposits the major yolk components in the ratio of their relative abundance in the accessible pool.

Asunto(s)

Proteínas Aviares/metabolismo , Complemento C3/metabolismo , Endocitosis , Lipoproteínas/metabolismo , Oocitos/metabolismo , Animales , Proteínas Aviares/genética , Línea Celular , Vesículas Cubiertas/metabolismo , Yema de Huevo/metabolismo , Femenino , Humanos , Técnicas In Vitro , Lipoproteínas/genética , Lipoproteínas VLDL/metabolismo , Oocitos/crecimiento & desarrollo , ARN Mensajero/metabolismo , Vitelogeninas/metabolismo , alfa-Macroglobulinas/metabolismo

Asunto(s)

Hiperlipidemias/diagnóstico , Hiperlipidemias/terapia , Lipoproteínas , Niño , Europa (Continente) , Femenino , Alemania , Humanos , Sociedades Médicas

RESUMEN

For many years, the low-density lipoprotein (LDL) receptor and the LDL receptor-related protein (LRP) have been considered to be prototypes of cargo receptors which deliver, via endocytosis, macromolecules into cells. However, the recent identification of additional members of this gene family and examination of their biology has revealed that at least some of these proteins are also signaling receptors. Very low density lipoprotein receptor and ApoER2 transmit the extracellular reelin signal into migrating neurons, and thus are key components of the reelin pathway which governs neuronal layering of the forebrain during embryonic brain development. LRP5 and LRP6 are integral components of the Wnt signaling pathway which is central to many processes of metazoan development, cell proliferation, and tumor formation. Adaptor proteins interacting with the cytosolic domains of these receptors might orchestrate their ability to deliver their cargo or a signal.

Asunto(s)

Endocitosis/fisiología , Receptores de LDL/fisiología , Transducción de Señal/fisiología , Animales , Proteínas del Huevo/fisiología , Humanos , Hiperlipoproteinemia Tipo II/metabolismo , Proteínas Relacionadas con Receptor de LDL , Lipoproteínas/metabolismo , Proteína-5 Relacionada con Receptor de Lipoproteína de Baja Densidad , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad , Receptores de Superficie Celular/fisiología , Receptores de LDL/química , Receptores de Lipoproteína/fisiología , Proteína Reelina

RESUMEN

Previously, we showed that mRNA for transglutaminase factor XIIIA (FXIIIA) is up-regulated in the hypertrophic zone of the growth plate of the chicken tibiotarsus, a well-characterized model of long bone development. In the present study, we have studied the distribution of the FXIIIA protein and of transglutaminase enzymatic activity in this growth plate, as well as in the cartilage of the epiphysis, which includes that of the articular surface. By immunohistochemical analysis, the protein is detected in the zone of maturation, where it is mostly intracellular, and in the hypertrophic zone, where it is present both intracellularly and in the extracellular matrix. The intracellular enzyme is mostly a zymogen, as determined with an antibody specific for the activation peptide. Externalization of FXIIIA is accompanied by enzyme activation. To study the pattern of transglutaminase activity, a synthetic transglutaminase substrate, rhodamine-conjugated tetrapeptide (Pro-Val-Lys-Gly), was used for pulse labeling in organ cultures. Intensive incorporation of the fluorescent substrate was observed throughout the hypertrophic zone and in the cells surrounding the forming blood vessels. The patterns of FXIIIA immunostaining and substrate incorporation overlap almost completely. The cartilaginous factor XIIIA is different from the plasma form in that, both intracellularly and extracellularly, it exists as a monomer, as determined by Western analysis, whereas the plasma form of FXIII is a tetrameric complex composed of both A and B subunits. We also identified FXIIIA and transglutaminase activity within the articular and condylar regions of the tarsus, suggesting a possible involvement of mechanical pressure and/or stress in the production of the molecule and subsequent cross-linking of the cartilage matrix. Thus, transglutaminases, in particular FXIIIA, are involved in the formation of long bones through its activity both in the hypertrophic region of the growth plate and in the formation of articular/epiphyseal cartilages.

Asunto(s)

Desarrollo Óseo , Cartílago/enzimología , Factor XIIIa/metabolismo , Placa de Crecimiento/enzimología , Osteogénesis , Animales , Cartílago/citología , Cartílago/crecimiento & desarrollo , Células Cultivadas , Embrión de Pollo , Colágeno Tipo X/metabolismo , Placa de Crecimiento/citología , Placa de Crecimiento/metabolismo , Inmunohistoquímica , Tarso Animal/citología , Tarso Animal/enzimología

RESUMEN

LR7/8B and ApoER2 are recently discovered members of the low density lipoprotein (LDL) receptor family. Although structurally different, these two proteins are derived from homologous genes in chicken and man by alternative splicing and contain 7 or 8 LDL receptor ligand-binding repeats. Here we present the cDNA for ApoER2 cloned from mouse brain and describe splice variants in the ligand binding domain of this protein, which are distinct from those present in man and chicken. The cloned cDNA is coding for a receptor with only five LDL receptor ligand-binding repeats, i.e. comprising repeats 1-3, 7, and 8. Reverse transcriptase-polymerase chain reaction analysis of mRNA from murine brain revealed the existence of two additional transcripts. One is lacking repeat 8, and in the other repeat 8 is substituted for by a 13-amino acid insertion with a consensus site for furin cleavage arising from an additional small exon present in the murine gene. None of the transcripts in the mouse, however, contain repeats 4-6. In murine placenta only the form containing repeats 1-3 and 7 and the furin cleavage site is detectable. Analysis of the corresponding region of the murine gene showed the existence of 6 exons coding for a total of 8 ligand binding repeats, with one exon encoding repeats 4-6. Exon trapping experiments demonstrated that this exon is constitutively spliced out in all murine transcripts. Thus, the murine ApoER2 gene codes for receptor variants harboring either 4 or 5 binding repeats only. Recombinant expression of the 5-repeat and 4-repeat variants showed that repeats 1-3, 7, and 8 are sufficient for binding of beta-very low density lipoprotein and reelin, but not for recognition of alpha(2)-macroglobulin, which binds to the avian homologue of ApoER2 harboring 8 ligand binding repeats.

Asunto(s)

Empalme Alternativo , Moléculas de Adhesión Celular Neuronal/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Receptores de Lipoproteína/genética , alfa-Macroglobulinas/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Clonación Molecular , ADN Complementario , Exones , Intrones , Proteínas Relacionadas con Receptor de LDL , Ligandos , Ratones , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso , Receptores de Lipoproteína/química , Receptores de Lipoproteína/metabolismo , Proteína Reelina , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Serina Endopeptidasas

RESUMEN

The discovery of an ever growing number of low density lipoprotein (LDL) receptor gene family members has triggered research into many different directions. Here we first summarize the results of classical studies on the role of the LDL receptor in cholesterol transport, the structure/function relationships delineated with the help of LDL receptor mutations in familial hypercholesterolemia, and the elegant way in which cells regulate cholesterol at the transcriptional level. The second part deals with a multifunctional, structurally very close relative, the very low density lipoprotein (VLDL) receptor. While it is involved in lipoprotein transport in certain tissues and species, detailed studies on its function have generated new knowledge about the growing spectrum of ligands and about exciting and unexpected aspects of receptor biology. In particular, these investigations have elucidated the roles of LDL receptor gene family members in ligand-mediated signal transduction. In the third part of this review article, we provide first insight into the roles of the VLDL receptor and of another small relative, the so-called apolipoprotein E receptor-2, in such signaling processes. These findings suggest that to date, only the tip of an iceberg has been uncovered.

Asunto(s)

Colesterol/metabolismo , Receptores de LDL/metabolismo , Receptores de Lipoproteína/metabolismo , Animales , Apolipoproteína E2 , Apolipoproteínas E/metabolismo , Transporte Biológico , Moléculas de Adhesión Celular Neuronal/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Humanos , Lipoproteínas VLDL/metabolismo , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad , Mutación , Proteínas del Tejido Nervioso , Empalme del ARN , Receptores de LDL/genética , Receptores de Lipoproteína/genética , Proteína Reelina , Serina Endopeptidasas , Transducción de Señal , Relación Estructura-Actividad

RESUMEN

Development of the follicle in egg-laying species such as the chicken is regulated by systemic factors as well as by the highly orchestrated interplay of differentially expressed genes within this organ. Differential mRNA display analysis of defined phases of follicle development resulted in the characterization of coagulation factor XIIIA. It is expressed and produced by cells of the theca externa in a highly regulated manner during distinct growth phases of the follicle. Transcripts for factor XIIIA are already detectable at the beginning of follicle development and peak at the end of phase 2. Protein levels, however, still increase during phase 3, peak shortly after ovulation, and persist until the postovulatory tissue is completely resorbed. Factor XIIIA is secreted as a monomer into the extracellular matrix of the theca externa and is not associated with factor XIIIB as is the case in plasma. Our data suggest that, due to its transglutaminase activity, factor XIIIA stabilizes the follicular wall by cross-linking matrix components. Thus, coagulation factor XIIIA might play a key role in coping with the massive mechanical stress exerted by the large amount of yolk accumulating during the rapid growth phase of the oocyte.

Asunto(s)

Folículo Ovárico/metabolismo , Células Tecales/metabolismo , Transglutaminasas/biosíntesis , Secuencia de Aminoácidos , Animales , Membrana Basal/metabolismo , Northern Blotting , Western Blotting , Cloruro de Calcio/farmacología , Línea Celular , Pollos , ADN Complementario/metabolismo , Ácido Edético/farmacología , Femenino , Perfilación de la Expresión Génica , Glutatión Transferasa/metabolismo , Células de la Granulosa/metabolismo , Microscopía Fluorescente , Datos de Secuencia Molecular , Ovulación/fisiología , Pruebas de Precipitina , ARN Mensajero/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Homología de Secuencia de Aminoácido , Factores de Tiempo , Transfección , Transglutaminasas/metabolismo

RESUMEN

The members of the low density lipoprotein (LDL) receptor gene family bind a broad spectrum of extracellular ligands. Traditionally, they had been regarded as mere cargo receptors that promote the endocytosis and lysosomal delivery of these ligands. However, recent genetic experiments in mice have revealed critical functions for two LDL receptor family members, the very low density lipoprotein receptor and the apoE receptor-2, in the transmission of extracellular signals and the activation of intracellular tyrosine kinases. This process regulates neuronal migration and is crucial for brain development. Signaling through these receptors requires the interaction of their cytoplasmic tails with the intracellular adaptor protein Disabled-1 (DAB1). Here, we identify an extended set of cytoplasmic proteins that might also participate in signal transmission by the LDL receptor gene family. Most of these novel proteins are adaptor or scaffold proteins that contain PID or PDZ domains and function in the regulation of mitogen-activated protein kinases, cell adhesion, vesicle trafficking, or neurotransmission. We show that binding of DAB1 interferes with receptor internalization suggesting a mechanism by which signaling through this class of receptors might be regulated. Taken together, these findings imply much broader physiological functions for the LDL receptor family than had previously been appreciated. They form the basis for the elucidation of the molecular pathways by which cells respond to the diversity of ligands that bind to these multifunctional receptors on the cell surface.

Asunto(s)

Citosol/metabolismo , Receptores de LDL/metabolismo , Secuencia de Aminoácidos , Animales , Encéfalo/embriología , Adhesión Celular , Comunicación Celular , Células Cultivadas , Cartilla de ADN/metabolismo , Endocitosis , Glutatión Transferasa/metabolismo , Hibridación in Situ , Riñón/metabolismo , Lipoproteínas LDL/metabolismo , Hígado/metabolismo , Ratones , Modelos Biológicos , Datos de Secuencia Molecular , Familia de Multigenes , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/metabolismo , Plásmidos/metabolismo , Estructura Terciaria de Proteína , Receptores de LDL/química , Receptores de LDL/genética , Proteínas Recombinantes de Fusión/metabolismo , Homología de Secuencia de Aminoácido , Transducción de Señal , Factores de Tiempo , Técnicas del Sistema de Dos Híbridos

RESUMEN

Correct positioning of neurons during embryonic development of the brain depends, among other processes, on the proper transmission of the reelin signal into the migrating cells via the interplay of its receptors with cytoplasmic signal transducers. Cellular components of this signaling pathway characterized to date are cell surface receptors for reelin like apolipoprotein E receptor 2 (ApoER2), very low density lipoprotein receptor (VLDLR), and cadherin-related neuronal receptors, and intracellular components like Disabled-1 and the nonreceptor tyrosine kinase Fyn, which bind to the intracellular domains of the ApoER2 and VLDL receptor or of cadherin-related neuronal receptors, respectively. Here we show that ApoER2, but not VLDLR, also binds the family of JNK-interacting proteins (JIPs), which act as molecular scaffolds for the JNK-signaling pathway. The ApoER2 binding domain on JIP-2 does not overlap with the binding sites for MLK3, MKK7, and JNK. These results suggest that ApoER2 is able to assemble a multiprotein complex containing Disabled-1 and JIPs, together with their binding partners, to the cell surface of neurons. This complex might participate in ApoER2-specific reelin signaling and thus would explain the different phenotype of mice lacking the ApoER2 from that of VLDLR-deficient mice.

Asunto(s)

Proteínas Adaptadoras Transductoras de Señales , Proteínas Portadoras/metabolismo , Moléculas de Adhesión Celular Neuronal/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Prolina/metabolismo , Receptores de Lipoproteína/metabolismo , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Northern Blotting , Encéfalo/metabolismo , Diferenciación Celular , Células Cultivadas , Citoplasma/química , ADN Complementario/metabolismo , Epidídimo/metabolismo , Glutatión Transferasa/metabolismo , Humanos , Inmunohistoquímica , Proteínas Relacionadas con Receptor de LDL , Masculino , Ratones , Microscopía Fluorescente , Modelos Biológicos , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso , Neuronas/citología , Unión Proteica , Proteína Reelina , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Serina Endopeptidasas , Transducción de Señal , Células Madre/metabolismo , Distribución Tisular , Técnicas del Sistema de Dos Híbridos

RESUMEN

Yolk is the major source of nutrients for the developing chicken embryo, but molecular details of the delivery mechanisms are largely unknown. During oogenesis in the chicken, the main yolk components vitellogenin and very low density lipoprotein (VLDL) are taken up into the oocytes via a member of the low density lipoprotein receptor gene family termed LR8 (Bujo, H., Hermann, M., Kaderli, M. O., Jacobsen, L., Sugawara, S., Nimpf, J., Yamamoto, T., and Schneider, W. J. (1994) EMBO J. 13, 5165-5175). This endocytosis is accompanied by partial degradation of the yolk precursor protein moieties; however, fragmentation does not abolish binding of VLDL to LR8. The receptor exists in two isoforms that differ by a so-called O-linked sugar domain; the shorter form (LR8-) is the major form in oocytes, and the longer protein (LR8+) predominates in somatic cells. Here we show that both LR8 isoforms are expressed at ratios that vary with embryonic age in the extraembryonic yolk sac, which mobilizes yolk for utilization by the embryo, and in the allantois, the embryo's catabolic sink. Stored yolk VLDL interacts with LR8 localized on the surface of the yolk sac endodermal endothelial cells (EEC), is internalized, and degraded, as demonstrated by the catabolism of fluorescently labeled VLDL in cultured EEC. Addition to the incubation medium of the 39-kDa receptor-associated protein, which inhibits all known LR8/ligand interactions, blocks the uptake of VLDL by EEC. The levels of endogenous receptor-associated protein correspond to those of LR8+ but not LR8-, suggesting that it may play a role in the modulation of surface presentation of LR8+. Importantly, EEC express significant levels of microsomal triglyceride transfer protein and protein disulfide isomerase, key components required for lipoprotein synthesis. Because the apolipoprotein pattern of VLDL isolated from the yolk sac-efferent omphalomesenteric vein is very different from that of yolk VLDL, these data strongly suggest that embryo plasma VLDL is resynthesized in the EEC. LR8 is a key mediator of a two-step pathway, which affects the uptake of VLDL from the yolk sac and the subsequent delivery of its components to the growing embryo.

Asunto(s)

Embrión no Mamífero/metabolismo , Receptores de LDL/metabolismo , Saco Vitelino/metabolismo , Empalme Alternativo , Animales , Secuencia de Bases , Embrión de Pollo , Cartilla de ADN , Femenino , Lipoproteínas VLDL/biosíntesis , Lipoproteínas VLDL/metabolismo

RESUMEN

Complement component 3 (C3) and alpha(2)-macroglobulin evolved from a common, evolutionarily old, ancestor gene. Low density lipoprotein-receptor-related protein/alpha(2)-macroglobulin receptor (LRP/alpha(2)MR), a member of the low density lipoprotein receptor family, is responsible for the clearance of alpha(2)-macroglobulin-protease complexes. In this study, we examined whether C3 has conserved affinity for LRP/alpha(2)MR. Ligand blot experiments with human (125)I-C3 on endosomal proteins show binding to a 600-kDa protein, indistinguishable from LRP/alpha(2)MR by the following criteria: it is competed by receptor-associated protein (the 39-kDa receptor-associated protein that impairs binding of all ligands to LRP/alpha(2)MR) and by lactoferrin and Pseudomonas exotoxin, other well known ligands of the multifunctional receptor. Binding of C3 is sensitive to reduction of the receptor and is Ca(2+)-dependent. All these features are typical for cysteine-rich binding repeats of the low density lipoprotein receptor family. In LRP/alpha(2)MR, they are found in four cassettes (2, 8, 10, and 11 repeats). Ligand blotting to chicken LR8 demonstrates that a single 8-fold repeat is sufficient for binding. Confocal microscopy visualizes initial surface labeling of human fibroblasts incubated with fluorescent labeled C3, which changes after 5 min to an intracellular vesicular staining pattern that is abolished in the presence of receptor-associated protein. Cell uptake is abolished in mouse fibroblasts deficient in LRP/alpha(2)MR. Native plasma C3 is not internalized. We demonstrate that the capacity to internalize C3 is saturable and exhibits a K(D) value of 17 nM. After intravenous injection, rat hepatocytes accumulate C3 in sedimentable vesicles with a density typical for endosomes. In conclusion, our ligand blot and uptake studies demonstrate the competence of the LRP/alpha(2)MR to bind and endocytose C3 and provide evidence for an LRP/alpha(2)MR-mediated system participating in C3 metabolism.

Asunto(s)

Complemento C3/metabolismo , Receptores Inmunológicos/fisiología , Receptores de LDL/metabolismo , alfa-Macroglobulinas/metabolismo , Animales , Activación de Complemento , Complemento C3/administración & dosificación , Endocitosis , Humanos , Cinética , Hígado/metabolismo , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad , Ratones , Unión Proteica , Ratas , Receptores Inmunológicos/metabolismo

RESUMEN

Layering of neurons in the cerebral cortex and cerebellum requires Reelin, an extracellular matrix protein, and mammalian Disabled (mDab1), a cytosolic protein that activates tyrosine kinases. Here, we report the requirement for two other proteins, cell surface receptors termed very low density lipoprotein receptor (VLDLR) and apolipoprotein E receptor 2 (ApoER2). Both receptors can bind mDab1 on their cytoplasmic tails and are expressed in cortical and cerebellar layers adjacent to layers that express Reelin. mDab1 expression is upregulated in knockout mice that lack both VLDLR and ApoER2. Inversion of cortical layers and absence of cerebellar foliation in these animals precisely mimic the phenotype of mice lacking Reelin or mDab1. These findings suggest that VLDLR and ApoER2 participate in transmitting the extracellular Reelin signal to intracellular signaling processes initiated by mDab1.

Asunto(s)

Moléculas de Adhesión Celular Neuronal/metabolismo , Dendritas/fisiología , Proteínas de la Matriz Extracelular/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Células de Purkinje/fisiología , Receptores de LDL/fisiología , Receptores de Lipoproteína/fisiología , Secuencia de Aminoácidos , Animales , Línea Celular , Movimiento Celular/fisiología , Corteza Cerebral/metabolismo , Corteza Cerebral/patología , Femenino , Proteínas Relacionadas con Receptor de LDL , Ratones , Ratones Noqueados , Ratones Mutantes , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/genética , Receptores de LDL/genética , Receptores de LDL/metabolismo , Receptores de Lipoproteína/genética , Receptores de Lipoproteína/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteína Reelina , Serina Endopeptidasas , Regulación hacia Arriba

RESUMEN

Apolipoprotein E isoforms affect the risk of developing Alzheimer's disease. Apolipoprotein E-associated risk may be related to its binding to and clearance by cell surface receptors, including members of the low-density lipoprotein receptor family. We examined the brain expression of the most recently identified member of this receptor family, apolipoprotein E receptor 2, in human brain and placenta. We analysed apolipoprotein E receptor 2 messenger RNA by reverse transcription-polymerase chain reaction and apolipoprotein E receptor 2 protein by immunohistochemistry. Four exons of the apolipoprotein E receptor 2 message were alternately spliced in both fetal and adult brain tissue. Exon 5, encoding three of the seven ligand binding repeats, was absent in the apolipoprotein E receptor 2 messenger RNA examined. Apolipoprotein E receptor 2 messages lacking exon 8, encoding an epidermal growth factor precursor repeat, exon 15, encoding the O-glycosylation region, or exon 18, encoding a cytoplasmic domain, were also present as minor splice variants in the brain and placenta. No differences were observed in the pattern of apolipoprotein E receptor 2 splicing between control and Alzheimer brains. Immunohistochemistry of mouse brain showed that apolipoprotein E receptor 2 was expressed in neurons throughout the brain, with strong expression in pyramidal neurons of the hippocampus, granule cells of the dentate gyrus, cortical neurons and Purkinje cells of the cerebellum. Thus, apolipoprotein E receptor 2 is the fourth apolipoprotein E receptor identified on neuronal cells.

Asunto(s)

Empalme Alternativo , Encéfalo/metabolismo , Receptores de Lipoproteína/genética , Receptores de Lipoproteína/metabolismo , Enfermedad de Alzheimer/metabolismo , Animales , Humanos , Inmunohistoquímica , Isomerismo , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad , Ratones , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa

RESUMEN

The interaction of the female germ cell with somatic cells during the development of the ovarian follicle in the chicken provides a prime system to study gene expression. Here, we have uncovered the involvement of clusterin, the function(s) of which is still poorly understood, in this complex process. As revealed by molecular cloning, chicken clusterin is a 428-residue protein that migrates at 70 kDa on SDS-polyacrylamide gel electrophoresis and possesses most of the structural features of its mammalian successors. However, in contrast to mammalian clusterin, the chicken protein appears not to be cleaved intracellularly into a disulfide-linked heterodimer; possibly as a consequence thereof, it is not secreted constitutively and is absent from the circulation, where most of clusterin is found in mammals. In the ovary, clusterin is a major product of the somatic granulosa cells, in a pattern correlating with the developmental phases of individual follicles. In that, transcript levels are high not only at onset of vitellogenesis, but also in atretic follicles and in the postovulatory follicle sac, i.e. in situations characterized by apoptotic events. Yolk of growing oocytes contains a 43-kDa truncated form of clusterin that does not appear to be synthesized within the oocyte. Rather, we here show for the first time that 70-kDa clusterin interacts not only with megalin, but also with two chicken oocyte-specific members of the low density lipoprotein receptor (LDLR) gene family. These receptors, termed LDLR-related protein with eight ligand binding repeats (LR8) and LDLR-related protein (380 kDa), likely internalize granulosa cell-derived 70-kDa clusterin, which may subsequently be processed to the 43-kDa product. Thus, chicken clusterin could serve as a marker for follicular atresia and resorption, and, based on its ability to bind several other proteins, it may serve as carrier for the receptor-mediated endocytosis into oocytes of components important for embryonic development, two hitherto unknown functions of this intriguing protein.

Asunto(s)

ADN/metabolismo , Glicoproteínas/fisiología , Chaperonas Moleculares , Oocitos/crecimiento & desarrollo , Folículo Ovárico/crecimiento & desarrollo , Receptores de LDL/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Pollos , Clusterina , Secuencia de Consenso , Femenino , Glicoproteínas/química , Glicoproteínas/genética , Glicosilación , Humanos , Datos de Secuencia Molecular , Peso Molecular , Codorniz , ARN Mensajero/metabolismo , Alineación de Secuencia

RESUMEN

With ever increasing sophistication in molecular biological approaches, the low-density lipoprotein receptor supergene family continues to grow rapidly. From the well-defined key role of these receptors in lipoprotein metabolism, the new members move the field into many different and diverse physiologic and developmental areas. We observe an expansion of the functional spectrum of the family members, which is due to 1) the binding to their extracellular domains of more and more components lacking homology to apolipoproteins, and 2) the recently uncovered interaction of the receptors' cytoplasmic tails with adaptor proteins that are part of signaling pathways. As this review attempts to describe, the task of delineation of the evolutionary history of the gene family may be aided by concepts that consider events, both divergent and convergent, within and between the intra- and extracellular domains.

Asunto(s)

Receptores de LDL/genética , Animales , Complejo Antigénico de Nefritis de Heymann , Humanos , Hiperlipoproteinemia Tipo II/genética , Glicoproteínas de Membrana/genética , Receptores de LDL/fisiología , Secuencias Repetitivas de Ácidos Nucleicos

RESUMEN

The discovery in 1992 of a member of the low density lipoprotein receptor (LDLR) family with eight ligand binding repeats (LR8) has raised more questions than have been answered to date. Here, we summarize the current status of knowledge about this intriguing molecule, generally termed VLDL receptor, at the molecular biological, cell biological, and physiological levels. On one hand, the wealth of reports concerning the role(s) of this receptor in lipoprotein metabolism in mammalian systems has revealed partially conflicting details, particularly in regards to its natural ligand(s) and site of action. On the other hand, molecular genetic and biochemical studies in the chicken have clearly demonstrated the multiple roles of LR8 in the physiology and reproduction of egg-laying species, and have generated insights into the evolutionary aspects of the LDLR gene family.

Asunto(s)

Lipoproteínas VLDL/química , Lipoproteínas VLDL/fisiología , Receptores de LDL/química , Receptores de LDL/fisiología , Animales , Sitios de Unión , Aves , Humanos , Ligandos , Mamíferos

RESUMEN

LR7/8B is a member of the low density lipoprotein receptor gene family that is specifically synthesized in the brain. Here we have functionally expressed in 293 cells the splice variant harboring eight ligand binding repeats (LR8B). As assessed by confocal microscopy, the expressed receptor is localized to the plasma membrane. Importantly, in cell binding experiments, we demonstrate that this protein is a receptor for activated alpha2-macroglobulin. Because to date low density lipoprotein receptor-related protein (LRP) has been shown to be the only alpha2-macroglobulin receptor in brain, we became interested in the expression pattern of both proteins at the cellular level in the brain. LR7/8B is expressed in large neurons and Purkinje cells of the cerebellum and in cells constituting brain barrier systems such as the epithelial cells of the choroid plexus, the arachnoidea, and the endothelium of penetrating blood vessels. Anti-LR7/8B antibody stains the plasma membrane, dendrites, and vesicular structures close to the cell membrane of neurons, especially of Purkinje cells. In contrast, LRP is present in patchy regions around large neurons and most prominently in the glomeruli of the stratum granulare of the cerebellum. This suggests that, contrary to LR7/8B, LRP is expressed in synaptic regions of the neurons; furthermore, there is a striking difference in the expression patterns of LR7/8B and LRP in the choroid plexus. Whereas LRP shows baso-lateral and apical localization in the epithelial cells, LR7/8B is restricted to the apical cell aspect facing the cerebrospinal fluid. Finally, these studies were extended to cultured primary rat neurons, where double immunofluorescence labeling with anti-LR7/8B and anti-microtubuli-associated protein 2 (MAP2) confirmed the somatodendritic expression of the receptor. Based upon these data, we propose that LR7/8B is involved in the clearance of alpha2-macroglobulin.proteinase complexes and/or of other substrates bound to alpha2-macroglobulin from the cerebrospinal fluid and from the surface of neurons.

Asunto(s)

Empalme Alternativo , Encéfalo/metabolismo , Familia de Multigenes , Receptores Inmunológicos/genética , Receptores de LDL/genética , Animales , Sitios de Unión , Línea Celular , Pollos , Variación Genética , Humanos , Inmunohistoquímica , Cinética , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad , Ratas , Receptores Inmunológicos/análisis , Receptores Inmunológicos/metabolismo , Receptores de LDL/análisis , Receptores de LDL/metabolismo , Proteínas Recombinantes/metabolismo , Secuencias Repetitivas de Aminoácido , Transfección , alfa-Macroglobulinas/metabolismo

RESUMEN

Oocyte development within avian ovarian follicles is an intricate process involving yolk deposition and the formation of extraoocytic matrices. Of these, the perivitelline membrane (pvm) not only plays a role in sperm binding but also provides mechanical support for the large oocyte's journey through the oviduct after ovulation. To date we have focused on the mechanisms for uptake of yolk precursors into oocytes of the chicken; now we extend our studies to a detailed analysis of the pvm. In the course of characterization of its major components, we obtained partial protein sequences; comparison with the GenBank database revealed that one of the pvm proteins is the homologue of mammalian zona pellucida glycoprotein 3 (ZP3), a key component in sperm binding. Following a nomenclature based on gene structure, the protein is referred to as chicken ZPC (chZPC). The chicken protein (444 residues) and murine ZP3 (424 residues) are highly conserved, with 41% of the amino acids identical. As shown by Northern blot analysis, the avian ZPC gene is expressed exclusively in the granulosa cells surrounding the oocyte, in contrast to murine ZP3, which is synthesized by the oocyte. Upon reaching a size larger than 1.5 mm in diameter, follicles accumulate chZPC in highly polarized fashion, i.e., in the space intercalated between the oocyte and the granulosa cells, as revealed by immunohistochemistry of follicle sections. ChZPC synthesis and secretion by granulosa cells was demonstrated directly by metabolic labeling and immunoprecipitation from the culture medium of granulosa cell sheets isolated ex vivo from follicles. Immunoblot analysis and glycosidase treatment of chZPC from preovulatory and freshly ovulated oocytes, as well as laid eggs, revealed that the primary product undergoes a two-step decrease in size from follicle to laid egg that is unlikely to be due to modification of the carbohydrate moiety.

Asunto(s)

Pollos , Proteínas del Huevo/biosíntesis , Células de la Granulosa/metabolismo , Glicoproteínas de Membrana/biosíntesis , Receptores de Superficie Celular , Secuencia de Aminoácidos , Animales , Northern Blotting , Secuencia Conservada , Proteínas del Huevo/química , Proteínas del Huevo/genética , Femenino , Expresión Génica , Humanos , Inmunohistoquímica , Masculino , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Ratones , Datos de Secuencia Molecular , Folículo Ovárico/química , Óvulo/química , Fragmentos de Péptidos/química , Homología de Secuencia , Membrana Vitelina/química , Glicoproteínas de la Zona Pelúcida

RESUMEN

The low density lipoprotein receptor-related protein (LRP) is synthesized as a proreceptor that undergoes post-translational proteolytic processing, yielding a noncovalently associated alphabeta dimer as the mature LRP. We tested the role of processing by creating a mutant in which the P1 residue (Arg3942) of the consensus site for furin cleavage (Arg-Asn-Arg-Arg3942 downward arrow) was replaced with Ser in chicken LRP. Transfection of the mutant LRP (designated LRP-RS) into a Chinese hamster ovary cell line lacking endogenous LRP resulted in expression of the unprocessed full-length proreceptor. Comparison of cell lines stably expressing either the wild-type LRP (LRP-wt) or the unprocessed LRP-RS showed that at comparable expression levels, both receptors restored the sensitivity of cellular protein synthesis to Pseudomonas exotoxin A (IC50 = 25 ng/ml). Subcellular fractionation and neuraminidase treatment showed that both LRP forms were transported to the plasma membrane. In addition, LRP-RS exhibited kinetics of binding, endocytosis, and degradation of methylamine-activated alpha2-macroglobulin that were identical to those of LRP-wt. The internalization rate constant was similar for LRP-wt (Ke = 0.259 min-1) and mutant LRP-RS (Ke = 0.252 min-1), suggesting that it takes about 4 min for the entire surface LRP pool to be internalized. Sorting of LRP from the endosomal compartment to lysosomes or recycling to the plasma membrane were also unaltered in mutant LRP-RS. Pulse-chase analysis showed that the lack of processing of LRP had no effect on the stability of its post-endoplasmic reticulum form or on the rate of its intracellular transit from the endoplasmic reticulum to the Golgi apparatus. However, the exit of mutant LRP from the endoplasmic reticulum was retarded by the Arg3942-to-Ser substitution, as evidenced by prolonged retention within the endoplasmic reticulum (t1/2 = 4 h for LRP-wt and t1/2 > 13 h for LRP-RS).

Asunto(s)

Endocitosis , Retículo Endoplásmico/metabolismo , Procesamiento Proteico-Postraduccional , Receptores Inmunológicos/metabolismo , Animales , Arginina/genética , Transporte Biológico , Pollos , Furina , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad , Mutación , Receptores Inmunológicos/genética , Proteínas Recombinantes/metabolismo , Subtilisinas/metabolismo , alfa-Macroglobulinas/metabolismo

RESUMEN

The laying hen's left ovary (the ovary on the right side regresses during embryonic development) at any given time contains 5-8 follicles with vitellogenic oocytes, i.e., female germ cells of increasing size ranging from 8 to approx. 30 mm in diameter. Between the 8mm and the 30 mm cell lie about 7 days of massive growth of the oocyte, which in essence occurs via accumulation of yolk. While the chemical composition of the oocyte content, i.e., the yolk, is well known, the origin(s) of this material, its mode(s) of accumulation in the oocyte, and the cell biological details of the oocyte's specialized machineries for maintenance of structural organization and stability have only recently been begun to be elucidated. Here, we outline recent findings in the following areas: (i) the role of low density lipoprotein receptor family members in the transport of yolk precursors from the plasma compartment into oocytes; these findings have implications for metabolic events in certain other tissues such as the brain; and (ii) the synthetic and structural specialization of the granulosa cells, which surround the oocyte within the ovarian follicle, as a model for epithelial cell biology.

Asunto(s)

División Celular/fisiología , Proteoglicanos de Heparán Sulfato , Oocitos/citología , Receptores de LDL/fisiología , Animales , Pollos , Proteínas del Huevo/análisis , Femenino , Heparitina Sulfato/fisiología , Glicoproteínas de Membrana/análisis , Glicoproteínas de Membrana/fisiología , Familia de Multigenes , Proteoglicanos/fisiología , Receptores de LDL/genética , Zona Pelúcida/fisiología