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PubMed1982

IgG induction in a human B cell line by red cell-mediated microinjection of the cytoplasm from T cell factor-stimulated B cells.

Generation of a cytoplasmic factor(s) that induced IgG secretion in nonstimulated cells was demonstrated in TRF-stimulated cells by using red cell-mediated microinjection. Injection of the cytoplasm from a TRF-stimulated B lymphoblastoid cell line (CESS) into nonstimulated cells induced an increase of IgG-producing cells. Injection of TRF itself did not induce an increase of IgG-producing cells. Active substance(s) in the cytoplasm were generated at 2 hr after TRF stimulation, and IgG-producing cells reached their maximum level at 40 hr after injection of the factor(s). DFP, but not actinomycin D, inhibited the generation of the cytoplasmic factor(s). The activity of the cytoplasmic factor(s) was not destroyed by RNase and not absorbed with anti-IgG. These results suggested that binding of TRF with its acceptors induced the generation of the cytoplasmic factor(s) involved in the transmission of TRF-mediated signals from membrane to nuclei.

Antibody Formation↗
PubMed1980

Hydrolysis and excretion of cytoplasmic cholesteryl esters by macrophages: stimulation by high density lipoprotein and other agents.

The ability of mouse peritoneal macrophages to hydrolyze and excrete cytoplasmic cholesteryl ester droplets was studied. The macrophages were loaded with cholesteryl esters by incubation with acetylated low density lipoprotein (acetyl-LDL), which is internalized by adsorptive endocytosis. The cholesteryl esters of acetyl-LDL are hydrolyzed within lysosomes and the liberated cholesterol is re-esterified in the cytoplasm where it accumulates as cytoplasmic cholesteryl ester droplets. Hydrolysis and excretion of these stored cholesteryl esters were quantified by gas-liquid chromatographic measurement of the content of free and esterified cholesterol in cells and in medium. After removal of acetyl-LDL from the culture medium, the cytoplasmic cholesteryl esters were rapidly hydrolyzed and large amounts of free cholesterol were excreted from the cells. Hydrolysis and excretion required a cholesterol acceptor in the culture medium. The following agents were shown to be effective as cholesterol acceptors: high density lipoprotein (HDL), whole serum, the density > 1.215 g/ml fraction of whole serum, intact erythrocytes, casein, and thyroglobulin. The following agents did not promote the hydrolysis and excretion of cholesteryl esters under these experimental conditions: LDL, serum albumin, serum gamma-globulins, and phosphatidylcholine/sphingomyelin liposomes. The results indicate that net hydrolysis of cytoplasmic cholesteryl esters in macrophages is coupled to the process of cholesterol excretion and that net hydrolysis does not occur unless an effective cholesterol acceptor is present in the culture medium.-Ho, Y. K., M. S. Brown, and J. L. Goldstein. Hydrolysis and excretion of cytoplasmic cholesteryl esters by macrophages: stimulation by high density lipoprotein and other agents.

Animals↗
PubMed1994

The high affinity Fc gamma receptor (CD64) induces phagocytosis in the absence of its cytoplasmic domain: the gamma subunit of Fc gamma RIIIA imparts phagocytic function to Fc gamma RI.

The high affinity Fc gamma receptor, Fc gamma RI, is unique among the three classes of macrophage Fc gamma receptors not only in its affinity for IgG, but also in the structure of its cytoplasmic domain. Fc gamma RIIA and the gamma subunit of Fc gamma RIIIA have tyrosine-containing motifs within their cytoplasmic domains that are phosphorylated when crosslinked and that are required for phagocytosis by COS-1 cell transfectants. In contrast to these other Fc gamma receptors, Fc gamma RI does not contain cytoplasmic tyrosines and does not induce phagocytosis in COS-1 transfectants. We transfected wild-type (WT) and mutant (MT) Fc gamma RI lacking the cytoplasmic domain into COS-1 cells and murine macrophages and assessed phagocytosis using IgG-coated red blood cells (RBCs) and RBCs conjugated with Fab anti-human Fc gamma RI monoclonal antibody (mAb). Fc gamma RI, in contrast to Fc gamma RIIA, did not induce phagocytosis in COS cells. However, both WT and MT Fc gamma RI induced phagocytosis in murine macrophages, and phagocytosis was inhibited by the tyrosine kinase inhibitor tyrphostin 23. Human monocytes also phagocytosed Fc gamma RI-targeted RBCs, and activation of Fc gamma RI on monocytes with Fab anti-Fc gamma RI induced phosphorylation of Fc gamma RII on tyrosine residues. However, Fc gamma RI activation of Fc gamma RI-Fc gamma RIIA COS-1 cotransfectants did not induce tyrosine phosphorylation of Fc gamma RIIA, and coexpression of Fc gamma RI and Fc gamma RIIA in COS cells did not confer Fc gamma RI phagocytic capability. In contrast, coexpression in COS-1 cells of Fc gamma RI with the gamma subunit of Fc gamma RIIIA conferred phagocytic function to both Fc gamma RI and the MT Fc gamma RI lacking the cytoplasmic domain. Thus, Fc gamma RI does not require its cytoplasmic domain to mediate a phagocytic signal and interacts with the gamma subunit of Fc gamma RIIIA to induce phagocytosis.

Animals↗
PubMed1994

Protein phosphorylation and control of chorion gene activation through temporal mobilization of a promoter DNA binding factor from the cytoplasm into the nucleus.

The transcriptional activation of high cysteine chorion genes in the follicular cells of the silkworm Bombyx mori occurs at the end of oogenesis and coincides with the appearance of a chorion promoter DNA binding factor, BCFI, in follicular cell nuclei. Follicular cells of vitellogenic and choriogenic follicles that do not express high cysteine chorion genes contain high levels of a latent form of BCFI in their cytoplasm. The abundance of the cytoplasmic factor, termed cBCFI, is dramatically reduced during late choriogenesis, coincident with the appearance of factor BCFI in the nucleus and the transcriptional activation of high cysteine genes. Mobility shift assays performed with partially proteolyzed nuclear and cytoplasmic extracts of follicular cells, DNA binding assays carried out in the presence of anti-BCFI antibodies, and electrophoretic analyses of the proteins present in the nuclear and cytoplasmic fractions of follicular cells and recognized by the same antibodies suggest that factor cBCFI represents a covalently modified version of BCFI. The DNA-binding sites of BCFI and cBCFI include a core sequence, AGATAA, but, while this sequence is sufficient for specific binding of BCFI, it only constitutes part of the DNA-binding site of cBCFI. Dephosphorylation of cBCFI results in a change of its binding specificity to that of BCFI. The cytoplasmic sequestration of cBCFI appears to be mediated by a phosphorylation-dependent, reversible association of this factor with an ancillary cytoplasmic factor.

Animals↗
PubMed1993

Class II-restricted presentation of a hen egg lysozyme determinant derived from endogenous antigen sequestered in the cytoplasm or endoplasmic reticulum of the antigen presenting cells.

A cDNA encoding a form of hen egg lysozyme (HEL) lacking a leader sequence and predicted to be localized in the cytoplasm, was transfected into MHC class II-positive B lymphoma cells. Cytoplasmically expressed HEL (cytHEL) had a half-life of less than 5 min and did not react with HEL specific mAb suggesting non-native conformation. Cells expressing cytoplasmic HEL, as well as cells previously reported to express a low level of HEL retained in the endoplasmic reticulum (ERHEL), constitutively presented the HEL determinant encoded by residues 46-61 to a sensitive class II-restricted T hybridoma (3A9). Constitutive presentation of HEL determinants was not detectable in cytHEL or ERHEL transfectants using T hybridomas with lower sensitivity to exogenous Ag. Constitutive presentation of HEL46-61 derived from cytoplasmic HEL was demonstrable in multiple transfected clones and was most obvious when a CMV rather than SV40 promoter was used to express the cytHEL gene. The presentation of HEL46-61 by cytHEL transfectants was not due to HEL reuptake by bystander cells because there was no biochemical evidence of cytHEL shedding and cytHEL supernatants added to indicator APC did not result in HEL46-61 presentation. Constitutive presentation of endogenous HEL46-61 by the cytHEL and ERHEL transfectants was inhibited by chloroquine, and recovery of presentation of endogenous HEL was slower in cytHEL compared with ERHEL transfectants. The findings indicate that class II-restricted presentation of Ag retained in the cytoplasm or endoplasmic reticulum does take place but probably requires abundant levels of intracellular Ag and is easily disrupted by lysosomotropic agents. These pathways of presentation may be important when high levels of foreign endoplasmic reticulum-retained or cytoplasmic Ag are present (e.g., viral infection), and during the acquisition of self-tolerance by highly sensitive developing T cells.

Amino Acid Sequence↗
PubMed1996

Cell fusion to study nuclear-cytoplasmic interactions in endothelial cell apoptosis.

Studies examining the regulation of nuclear rearrangements during apoptosis have led to conflicting results. Cytoplasmic control of nuclear events has been strongly suggested by cell-free experimental systems. In contrast, strict cytoplasmic control cannot account for the results of fibroblast-thymocyte fusion experiments in which dexamethasone induction of polykaryons led only to thymocyte nuclear apoptosis. Unresolved by these fusion studies was whether fibroblast nuclei were indifferent to heterologous cytoplasmic signals. Our objective was to resolve this discrepancy using cell fusion in a homologous system. Our strategy was to fuse endothelial cells with high levels of susceptibility to the induction of apoptosis (log phase cells arrested in G1 for 48 hours by isoleucine deprivation) with those manifesting low levels of susceptibility (serum-deprived, G0). Resultant fused and unfused cells were induced to undergo apoptosis by incubation with tumor necrosis factor-alpha and cycloheximide. Depending on the parental cell of origin, between 14 and 30% of dikaryons contained one apoptotic and one intact nucleus, indicating that strict cytoplasmic control was not occurring. In accord with this, the total frequency of nuclear apoptosis was unchanged after fusion. However, the distribution of apoptotic nuclei revealed a pronounced cytoplasmic influence, with a two- to fivefold increase in coordinate nuclear behavior. This pattern of nuclear apoptosis was consistent with a model of control in which both the state of nuclear susceptibility to apoptosis and expression of cytoplasmic pro-apoptotic regulators determined whether nuclear apoptosis would eventuate.

Animals↗
PubMed1996

Changes in the cytoplasmic structure of CTLs during target cell recognition and killing.

CTL play a critical role in immune defense by recognizing and killing virally infected or tumor cells. In this report, the structure of cytoplasm within living CTL was monitored during CTL killing of target cells. Living CTL were simultaneously loaded with fluorescent 70,000- and 10,000-kDa dextran particles. The relative distribution of the large and small dextrans within CTL revealed subcellular heterogeneities in the submicroscopic structure of cytoplasm. Localized alterations in cytoplasmic structure correlated with specific events during CTL killing. Recognition of target cells was accompanied by a transient increase in large dextran accessibility over a broad front near the interface between CTL and target cells. This region narrowed to a smaller area from which pseudopodia were extended toward the target. During extension, there was a large difference between regions of high dextran accessibility within the pseudopod and more structured cytoplasm within the cell body. Areas undergoing structural changes showed localized foci of high dextran accessibility. During retraction, cytoplasmic structure became gradually more uniform throughout the protrusion and cell body. These observations revealed subcellular regions undergoing major changes during early stages of the killing response, and addressed the role of cytoplasmic solation in controlling CTL morphology. They support mechanisms of pseudopod extension driven by hydrostatic pressure and demonstrate a precise regulation of cortical structure to control the direction of pseudopod extension.

Animals↗
PubMed1996

Nuclear and cytoplasmic actin in dinoflagellates.

Experiments using monoclonal and polyclonal anti-actin antibodies allowed us to demonstrate the presence of F- or G-actin in original protists, dinoflagellates, either by biochemistry, immunofluorescence and in TEM. SDS-PAGE electrophoresis and immunoblottings made either from total or nuclear protein extracts revealed the presence of a 44-kDa band reacting with monoclonal anti-actin antibody in two species, Prorocentrum micans and Crypthecodinium cohnii, and thus demonstrated the presence of actin in nuclear and cytoplasmic fractions. After squash preparation of P micans cells, actin was identified within the nucleus and in some regions of the cytoplasm by immunofluorescence microscopy. Labelling of both the nucleolus and the centrosome region was evident together with amorphous nucleoplasmic material surrounding the chromosomes. The use of cryosections of intact P micans and C cohnii cells for immunofluorescence along with staining with DAPI to delineate the chromosomes themselves, yielded finer resolution of the intranuclear network labelling pattern and allowed us to complete our observations, in particular on the cytoplasmic labelling. In P micans, in addition to the centrosome region, the cytoplasmic channels passing through the nucleus in dividing cells are labelled. In C cohnii, the cortex, the centrosome region, the cytoplasmic channels, the region surrounding the nucleus, the filaments linking it to the cortex and the cleavage furrow are also labelled. In the nucleus of the two species, there is a prominent "weft' of fine actin filaments in the nucleoplasm forming a matrix of varying density around the persistent chromosomes. This actin matrix, of unknown function, is most conspicuous at the end of the S-phase of the cell cycle. Fluorescent derivatives of phalloidin, used as diagnostic cytochemical probes for polymeric actin (F-actin), gave similar results. Positive TEM immunolabelling of intranuclear actin confirms its presence in the nucleoplasm, in the nucleolus where the preribosomal region is labelled while C cohnii chromosomes are unlabelled and the P micans chromosomes very slightly. In the cytoplasm, lips of the cleavage furrow and kinetosome regions are labelled as well as the centrosome region. The possible functions of this protein located in several compartments of dinoflagellate cells are discussed.

Actins↗
PubMed1998

The Fanconi anemia group C gene product is located in both the nucleus and cytoplasm of human cells.

The Fanconi anemia (FA) complementation group C (FAC) protein gene encodes a cytoplasmic protein with a predicted Mr of 63,000. The protein's function is unknown, but it has been hypothesized that it either mediates resistance to DNA cross-linking agents or facilitates repair after exposure to such factors. The protein also plays a permissive role in the growth of colony-forming unit-granulocyte/macrophage (CFU-GM), burst-forming unit-erythroid (BFU-E), and CFU-erythroid (CFU-E). Attributing a specific function to this protein requires an understanding of its intracellular location. Recognizing that prior study has established the functional importance of its cytoplasmic location, we tested the hypothesis that FAC protein can also be found in the nucleus. Purified recombinant Escherichia coli-derived FAC antigens were used to create antisera able to specifically identify an Mr = 58,000 protein in lysates from human Epstein-Barr virus (EBV)-transformed cell lines by immunoblot analysis. Subcellular fractionation of the cell lysates followed by immunoblot analysis revealed that the majority of the FAC protein was cytoplasmic, as reported previously; however, approximately 10% of FAC protein was reproducibly detected in nuclear fractions. These results were reproducible by two different fractionation methods, and included markers to control for contamination of nuclear fractions by cytoplasmic proteins. Moreover, confocal image analysis of human 293 cells engineered to express FAC clearly demonstrated that FAC protein is located in both cytoplasmic and nuclear compartments, consistent with data obtained from fractionation of the FA cell lines. Finally, complementation of the FAC defect using retroviral-mediated gene transfer resulted in a substantial increase in nuclear FAC protein. Therefore, while cytoplasmic localization of this protein appears to be functionally important, it may also exert some essential nuclear function.

Cell Cycle Proteins↗
PubMed1998

Dominant-negative effect of the lymphocyte function-associated antigen-1 beta (CD18) cytoplasmic domain on leukocyte adhesion to ICAM-1 and fibronectin.

The cytoplasmic domains of LFA-1 (CD11a/CD18) are thought to play an important role in the regulation of LFA-1 function. To further elucidate the role of the LFA-1 cytoplasmic domains, we transfected chimeric proteins consisting of the extracellular domain of CD4 fused with the transmembrane and cytoplasmic domains of LFA-1 into T and B cell lines, EL-4 and A20, respectively, and examined their effects on LFA-1-mediated cell adhesion. The CD4/18, but not CD4/11a, chimera profoundly inhibited LFA-1-mediated cell adhesion to ICAM-1, as well as cell spreading following cell adhesion. Unexpectedly, cell adhesion to fibronectin was also inhibited by the CD4/18 chimera. The CD4/18 chimera did not affect the expression of endogenous LFA-1 or the association of CD11a and CD18. Truncation of the carboxyl-terminal 13 amino acid residues of the CD18 cytoplasmic domain of the chimera completely abrogated the inhibitory effect on LFA-1. Among these amino acid residues, the carboxyl-terminal six residues were dispensable for the inhibitory effect in EL-4 cells, whereas it significantly reduced the inhibitory activity of CD4/18 in A20 cells. A larger truncation of the CD18 cytoplasmic domain was needed to fully abrogate the inhibitory effects of CD4/18 on the adhesion to fibronectin. These results show that 1) the CD4/18 chimera has dominant-negative effects on cell adhesion mediated by LFA-1 as well as fibronectin receptors, and 2) amino acid residues of the CD18 cytoplasmic domain involved in the inhibition of LFA-1 seem to be different from those for fibronectin receptors.

Amino Acid Sequence↗
PubMed1998

Conformational alterations resulting from mutations in cytoplasmic domains of the alpha subunit of the Na,K-ATPase.

This paper summarizes experiments concerned with the functional consequences of mutations in cytoplasmic regions of the alpha 1 subunit of the Na,K-ATPase, in particular the amino terminus, the first cytoplasmic loop between transmembrane segments M2 and M3, and the major cytoplasmic loop between M4 and M5. In the first mutation (alpha 1M32), 32 residues were removed from the N-terminus. The second mutation (E233K) was in the putative beta strand of M2-M3 loop and the third, comprised the replacement of the amino terminal half of loop M4-M5 of the Na,K-ATPase with the homologous segment (residues 356-519) of the gastric H,K-ATPase. The first two mutations, either separately or in combination (alpha 1M32E233K), shift the equilibrium between the major conformational states of the enzyme, E1 and E2, in favor of E1 as manifested by increased apparent affinity for ATP, lower catalytic turnover, and decreased sensitivity to inhibition by vanadate. The striking changes observed with alpha 1M32E233K suggests interactions between the N-terminus, the beta-strand in the M2-M3 loop and the catalytic phosphorylation site. The behavior of these mutants contrasts with that of least one mutant involving substitution of a residue in the putative cation binding pocket, namely S775A in the fifth transmembrane segment (Arguello, J.M., & Lingrel, J. B. J. Biol. Chem. 270: 22764-22771, 1995). Although its K+/ATP antagonism resembles that of the foregoing cytoplasmic mutants, its vanadate sensitivity is unaltered suggesting that changes in apparent affinity for ATP are secondary to changes in K+ ligation. The question of cation selectivity, in particular that of Na+ versus protons, has been addressed in structure/function analysis of a cytoplasmic chimera involving the M4-M5 loop. Transport studies performed in the presence or absence of Na+ and at low versus high pH indicate a marked alteration in cation affinity and/or selectivity. This results suggests coupling of an alteration in the large M4-M5 cytoplasmic domain to cation binding in, presumably, the juxtapositioned transmembrane domain.

Amino Acid Substitution↗
PubMed1998

Formation and localization of cytoplasmic domains in leech and ascidian zygotes.

Leech and ascidian embryos are well suited for the study of certain developmental processes. Although leeches and ascidians belong to different bilateralia groups (protostomes and deuterostomes, respectively) they share important developmental features and, in particular, the determinate character of their embryogenesis. In both types of embryos this property is related to the presence of specific cytoplasmic domains that are selectively allocated to different blastomeres during cleavage. In this review leech and ascidian eggs and zygotes are compared in terms of the structure of these cytoplasmic domains and of the cellular mechanisms involved in their formation and localization. During meiosis the zygote of leeches and ascidians undergo stereotypic actin-dependent contraction movements related to both the emission of the polar bodies and the formation and relocalization of cytoplasmic domains. After completion of meiosis, during first interphase, monaster microtubules nucleated from the sperm-derived centrosome play a key role in pronuclear migration. In addition, these astral microtubules direct the relocalization of cytoplasmic domains and the translocation and accumulation of organelles in the interior of the zygote. Microtubules and microfilaments, on the other hand, are involved in cortical reorganizations and organelle translocations in both zygote species during interphase and cleavage divisions. In the case of leech zygotes, this process leads to formation of characteristic polar cytoplasmic domains called teloplasms. These domains are selectively inherited by teloblasts, precursor stem cells of ectodermal and mesodermal tissues in the leech embryo. In the ascidian zygote, the cytoplasmic movements observed during interphase and mitosis lead to relocalization of the bulk of a mitochondria-rich domain, called the myoplasm, along with an endoplasmic reticulum-rich domain towards the future posterior pole of the embryo. The myoplasm is inherited by a subset of posterior blastomeres committed to become the primary muscle cells of the ascidian tadpole.

Animals↗
PubMed2000

Scanning EM of resting gastric parietal cells reveals a network of cytoplasmic tubules and cisternae connected to the intracellular canaliculus.

Using a recently developed fixation technique for parietal cells (Sugai et al., Acta Anat Nippon 1995:70:S79, 1999:74:S101), we have reinvestigated the organization of the cytoplasmic membrane system in the resting stomach by ultra-high-resolution scanning electron microscopy (SEM). Rat gastric mucosae were microwave-fixed in cacodylate buffer [334 milliosmoles/kg H(2)O (mOsm)], to which 1.0% glutaraldehyde and 0.5% formaldehyde were added. Specimens examined by transmission electron microscopy (TEM) of thin sections revealed cytoplasm packed with tubular membranes similar to images detected by rapid-freeze/freeze-substitution fixation which is generally considered to cause minimal structural alterations. To render the cytoplasmic membranes visible by SEM, fixed mucosae were frozen, fractured, and the exposed cytoplasm of parietal cells was macerated by the aldehyde-osmium-DMSO-osmium procedure. With much of the cell matrix and filaments removed, SEM revealed numerous 30-60 nm tubules which formed a meshwork and also small cisternae. The cytoplasmic surface of the tubules was smooth while some cisternal areas had attached polyribosomes. Vesicles or isolated tubules were not found in appropriately macerated parietal cells. The cytoplasmic surface of the intracellular canaliculus was smooth except for round openings representing the bases of macerated microvilli. In favorable sites connections of the tubular membranes to the canaliculi were clearly visible. Stereo pair views were particularly useful to demonstrate these continuities. Connections between these two membrane compartments suggest the probability of rapid membrane transposition.

Animals↗
PubMed1992

The internalization signal in the cytoplasmic tail of lysosomal acid phosphatase consists of the hexapeptide PGYRHV.

Lysosomal acid phosphatase (LAP) is rapidly internalized from the cell surface due to a tyrosine-containing internalization signal in its 19 amino acid cytoplasmic tail. Measuring the internalization of a series of LAP cytoplasmic tail truncation and substitution mutants revealed that the N-terminal 12 amino acids of the cytoplasmic tail are sufficient for rapid endocytosis and that the hexapeptide 411-PGYRHV-416 is the tyrosine-containing internalization signal. Truncation and substitution mutants of amino acid residues following Val416 can prevent internalization even though these residues do not belong to the internalization signal. It was shown recently that part of the LAP cytoplasmic tail peptide corresponding to 410-PPGY-413 forms a well-ordered beta turn structure in solution. Two-dimensional NMR spectroscopy of two modified LAP tail peptides, in which the single tyrosine was substituted either by phenylalanine or by alanine, revealed that the tendency to form a beta turn is reduced by 25% in the phenylalanine-containing peptide and by approximately 50% in the alanine-containing mutant peptide. Our results suggest, that in the short cytoplasmic tail of LAP tyrosine is required for stabilization of the right turn and that the aromatic ring system of the tyrosine residue is a contact point to the putative cytoplasmic receptor.

Acid Phosphatase↗
PubMed1983

Evidence that a variety of cultured cells secrete protease nexin and produce a distinct cytoplasmic serine protease-binding factor.

Four criteria were used to examine serum-free conditioned cell culture medium for protease nexin (PN):(1) formation of SDS-stable approximately 77 K Da complexes between a medium component and [125I]thrombin; (2) acceleration by heparin of the rate of formation of these complexes; (3) cellular binding of these complexes; and (4) inhibition by heparin of the cellular binding of complexes. Listed in order of decreasing PN production, PN was detected in media conditioned by the following cell types: human foreskin fibroblasts (0.18 micrograms/10(6) cells), rat embryo heart muscle cells (0.13 micrograms/10(6) cells), mouse myotubes (0.1 micrograms/10(6) cells), monkey kidney epithelial cells, human fibrosarcoma cells, human lung fibroblasts, simian virus 40 (SV-40)-transformed human fibroblasts, human epidermoid carcinoma cells, bovine aortic endothelial cells (only after phorbol ester treatment), and mouse myoblasts. No PN was found in medium conditioned by mouse 3T3 cells, SV40 virus-transformed 3T3 cells, human lymphoblasts, or mouse leukemia cells. Eleven of the cell types examined for secretion of PN were also examined for the presence of cytoplasmic thrombin-binding factors. Lysates from all of these cell types contained a factor that formed approximately 60-65 K Da sodium dodecyl sulfate (SDS)-stable complexes with [125I] thrombin. This MW is significantly lower than that of [125I] thrombin-PN complexes, indicating that the factor is distinct from PN. Nevertheless, PN and the cytoplasmic factor share similarities. Production of both PN (by HF cells and WI-26 cells) and the cytoplasmic factor (by HF cells and 3T3 cells) are stimulated by epidermal growth factor and phorbol myristate acetate. Also, both PN and the cytoplasmic factor complex trypsin, plasmin, urokinase, and thrombin, but not pancreatic elastase. Because a number of the cells that produce PN or the cytoplasmic serine protease-binding factor are known to produce plasminogen activators, both PN and the cytoplasmic factor could regulate plasminogen activator activity.

Amyloid beta-Protein Precursor↗
PubMed2003

Rapsyn-mediated clustering of acetylcholine receptor subunits requires the major cytoplasmic loop of the receptor subunits.

During synaptogenesis at the neuromuscular junction, nicotinic acetylcholine receptors (AChRs) are organized into high-density postsynaptic clusters that are critical for efficient synaptic transmission. Rapsyn, an AChR associated cytoplasmic protein, is essential for the aggregation and immobilization of AChRs at the neuromuscular junction. Previous studies have shown that when expressed in nonmuscle cells, both assembled and unassembled AChR subunits are clustered by rapsyn, and the clustering of the alpha subunit is dependent on its major cytoplasmic loop. In the present study, we investigated the mechanism of rapsyn-induced clustering of the AChR beta, gamma, and delta subunits by testing mutant subunits for the ability to cocluster with rapsyn in transfected QT6 cells. For each subunit, deletion of the major cytoplasmic loop, between the third and fourth transmembrane domains, dramatically reduced coclustering with rapsyn. Furthermore, each major cytoplasmic loop was sufficient to mediate clustering of an unrelated transmembrane protein. The AChR subunit mutants lacking the major cytoplasmic loops could assemble into alphadelta dimers, but these were poorly clustered by rapsyn unless at least one mutant was replaced with its wild-type counterpart. These results demonstrate that the major cytoplasmic loop of each AChR subunit is both necessary and sufficient for mediating efficient clustering by rapsyn, and that only one such domain is required for rapsyn-mediated clustering of an assembly intermediate, the alphadelta dimer.

Animals↗
PubMed1995

High affinity chimeric human granulocyte-macrophage colony-stimulating factor receptor carrying the cytoplasmic domain of the beta subunit but not the alpha subunit transduces growth promoting signals in Ba/F3 cells.

Granulocyte-macrophage colony-stimulating factor receptor (GMR) is composed of two distinct subunits alpha and beta, and the cytoplasmic domains of both subunits are essential to transduce signals. We further analyzed the role of the cytoplasmic domain of each subunit by constructing chimeric subunits, designated alpha/beta and beta/alpha, by exchanging cytoplasmic domains of the alpha and beta subunits of hGMR. Reconstituted high-affinity chimeric hGMRs, hGMR(alpha/beta,beta/alpha) and hGMR(alpha/beta,beta), as well as the wild type hGMR(alpha,beta), transduced signals in Ba/F3 cells. These observations indicate that the original configuration between the extracellular and the cytoplasmic domains of the hGMR(alpha,beta) subunits is not obligatory, and that hGMR(alpha/beta,beta) transduced signals through the cytoplasmic domain of the beta subunit in an oligomeric form, without involvement of the cytoplasmic domain of the alpha subunit.

Animals↗
PubMed2001

Functional comparison of the alpha3A and alpha3B cytoplasmic domain variants of the chicken alpha3 integrin subunit.

Integrin alpha3beta1 can be alternatively spliced to generate alpha3A and alpha3B cytoplasmic domain variants that are conserved among vertebrates. To identify distinct functions of these variants, we transfected cells with intact alpha3 integrins or chimeric receptors. alpha3Abeta1 and alpha3Bbeta1 each localized to focal contacts in keratinocytes on an extracellular matrix rich in laminin-5, to which both are known to bind with high affinity. However, alpha3B accumulated intracellularly in keratinocytes on collagen, suggesting that laminin binding may stabilize alpha3Bbeta1 surface expression. Neither alpha3 cytoplasmic domain affected recruitment of chimeric alpha5 integrins to fibronectin-induced focal contacts, and either substituted for the alpha5 cytoplasmic domain in alpha5beta1-mediated cell migration. However, the alpha5/alpha3B chimera localized to cell-cell borders in MDCK or CHO cells to a lesser extent than did the alpha5/alpha3A chimera. To determine whether the alpha3 cytoplasmic domains conferred distinct localization to a nonintegrin protein, we transfected cells with interleukin-2 receptor (IL-2R) chimeras containing the alpha3 cytoplasmic domains. The IL-2R/alpha3A chimera was expressed efficiently on the cell surface, while the IL-2R/alpha3B chimera accumulated intracellularly. Our findings suggest that the alpha3B cytoplasmic domain harbors a retention signal that is regulated in an intact integrin and can alter cell surface expression and distribution of alpha3beta1.

Alternative Splicing↗