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At least 19 recordsLinked to original sources

PubMed1992

The cystic fibrosis transmembrane regulator is present and functional in endosomes. Role as a determinant of endosomal pH.

Cystic fibrosis is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), which lead to defective Cl- conductance in epithelial cells. While the CFTR gene product has been detected in the plasma membrane, its presence and functional role in the membranes of intracellular compartments remain to be established. The purpose of the present experiments was to functionally localize CFTR in the endosomal membrane and to test the role of the associated Cl- conductance in the regulation of endosomal pH (pH(en)). When using conductive protonophores, the net H+ flux across the endosomal membrane of Chinese hamster ovary (CHO) cells is limited by the movement of counterions. Thus, ionic permeability could be estimated indirectly, from the changes in pH(en) determined fluorimetrically. Measurements in situ and in a cell-free microsomal preparation indicate the presence of a protein kinase A (PKA)-activated anion conductance in endosomes from CHO cells transfected with CFTR, but not in endosomes from wild-type or mock-transfected cells. In endosomes isolated from CFTR-expressing cells, the stimulatory effect of PKA was diminished by a specific peptide inhibitor of PKA, by alkaline phosphatase treatment or by a monoclonal antibody against the second nucleotide binding fold of CFTR. Increasing counterion permeability by phosphorylation of CFTR or by addition of valinomycin failed to alter the rate or extent of endosomal acidification in situ. Our observations indicate that functional CFTR, susceptible to activation by PKA, is present in endosomes of transfected CHO cells. More importantly, the data suggest that factors other than counterion permeability are the major determinants of pH(en).

Alkaline Phosphatase↗

PubMedDate not supplied

Kinetics of endosomal acidification in Dictyostelium discoideum amoebae. 31P-NMR evidence for a very acidic early endosomal compartment.

We have examined the pH of the various endosomal compartments in the amoebae of the cellular slime mould Dictyostelium discoideum. This was accomplished both by fluorescence and by in vivo 31P-NMR methods. The fluid-phase marker, fluorescein-labeled dextran, was fed to the amoebae to report the average pH of their endocytic vesicles. During the progressive loading of successive endosomal compartments, we observed an early acidification down to a minimum value of pH < or = 5.3 after 30 min at 20 degrees C followed by an increase to an average pH of 5.8 when all the endosomal compartments were loaded by the fluid-phase marker. The weak fluorescence intensity of FITC-dextran at acidic pH precluded a more detailed investigation and we checked various phosphonate compounds as potential 31P-NMR pH probes for the endosomal compartments. Two molecules, aminomethylphosphonate and 2-aminoethylphosphonate, were selected for this study because of the large amplitudes of their chemical shift variation with pH (2 and 2.5 ppm, respectively) and their acidic pKs of 5.5 and 6.3, respectively. They were only moderately toxic (IC50% approximately 10 mM) towards both the axenic growth and the differentiation program of Dictyostelium amoebae. Internalization of the two aminophosphonates occurred only through the fluid-phase pinocytosis pathway as revealed by the full inhibition of their entry with 1 mM vanadate or 7.5 mM caffeine, two previously characterized inhibitors of endocytosis in Dictyostelium. We found that in vivo 31P-NMR of amoebae suspensions incubated with the aminophosphonates allowed the detection of three distinct intracellular compartments at pH 4.3, 5.8-6.0 and 7.3. Kinetics of aminophosphonate entry were analyzed and the results allowed us to reconstruct the time course for the acidification sequence during endocytosis. The data are consistent with the hypothesis that in Dictyostelium amoebae phosphonates occupy a highly acidic early endosomal compartment (t1/2 = 18 min; pH 4.3) before reaching a less acidic late endosomal/prelysosomal compartment (pH 5.8-6.0) from where they are immediately transported to, and trapped in, the cytoplasm (pH 7.3).

Acids↗

PubMed1992

Inhibition of endosomal proteolytic activity by leupeptin blocks surface expression of MHC class II molecules and their conversion to SDS resistance alpha beta heterodimers in endosomes.

The biosynthesis of MHC Class II molecules starts with the assembly of the alpha and beta subunits and the invariant chain. Intracellular transport of Class II molecules was followed in pulse-chase experiments of a human Epstein-Barr virus-transformed B lymphoblastoid cell line. Entry of Class II molecules into the endocytotic pathway and their cell surface appearance were monitored using neuraminidase as a fluid endocytotic marker and as a surface probe, respectively. In the course of intracellular transport, the Class II associated invariant chain is removed by proteases located in the endosomal pathway. Here, we show that leupeptin inhibits not only invariant chain breakdown, but also surface deposition of newly synthesized Class II molecules. Class II molecules display remarkable resistance to SDS at ambient temperature when occupied by peptide. We exploit this property to show that peptide binding precedes surface expression, and takes place in the course of intracellular transport through an endosomal compartment. Leupeptin blocks the conversion of Class II molecules to an SDS resistant complex.

Animals↗

PubMed1992

Apical endosomes isolated from kidney collecting duct principal cells lack subunits of the proton pumping ATPase.

Endocytic vesicles that are involved in the vasopressin-stimulated recycling of water channels to and from the apical membrane of kidney collecting duct principal cells were isolated from rat renal papilla by differential and Percoll density gradient centrifugation. Fluorescence quenching measurements showed that the isolated vesicles maintained a high, HgCl2-sensitive water permeability, consistent with the presence of vasopressin-sensitive water channels. They did not, however, exhibit ATP-dependent luminal acidification, nor any N-ethylmaleimide-sensitive ATPase activity, properties that are characteristic of most acidic endosomal compartments. Western blotting with specific antibodies showed that the 31- and 70-kD cytoplasmically oriented subunits of the vacuolar proton pump were not detectable in these apical endosomes from the papilla, whereas they were present in endosomes prepared in parallel from the cortex. In contrast, the 56-kD subunit of the proton pump was abundant in papillary endosomes, and was localized at the apical pole of principal cells by immunocytochemistry. Finally, an antibody that recognizes the 16-kD transmembrane subunit of oat tonoplast ATPase cross-reacted with a distinct 16-kD band in cortical endosomes, but no 16-kD band was detectable in endosomes from the papilla. This antibody also recognized a 16-kD band in affinity-purified H+ ATPase preparations from bovine kidney medulla. Therefore, early endosomes derived from the apical plasma membrane of collecting duct principal cells fail to acidify because they lack functionally important subunits of a vacuolar-type proton pumping ATPase, including the 16-kD transmembrane domain that serves as the proton-conducting channel, and the 70-kD cytoplasmic subunit that contains the ATPase catalytic site. This specialized, non-acidic early endosomal compartment appears to be involved primarily in the hormonally induced recycling of water channels to and from the apical plasma membrane of vasopressin-sensitive cells in the kidney collecting duct.

Animals↗

PubMed1992

Second messengers regulate endosomal acidification in Swiss 3T3 fibroblasts.

Acidification of the endosomal pathway is important for ligand and receptor sorting, toxin activation, and protein degradation by lysosomal acid hydrolases. Fluorescent probes and imaging methods were developed to measure pH to better than 0.2 U accuracy in individual endocytic vesicles in Swiss 3T3 fibroblasts. Endosomes were pulse labeled with transferrin (Tf), alpha 2-macroglobulin (alpha 2M), or dextran, each conjugated with tetramethylrhodamine and carboxyfluorescein (for pH 5-8) or dichlorocarboxyfluorescein (for pH 4-6); pH in individual labeled vesicles was measured by ratio imaging using a cooled CCD camera and novel image analysis software. Tf-labeled endosomes acidified to pH 6.2 +/- 0.1 with a t1/2 of 4 min at 37 degrees C, and remained small and near the cell periphery. Dextran- and alpha 2M-labeled endosomes acidified to pH 4.7 +/- 0.2, becoming larger and moving toward the nucleus over 30 min; approximately 15% of alpha 2M-labeled endosomes were strongly acidic (pH less than 5.5) at only 1 min after labeling. Replacement of external Cl by NO3 or isethionate strongly and reversibly inhibited acidification. Addition of ouabain (1 mM) at the time of labeling strongly enhanced acidification in the first 5 min; Tf-labeled endosomes acidified to pH 5.3 without a change in morphology. Activation of phospholipase C by vasopressin (50 nM) enhanced acidification of early endosomes; activation of protein kinase C by PMA (100 nM) enhanced acidification strongly, whereas elevation of intracellular Ca by A23187 (1 microM) had no effect on acidification. Activation of protein kinase A by CPT-cAMP (0.5 mM) or forskolin (50 microM) inhibited acidification. Lysosomal pH was not affected by ouabain or the protein kinase activators. These results establish a methodology for quantitative measurement of pH in individual endocytic vesicles, and demonstrate that acidification of endosomes labeled with Tf and alpha 2M (receptor-mediated endocytosis) and dextran (fluid-phase endocytosis) is sensitive to intracellular anion composition, Na/K pump inhibition, and multiple intracellular second messengers.

3T3 Cells↗

PubMedDate not supplied

Pharmacological doses of insulin equalize insulin receptor phosphotyrosine content but not tyrosine kinase activity in plasmalemmal and endosomal membranes.

Following insulin administration to intact rats, the insulin receptor kinase activity of subsequently isolated cell fractions was significantly augmented. Of interest was the observation that the endosomal insulin receptor tyrosine kinase displayed four- to six-fold greater autophosphorylation activity than that of plasma membrane. Surprisingly, the endosomal insulin receptor tyrosine kinase displayed a decrease in beta-subunit phosphotyrosine content compared with that seen in the plasma membrane. These observations prompted the suggestion that insulin receptor tyrosine kinase phosphotyrosine dephosphorylation mediated by an endosome-specific phosphotyrosine phosphatase(s) yields activation of the endosomal insulin receptor tyrosine kinase. In a previous study we examined the effect of subsaturating doses of injected insulin. In this work we evaluated insulin receptor tyrosine kinase activity and phosphotyrosine content in plasma membrane and endosomes after a receptor-saturating pharmacological dose of insulin (150 micrograms/100 g body weight). At this dose the phosphotyrosine content per receptor was reduced compared with that seen earlier at insulin doses of 1.5 and 15 micrograms/100 g body weight. Endosomal insulin receptor tyrosine kinase was greater than that seen at the lower nonsaturating insulin doses. Furthermore, endosomal insulin receptor tyrosine kinase activity exceeded that of the plasma membrane, despite retaining about the same phosphotyrosine content per receptor. These data are consistent with the view that insulin receptor tyrosine kinase activity may be regulated by a particular pattern of phosphotyrosine content on the beta-subunit wherein both activating and inhibitory phosphotyrosine residues play a role.

Amino Acid Sequence↗

PubMed1992

Lumenal labeling of rat hepatocyte early endosomes. Presence of multiple membrane receptors and the Na+,K(+)-ATPase.

We used lactoperoxidase-mediated iodination to investigate the lumenal polypeptide composition of rat hepatocyte endosomes. A chemical conjugate of asialoorosomucoid and lactoperoxidase that binds specifically to hepatocyte asialoglycoprotein receptors was perfused through isolated rat livers at 16 degrees C in the presence of mannan, resulting in the accumulation of ligand in early endosomes. Endosome containing low density vesicle fractions were subsequently isolated from sucrose gradients of microsomes, and the lactoperoxidase moiety was used to catalyze the iodination of lumenal-facing proteins. After gel electrophoresis, 125I-labeled early endosomes reproducibly showed a distinct 125I-polypeptide profile containing prominently labeled bands migrating at 43, 52, 58, 90, 110, 135, 230, and greater than 300 kDa. The asialoglycoprotein receptor (43-, 52-, and 58-kDa subunits) was by far the predominantly labeled protein even when iodinations were performed under conditions of receptor-ligand dissociation, and we conclude that it is the most abundant hepatocyte early endosomal protein. Furthermore, the iodination profile of the three asialoglycoprotein receptor subunits differed strikingly from their chemical amounts. Using immunoprecipitation, we directly identified the Na+,K(+)-ATPase; to our knowledge, this is the first biochemical evidence for the Na+,K(+)-ATPase in rat hepatocyte early endosomes. We also directly identified receptors for mannose 6-phosphate, epidermal growth factor, transferrin, and polymeric IgA in 125I-labeled early endosomes.

Animals↗

PubMed1992

Association of the tyrosine phosphorylated epidermal growth factor receptor with a 55-kD tyrosine phosphorylated protein at the cell surface and in endosomes.

After the intraportal injection of EGF, the EGF receptor (EGFR) is rapidly internalized into hepatic endosomes where it remains largely receptor bound (Lai et al., 1989. J. Cell Biol. 109:2751-2760). In the present study, we evaluated the phosphotyrosine content of EGFRs at the cell surface and in endosomes in order to assess the consequences of internalization. Quantitative estimates of specific radioactivity of the EGFR in these two compartments revealed that tyrosine phosphorylation of the EGFR was observed at the cell surface within 30 s of ligand administration. However, the EGFR was also highly phosphorylated in endosomes reaching levels of tyrosine phosphorylation significantly higher than those of the cell surface receptor at 5 and 15 min after EGF injection. A 55-kD tyrosine phosphorylated polypeptide (pyp55) was observed in association with the EGFR at the cell surface within 30 s of EGF injection. The protein was also found in association with the EGFR in endosomes as evidenced by coprecipitation studies using a mAb to the EGFR as well as by coelution with the EGR in gel permeation chromatography. Limited proteolysis of isolated endosomes indicated that the tyrosine phosphorylated domains of the EGFR and associated pyp55 were cytosolically oriented while internalized EGF was intraluminal. The identification of pyp55 in association with EGFR in both hepatic plasma membranes and endosomes may be relevant to EGFR function and/or trafficking of the EGFR.

Amino Acid Sequence↗

PubMed2026

The VPS9-family GEF VINE activates Ypt10 in a late endosomal Rab cascade.

Rab GTPase cascades drive endosomal membrane maturation by sequentially activating and inactivating Rab proteins. These transitions in Rab signaling require the coordinated actions of guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). The yeast VINE complex is an endosomal VPS9-family GEF that stimulates a GAP to inactivate the Rab5 homolog Vps21, suggesting a role for VINE in coordinating Rab transitions. Here we report that VINE acts through its catalytic GEF domain to positively regulate the Rab5-related GTPase Ypt10 and establish a pool of Ypt10 at late endosomes. Ypt10 activation occurs downstream of Vps21 activity, placing Ypt10 within a late endosomal Rab cascade. Genome-wide protein-protein interaction screens revealed a VINE-dependent proximity interaction between Ypt10 and the GEF Mon1-Ccz1. Our data suggest that VINE and Ypt10 regulate late endosomal recruitment of Mon1-Ccz1 to enhance the activation of its substrate, the Rab7 homolog Ypt7. Together, these findings define a Vps21-VINE-Ypt10 regulatory module that adds a layer of control within the late endosomal Vps21-to-Ypt7 cascade and establish VINE as a dual Rab regulator. Through opposing activities on Vps21 and Ypt10, VINE may couple Rab5 inactivation to Mon1-Ccz1 recruitment to provide more precise control of degradative protein traffic to the vacuole.

Journal Article↗

PubMed1992

Protein kinase A dependent membrane protein phosphorylation and chloride conductance in endosomal vesicles from kidney cortex.

Regulation of Cl conductance by protein kinase A may play a role in control of endosomal acidification [Bae, H.-R., & Verkman, A. S. (1990) Nature, 348, 637-639]. To investigate the mechanism of kinase A action, cell-free measurements of Cl transport and membrane protein phosphorylation were carried out in apical endocytic vesicles from rabbit kidney proximal tubule. Cl transport was measured by a stopped-flow quenching assay in endosomes labeled in vivo with the fluorescent Cl indicator 6-methoxy-N-(3-sulfopropyl)quinolinium. Phosphorylation was studied in a purified endosomal preparation by SDS-PAGE and autoradiography of membrane proteins labeled by [gamma-32P]ATP. Endosomes had a permeability (PCl) for conductive Cl transport of 3.1 x 10(-8) cm/s at 23 degrees C which was stilbene inhibitable. PCl was increased by 90 +/- 20% by a 10-min preincubation with the catalytic subunit of kinase A (PKA, 10 units/mL) and MgATP (0.5 mM) with anion selectivity Cl greater than I greater than Br. The increase in PCl was blocked by 100 microM N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide (H-8) and was reversed by addition of alkaline phosphatase (AP, 40 units/mL) after incubation with PKA and MgATP; the increase in PCl was not blocked by pretreatment with AP.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Evidence of a role for heterotrimeric GTP-binding proteins in endosome fusion.

Guanosine triphosphate (GTP)-binding proteins are required for intracellular vesicular transport. Mastoparan is a peptide component of wasp venom that increases nucleotide exchange in some classes of G alpha subunits of regulatory heterotrimeric GTP-binding proteins (G proteins). Mastoparan and other compounds that increase nucleotide exchange by G proteins inhibited endosome fusion in vitro and reversed the effects of guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S), a nonhydrolyzable GTP analog. Addition of beta gamma subunits of G proteins to the fusion assay antagonized the stimulatory effect of GTP-gamma-S, confirming the participation of G proteins. These results indicate that GTP-binding proteins are required for endosome fusion and in particular that a G protein is involved. Given the function of G proteins in signal transduction, these findings may provide insight into the mechanism by which endosomal vesicles become competent for fusion after their formation at the cell surface.

Biological Transport↗

PubMed1992

Association of p60c-src with endosomal membranes in mammalian fibroblasts.

We have examined the subcellular localization of p60c-src in mammalian fibroblasts. Analysis of indirect immunofluorescence by three-dimensional optical sectioning microscopy revealed a granular cytoplasmic staining that co-localized with the microtubule organizing center. Immunofluorescence experiments with antibodies against a number of membrane markers demonstrated a striking co-localization between p60c-src and the cation-dependent mannose-6-phosphate receptor (CI-MPR), a marker that identifies endosomes. Both p60c-src and the CI-MPR were found to cluster at the spindle poles throughout mitosis. In addition, treatment of interphase and mitotic cells with brefeldin A resulted in a clustering of p60c-src and CI-MPR at a peri-centriolar position. Biochemical fractionation of cellular membranes showed that a major proportion of p60c-src co-enriched with endocytic membranes. Treatment of membranes containing HRP to alter their apparent density also altered the density of p60c-src-containing membranes. Similar density shift experiments with total cellular membranes revealed that the majority of membrane-associated p60c-src in the cell is associated with endosomes, while very little is associated with plasma membranes. These results support a role for p60c-src in the regulation of endosomal membranes and protein trafficking.

Animals↗

PubMed2026

Abnormal ClC-3/TMEM9-mediated endosomal ion transport in CLCN3-associated neurodevelopmental disease.

Endolysosomal abnormalities are particularly detrimental to the nervous system and have been implicated in neuropsychiatric disorders. Key regulators of the lysosomal and endosomal luminal ion homeostasis are CLC chloride/proton exchangers. We report 15 individuals carrying variants in CLCN3, encoding a ubiquitous endosomal 2Cl-/H+ exchanger, and provide updated clinical information for 5 previously reported individuals. Subjects displayed a broad spectrum of neuropsychiatric symptoms, including developmental delay, intellectual disability, and epilepsy. To reveal the pathogenic mechanism, we investigated ClC-3 variants-mediated ion transport and its regulation by the recently discovered inhibitory beta subunit TMEM9. 12/20 missense variants exhibited altered properties and fell into two classes: those affecting the region binding inhibitory TMEM9 carboxy-termini, and those that broaden the voltage range over which ClC-3 conducts ions. Surprisingly, the latter variants also attenuated TMEM9-mediated inhibition. Both classes produced a toxic gain-of-function, as evident from endolysosomal vacuolization by mutant ClC-3/TMEM9 overexpression. Our results expand the genetic and clinical spectrum of CLCN3-related disease, provide a solid basis for genetic counseling, and uncover an unexpected link between gating-associated conformational changes and inhibition by TMEM9.

Chloride Channels↗

PubMed1992

Release of endosomal content induced by plasma membrane tension: video image intensification time lapse analysis.

Rhodamine-labeled vinculin microinjected into chicken embryo fibroblasts and rhodamine-labeled alpha 2-macroglobulin added to the fibroblast culture medium were sequestered in endocytotic vesicles and digested. When a sealed microcapillary coated with fibronectin or polylysine was attached to the fibroblasts and pulled at speeds of 100-200 microns/h, stretching the plasma membrane, a variable fraction of the endosomes released the rhodamine label. Release from individual vesicles was rapid, reaching completion in less than 30 to 120 s. The microfilament disrupting agent cytochalasin B prevented release, as did the microtubule stabilizing drug taxol. Colcemide, which disrupts microtubules, did not inhibit the release. Release was dependent on extracellular calcium, as it was prevented by 10 mM EGTA in the incubation medium. We postulate that opening of vesicular channels consequent to centripetal transmission of tension generated in the plasma membrane along microfilaments may be a mechanism of release of endosomal content.

Alkaloids↗

PubMed1992

The morphology but not the function of endosomes and lysosomes is altered by brefeldin A.

Brefeldin A (BFA) induces the formation of an extensively fused network of membranes derived from the trans-Golgi network (TGN) and early endosomes (EE). We describe in detail here the unaffected passage of endocytosed material through the fused TGN/EE compartments to lysosomes in BFA-treated cells. We also confirmed that BFA caused the formation of tubular lysosomes, although the kinetics and extent of tubulation varied greatly between different cell types. The BFA-induced tubular lysosomes were often seen to form simple networks. Formation of tubular lysosomes was microtubule-mediated and energy-dependent; interestingly, however, maintenance of the tubulated lysosomes only required microtubules and was insensitive to energy poisons. Upon removal of BFA, the tubular lysosomes rapidly recovered in an energy-dependent process. In most cell types examined, the extensive TGN/EE network is ephemeral, eventually collapsing into a compact cluster of tubulo-vesicular membranes in a process that precedes the formation of tubular lysosomes. However, in primary bovine testicular cells, the BFA-induced TGN/EE network was remarkably stable (for > 12 h). During this time, the TGN/EE network coexisted with tubular lysosomes, however, the two compartments remained completely separate. These results show that BFA has multiple, profound effects on the morphology of various compartments of the endosome-lysosome system. In spite of these changes, endocytic traffic can continue through the altered compartments suggesting that transport occurs through noncoated vesicles or through vesicles that are insensitive to BFA.

Adenosine Triphosphate↗

PubMed1992

Ligand-regulated internalization and recycling of human beta 2-adrenergic receptors between the plasma membrane and endosomes containing transferrin receptors.

Agonist-regulated redistribution of human beta 2-adrenergic receptors was examined in 293 cells. A specific antiserum recognizing the carboxyl-terminal hydrophilic domain of the receptor was developed, characterized, and used for immunocytochemical localization of receptors in fixed cells by conventional fluorescence and confocal fluorescence microscopy. The beta-adrenergic agonist isoproterenol induced redistribution of receptors from the surface of cells into small (less than 1 micron diameter) punctuate accumulations which were detected in cells within 2 min of agonist addition. The time course of receptor redistribution paralleled that of receptor sequestration measured by ligand binding, and receptor redistribution was reversible in the presence of the beta-adrenergic antagonist alprenolol. Optical sections imaged through cells by confocal microscopy localized receptor accumulations within the cytoplasm. To address the question of receptor internalization further, a mutant receptor possessing an engineered antigenic epitope in the amino-terminal hydrophilic domain was constructed, transfected into cells, and localized using both a monoclonal antibody recognizing the epitope tag (receptor ectodomain) and an antiserum recognizing the carboxyl terminus (receptor endodomain). In untreated cells most receptor antigen was detected at the cell surface, as assessed by accessibility to ectodomain antibodies in unpermeabilized specimens. In isoproterenol-treated cells, however, little receptor antigen was detected at the cell surface. Punctate receptor accumulations present in isoproterenol-treated cells were labeled by antibodies only following permeabilization of cells, as expected if these receptor accumulations were intracellular. Finally, internalized beta-adrenergic receptors colocalized with transferrin receptors, which are markers of endosomal membranes. These data provide several lines of evidence establishing that beta-adrenergic receptors undergo ligand-regulated internalization, they suggest that internalized receptors may be recycled back to the cell surface, and they provide the first direct indication that these processes involve the same endosomal membrane system passaged by constitutively recycling receptors.

Alprenolol↗

PubMed1992

The dephosphorylation of insulin and epidermal growth factor receptors. Role of endosome-associated phosphotyrosine phosphatase(s).

The autophosphorylation, from [gamma-32P]ATP, of insulin and epidermal growth factor receptors in rat liver endosomes peaked at 2-5 min and declined thereafter. When autophosphorylation from either [gamma-32P]ATP or unlabeled ATP was stopped after 5 min by adding excess EDTA +/- ATP, the phosphotyrosine (PY) content of each receptor decreased at 37 degrees C with a t 1/2 of 1.6 min. This was equally so whether the PY content of 32P-labeled receptors was analyzed by autoradiography of KOH-treated gels or by Western blotting with PY antibodies of immunoprecipitated receptors. The dephosphorylation reaction was strictly dependent on the presence of sulfhydryl, was unaffected by the addition of rat liver cytosol, and was temperature-dependent. The phosphotyrosine phosphatase(s) (PTPase(s)) appeared to be tightly anchored to the endosomal membrane, since the dephosphorylation reaction was unaffected by sodium carbonate and 0.6 M KCl treatments. However, treatment with Triton X-100 abolished dephosphorylation, implying an intimate association between the PTPase(s) and its substrate in an intact membrane environment. The powerful insulinomimetic agent pervanadate was the most potent inhibitor (50% inhibition at 1 microM). Increasing the dose of injected ligand augmented the rate of insulin and decreased that of EGF receptor dephosphorylation, respectively. Immunoblotting with specific antibodies failed to identify PTPase 1B or T-cell PTPase in ENs, whereas positive signals were seen in plasma membrane. These studies indicate that the phosphorylation state of receptor tyrosine kinases is dynamically regulated, with dephosphorylation, by closely associated PTPase(s), playing an important role.

Adenosine Triphosphate↗

PubMed1992

High-efficiency receptor-mediated delivery of small and large (48 kilobase gene constructs using the endosome-disruption activity of defective or chemically inactivated adenovirus particles.

One limit to successful receptor-mediated gene delivery is the exit of the endocytosed material from the endosome. We demonstrate here the delivery of marker genes to tissue culture cells using a modification of the receptor-mediated gene delivery technique that exploits the endosomolytic activity of defective adenovirus particles. In particular, greater than 90% of the transfected-cell population is found to express a beta-galactosidase gene, and, most importantly, this high level of expression can be obtained with psoralen-inactivated virus particles. Furthermore, because the delivered gene is not carried within the genome of the adenovirus particle, the size constraints are relieved, and we can, therefore, show the delivery of a 48-kilobase cosmid DNA molecule.

Adenoviruses, Human↗