Differential and analytical subfraction of rat liver components internalizing insulin and prolactin

Bergeron, John J.M.; Searle, Norman R.; Khan, Masood N.; Posner, Barry I.

doi:10.1021/bi00355a046

Cited by 51 publications

(32 citation statements)

References 35 publications

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“…These E 2 b binding-sites co-purify with 5'-nucleotidase, a plasma membrane-marker enzyme, and appear to be free from signi®cant contamination by cytosol or nuclei. The plasma membrane E 2 b binding-sites constitute about 20% of total cell binding-sites for the steroid, a level of membrane concentration comparable to that found for other known transmembrane hormone receptors (Bergeron et al, 1986). In addition, monoclonal antibodies against the LBD of nuclear ER can identify membrane-associated ER in MCF-7 cells, a ®nding consistent with studies with other cell types (Pappas et al, 1995;Razandi et al, 1999;Russell et al, 2000).…”

Section: Discussionsupporting

confidence: 79%

Membrane-associated binding sites for estrogen contribute to growth regulation of human breast cancer cells

2001

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Membrane-associated binding sites for estrogen may mediate rapid eects of estradiol-17b that contribute to proliferation of human breast cancers. After controlled homogenization and fractionation of MCF-7 breast cancer cells, the bulk of speci®c estradiol binding is found in nuclear fractions. However, a signi®cant portion of speci®c, high-anity estradiol-17b binding-sites are also enriched in plasma membranes. These estradiol binding-sites co-purify with 5'-nucleotidase, a plasma membrane-marker enzyme, and are free from major contamination by cytosol or nuclei. Electrophoresis of membrane fractions allowed detection of a primary 67-kDa protein and a secondary 46-kDa protein recognized by estradiol-17b and by a monoclonal antibody directed to the ligand-binding domain of the nuclear form of estrogen receptor. Estrogen-induced growth of MCF-7 breast cancer cells in vitro was blocked by treatment with the antibody to estrogen receptor and correlated closely with acute hormonal activation of mitogenactivated protein kinase and Akt kinase signaling. Estrogen-promoted growth of human breast cancer xenografts in nude mice was also signi®cantly reduced by treatment in vivo with the estrogen receptor antibody. Thus, membrane-associated forms of estrogen receptor may play a role in promoting intracellular signaling for hormone-mediated proliferation and survival of breast cancers and oer a new target for antitumor therapy. Oncogene (2001) 20, 5420 ± 5430.

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Section: Discussionsupporting

confidence: 79%

Membrane-associated binding sites for estrogen contribute to growth regulation of human breast cancer cells

2001

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“…2B). The G/E fraction used in this study was shown to contain Golgi elements but to be essentially free of plasmalemma and other subcellular constituents (47,48,57). This was affirmed here by the detection of the slower migrating Cdk2 isoform in the cytosolic fraction, which was barely detected in the G/E and PM fractions ( Fig.…”

Section: Resultssupporting

confidence: 70%

Characterization of Cdk2-Cyclin E Complexes in Plasma Membrane and Endosomes of Liver Parenchyma

Gaulin¹,

Fiset²,

Fortier

et al. 2000

Journal of Biological Chemistry

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Rat liver parenchyma Golgi/endosomes fractions harbor a tyrosine-phosphorylated 34-kDa protein. Screening of Golgi, endosomes (ENs), plasmalemma (PM), and cytosolic (Cyt) fractions revealed the presence of the mitotic kinase Cdk2 in ENs, PM, and Cyt. The fluid phase endocytic marker horseradish peroxidase gained access to the endosomal Cdk2, confirming its localization. Cdk2 was shown to be associated to cyclin E and was active in ENs and PM fractions. The administration of a single dose of insulin (1.5 g/100 g, body weight) induced a time-dependent activation of the insulin receptor kinase in these structures. Insulin receptor-kinase activation was followed by the inhibition of immunoprecipitated Cdk2-cyclin E kinase activity in PM and the progressive disappearance of cyclin E. In marked contrast, no such effect was observed in ENs. The injection of a phosphotyrosyl phosphatase inhibitor (bpV-(phen)) increased the levels of cyclin E in ENs and PM. A massive recruitment of p27 kip1 was observed in the Cdk2-cyclin E complexes isolated from PM and Cyt but not from ENs. In vitro, Cdk2-cyclin E complexes have the capacity to inhibit the formation of hybrid structures containing horseradish peroxidase and radioiodinated epidermal growth factor. Therefore, in the PM and ENs of adult rat liver, an active and regulated pool of the mitotic kinase Cdk2-cyclin E and some yet to be defined effectors are present. Cdk2 may contribute to the modulation of transport events and/or maintenance of the topology of endocytic elements.

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“…The ability of chloroquine to inhibit endosomal insulin degradation in vivo [5,8,151 and in cell-free studies [12, 151 prompted us to examine whether this drug affected the pH dependence of insulin degradation. Chloroquine and the chemically related compound quinacrine were found to cause a leftward shift in the plot of insulin degradation versus pH, opposite to that observed with ATP.…”

Section: Discussionmentioning

confidence: 99%

Degradation of insulin in isolated liver endosomes is functionally linked to ATP‐dependent endosomal acidification

Desbuquois¹,

Janicot²,

Dupuis³

1990

European Journal of Biochemistry

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The degradation of insulin in isolated liver endosomes and the relationships of this process with ATP-dependent endosomal acidification have been studied. Incubation of endosomal fractions containing 1251-insulin in isotonic KCl at 30°C resulted in a rapid loss of insulin integrity as judged from trichloroacetic acid precipitability, Sephadex G-50 chromatography, immunoreactivity and receptor binding ability, with a maximum at pH 5 -6 ( t 1 , 2 : 10, 10, 6 and 6 min, respectively). On a log/log plot, the amount of acid-soluble products generated was linearly related to the amount of insulin associated with endosomes (slope, 0.80). Upon incubation, virtually all acid-soluble products diffused out of endosomes as judged from their solubility in aqueous poly(ethyleneglyco1). In permeabilized endosomes, intact insulin was also released in part extraluminally, but only when degradation was inhibited did this release increase with lowering pH. ATP shifted the pH for maximal insulin degradation to about 7.5 -8.5 and caused endosomal acidification as judged from the uptake of acridine orange and the fluorescence of internalized fluorescein-labeled dextran and galactosylated bovine serum albumin (dpH about 0.8 -0.9). GTP, ITP and UTP exerted comparable effects but with lower potencies. The ability of ATP to alter the pH dependence of insulin degradation was maximal in the presence of CI-, other anions being less effective (Br-> gluconate = SO:-> NO; = sucrose = mannitol) and/or inhibitory (NO;). Na', K + and Li' supported more effectively ATP-dependent insulin degradation than did choline. Divalent cations were required for the ATP effect (Mg2Little or no effects of ATP occurred in the presence of proton ionophores such as monensin and carbonyl cyanide chlorophenylhydrazone, and inhibitors of the proton ATPase such as N-ethylmaleimide. The abilities of nucleotides, ions and inhibitors to support or inhibit ATP-dependent insulin degradation were well correlated with their abilities to affect ATP-dependent acidification. The acidotropic agents chloroquine and quinacrine caused a leftward shift in the pH dependence of insulin degradation and a decrease in maximal degradation; in the presence of ATP, chloroquine almost completely inhibited degradation at pH 5 -9. It is concluded that ATP-dependent acidification, in part by enhancing the dissociation of the insulinreceptor complex, is required for optimum degradation of insulin within liver endosomes.Biochemical and morphological studies on intact liver and isolated hepatocytes have shown that, following binding to its receptor at the cell surface, insulin undergoes rapid endocytosis along with its receptor (reviewed in [l -31). Subsequently, the receptor is recycled in part to the cell surface, while most of the insulin is degraded within the cell. Several lines of evidence indicate that, although initially thought to occur in lysosomes, degradation of internalized insulin occurs mainly in endosomes. First, little association of insulin with authentical lysosomes is demonstr...

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Differential and analytical subfraction of rat liver components internalizing insulin and prolactin

Cited by 51 publications

References 35 publications

Membrane-associated binding sites for estrogen contribute to growth regulation of human breast cancer cells

Membrane-associated binding sites for estrogen contribute to growth regulation of human breast cancer cells

Characterization of Cdk2-Cyclin E Complexes in Plasma Membrane and Endosomes of Liver Parenchyma

Degradation of insulin in isolated liver endosomes is functionally linked to ATP‐dependent endosomal acidification

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