Chemical crosslinking: A useful tool for evaluations of steroid receptor structures and their functional states in intact cells

Rossini, Gian Paolo; Masci, Giovanni

doi:10.1016/0022-4731(89)90109-x

Cited by 9 publications

(3 citation statements)

References 11 publications

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“…Two other possibilities are that DSS might not be appropriate to cross-link intracellular PLA # -receptor complexes because of a lack of diffusion in adequate concentrations into U III cells, or due to its chemical structure. Both possibilities, however, could be also excluded, as we could not detect significant levels of intracellular PLA # -receptor complexes when crosslinking of U III cells treated with iodinated PLA # at 37 mC was repeated using two different reagents, glutaraldehyde and DSP, under conditions which have been shown to efficiently cross-link intracellular components in several cell lines [23,[33][34][35]41,42].…”

Section: Discussionmentioning

confidence: 99%

“…These findings would indicate that only a small fraction of receptor-bound PLA # in U III cells incubated at 37 mC is resistant to the acidic wash and is then intracellular, but may also simply reflect the inefficacy of DSS in stabilizing intracellular PLA # -receptor complexes. In order to check this latter possibility, we repeated the experiment using two different cross-linkers, glutaraldehyde and DSP, under conditions which have been shown to efficiently stabilize intracellular protein-protein interactions [23,[33][34][35]. The results we obtained by treatment of intact U III cells with these two crosslinkers were identical to those observed with DSS (results not shown), confirming the conclusion that very low levels of PLA # -receptor complexes can be detected inside U III cells incubated with ligand at 37 mC.…”

Section: Most Pla 2 Internalized In U III Cells Is Detected As Free Lmentioning

confidence: 99%

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Binding and internalization of extracellular type-I phospholipase A2 in uterine stromal cells

Rossini

Fayard

Tessier

et al. 1996

Biochemical Journal

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The cellular uptake of extracellular type-I phospholipase A2 (PLA2) was investigated in rat uterine stromal cells (UIII) in culture, which were found to express the high-affinity binding site for mammalian type-I PLA2, with a measured KD of 6.4 nM, a Bmax of 0.1–1 pmol/mg of DNA at 4 °C, and a molecular mass of about 200 kDa. When UIII cells were treated with type-I PLA2 at 37 °C, the ligand specifically associated with the cells increased, reaching a plateau after 90 min of incubation, whose level was about 5-fold higher than that measured if cells were maintained at 4 °C. We could determine that the PLA2 was bound to plasma membrane receptors which were responsible for internalization of the ligand, and that the binding sites were still suitable for binding at the level of plasma membrane during UIII cell incubation at 37 °C. Proteolysis of internalized PLA2 could be clearly detected only after 90 min of UIII cell incubation with the ligand at 37 °C, and most of the intracellular PLA2 consisted of the apparently intact 14 kDa enzyme. By cross-linking studies, we found that most of the internalized PLA2 was not associated with the receptor, supporting the conclusion that in our experimental system a single pool of membrane receptors for mammalian type-I PLA2 undergoes cycles of ligand binding, intracellular transfer and release of PLA2, followed by restoration of binding sites on the plasma membrane. We calculated that the rate of internalization of the ligand by one receptor molecule in UIII cells at 37 °C is about three molecules of type-I PLA2 per h.

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Section: Discussionmentioning

confidence: 99%

Section: Most Pla 2 Internalized In U III Cells Is Detected As Free Lmentioning

confidence: 99%

Binding and internalization of extracellular type-I phospholipase A2 in uterine stromal cells

Rossini

Fayard

Tessier

et al. 1996

Biochemical Journal

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“…Some have suggested that aldehyde fixatives might cause artifactual localization of steroid hormone receptors in nuclei [32] and others have suggested that detergent permeabilization procedures might preferen tially redistribute hormone receptors to cytoplasmic com partments [33]. Brink et al [33] suggested that as little as 0.1% glutaraldehyde prevents the washout of loosely bound receptors from cell nuclei, possibly by covalently linking the receptors to neighboring proteins [34], Al though a cytoplasmic localization of unoccupied glucocor ticoid receptors was reported in glutaraldehyde fixed cells, this occurred when detergents were included with the fixa tive [33], In the current study, animals were perfused with 0.2% glutaraldehyde (with 4% paraformaldehyde) and postfixed in the same solution for 6 h. Permeabilization procedures were not used until after tissue sections were cut by Vibratome. If the estrogen receptor is covalently linked to neighboring proteins by glutaraldehyde in brain cells, these procedures would make it less likely that the receptor was redistributed after the animals were sacri ficed.…”

Section: Discussionmentioning

confidence: 99%

Sex and Regional Differences in Intracellular Localization of Estrogen Receptor Immunoreactivity in Adult Ferret Forebrain

et al. 1993

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Estrogen receptors were visualized in adult ferret brains using the H222 estrogen receptor antibody and immunocytochemical techniques. H222 immunoreactive (H222ir) cell nuclei were present in many forebrain regions in gonadectomized ferrets of both sexes. In many instances, H222ir cells also had immunoreaction product in their processes. All cells with H222ir processes also contained H222ir nuclei. More H222ir processes were observed in females in the medial and lateral preoptic area/anterior hypothalamus, and at the level of the descending fornix and caudal anterior commissure. Quantitative image analysis confirmed that females had significantly more (approximately 50%) extranuclear H222 immunoreaction product than males in cells in the magnocellular or preoptic subnuclei of the bed nucleus of the stria terminalis. Cells in the principal subnucleus of the bed nucleus of the stria terminalis and ventrolateral septum were notable for the relative paucity of H222ir processes. Sex differences in the intracellular extranuclear distribution of estrogen receptor protein in particular brain regions might contribute to the differential regulation of estrogen-dependent functions in the two sexes.

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Transformation of glucocorticoid-receptor complexes is accompanied by dissociation of oligomers in intact cells

Rossini¹,

Malaguti²

1990

Biochemical and Biophysical Research Communications

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Chemical crosslinking: A useful tool for evaluations of steroid receptor structures and their functional states in intact cells

Cited by 9 publications

References 11 publications

Binding and internalization of extracellular type-I phospholipase A2 in uterine stromal cells

Binding and internalization of extracellular type-I phospholipase A2 in uterine stromal cells

Sex and Regional Differences in Intracellular Localization of Estrogen Receptor Immunoreactivity in Adult Ferret Forebrain

Transformation of glucocorticoid-receptor complexes is accompanied by dissociation of oligomers in intact cells

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