Vanadate modulates the activity of a subpopulation of asialoglycoprotein receptors on isolated rat hepatocytes: active surface receptors are internalized and replaced by inactive receptors

In the absence of ligand, sodium vanadate causes a time- and dose-dependent loss of up to approximately 50% of the surface galactosyl receptor (GalR) activity in rat hepatocytes at 37 degrees C. The effect on total (surface plus intracellular) GalR activity is also dependent on exposure time and vanadate concentration. At less than 1 mM, vanadate induces a transient decrease and then partial recovery of cell surface GalR activity. At greater than 3 mM vanadate, surface GalR activity decreases rapidly (t1/2 approximately 2 min). Lost surface activity is initially recovered in digitonin-permeabilized cells, indicating that active surface GalRs redistribute to the cell interior. However, an antibody assay for GalR protein showed that although surface activity decreased, there was no decrease in surface receptor protein. The active intracellular GalRs then slowly inactivate over 30-60 min. With 8 mM vanadate, the loss of both surface and total cellular GalR activity is more rapid and coincident; no lag is observed. Maximal activity loss, however, was still only approximately 50%. Again, no net change was seen in the distribution of GalR protein between the cell surface and the interior. These results indicate that vanadate causes active GalRs to move from the surface to the inside and be replaced by inactive receptors moving from the inside to the cell surface. The Gal receptor system is comprised of two functionally different receptor subpopulations that operate via two distinct intracellular pathways. Only the State 2 GalRs, which recycle constitutively, are sensitive to modulation by vanadate. Consistent with this, vanadate inhibits the endocytosis of 125I-asialoorosomucoid (ASOR) only partially. The rate of uptake and the steady state level of ASOR intracellular accumulation were maximally inhibited by 50 and 70%, respectively, at 0.2 mM vanadate. The rate and extent of degradation of 125I-ASOR were also inhibited by 50-70%. Residual ASOR uptake and degradation is accounted for by the minor vanadate-resistant State 1 Gal receptor pathway.     

ATP-dependent inactivation and reactivation of constitutively recycling galactosyl receptors in isolated rat hepatocytes

Isolated rat hepatocytes depleted of ATP with NaN3 without ligand lose galactosyl (Gal) receptors from the cell surface and accumulate inactive receptors within the cell [McAbee, D. D., & Weigel, P. H. (1987) J. Biol. Chem. 262, 1942-1945]. Here, we describe the kinetics of receptor redistribution and inactivation after ATP depletion with NaN3 and of receptor redistribution and reactivation after ATP recovery. Only intact cells (greater than 98% viable) isolated from Percoll gradients were assayed. Gal receptor activity and protein were measured by the binding of 125I-asialoorosomucoid (125I-ASOR) and 125I-anti-Gal receptor IgG (125I-IgGR), respectively, at 4 degrees C. Surface and total (surface and intracellular) cellular Gal receptors were measured in the absence or presence, respectively, of digitonin. Following ATP depletion, 60-70% of Gal receptor activity and protein were lost from cell surfaces with first-order kinetics (t1/2 = 6.5 min, k = 0.107 min-1) at an initial rate of 11,000 125I-ASOR binding sites cell-1 min-1. Lost cell-surface Gal receptors were transiently recovered still active inside the cell. After a short lag, total cellular receptor inactivation then proceeded with first-order kinetics (t1/2 = 13 min, k = 0.053 min-1) at an initial rate of 14,000 125I-ASOR binding sites cell-1 min-1. Up to half of all cellular Gal receptors were inactivated by 40 min. 125I-IgGR binding to NaN3-treated, permeable cells, however, was virtually constant. The distribution of total cellular receptors changed from 35% on the cell surface initially to 10% after 40 min of ATP depletion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nonionic detergents increase the stoichiometry of ligand binding to the rat hepatic galactosyl receptor

When digitonin is used to expose intracellular galactosyl (Gal) receptors in isolated rat hepatocytes, only about half of the binding activity for 125I-asialoorosomucoid (ASOR) is found as compared to cells solubilized with Triton X-100. The increased ligand binding in the presence of detergent is not due to a decrease in Kd but could be due either to an increase in the number of ASORs bound per receptor or to exposure of additional receptors. Several experiments support the former explanation. No additional activity is exposed even when 80% of the total cell protein is solubilized with 0.4% digitonin. It is, therefore, unlikely that receptors are in intracellular compartments not permeabilized by digitonin and inaccessible to 125I-ASOR. Digitonin-treated cells are not solubilized by Triton X-100 if they are first treated with glutaraldehyde under conditions that retain specific binding activity. 125I-ASOR binding to these permeabilized/fixed cells increases about 2-fold in the presence of Triton X-100 and a variety of other detergents (e.g., Triton X-114, Nonidet P-40, Brij-58, and octyl glucoside) but not with the Tween series, saponin, or other detergents. When these fixed cells are washed to remove detergent, 125I-ASOR binding decreases almost to the initial level. Affinity-purified Gal receptor linked to Sepharose 4B binds approximately twice as much 125I-ASOR in the presence of Triton X-100 as in its absence. The results suggest that the increase in Gal receptor activity in the presence of nonionic detergents is due to an increase in the valency of the receptor rather than to exposure of additional receptors.     

Subcellular distribution of enzymes determined by rapid digitonin fractionation of isolated hepatocytes

Conditions were determined for rapid separation of cytosolic and mitochondrial compartments by digitonin fractionation of rat hepatocytes. The minimum time required for separation of mitochondrial and cytosolic enzyme markers decreased rapidly with increasing temperature. Kyro EOB, a non-ionic detergent, increases the release of cytosolic enzymes, particularly at lower temperatures. Experimental procedures are described for greater than 90% release of cytosolic enzymes and less than 2% release of mitochondrial enzymes in 3s. By using appropriate concentrations of digitonin and Kyro EOB in a fractionation medium maintained at 1°C and a minimum time of exposure to the medium, nearly separate patterns of release were obtained for enzyme markers for the cytosol, mitochondrial matrix and mitochondrial intermembrane space. The distribution of enzymes that exist in more than one of these compartments was quantified by comparing their rates of release with those of marker enzymes. The cytosol/mitochondrial-matrix distributions for such enzymes in hepatocytes from starved rats were 16%/84% for aspartate aminotransferase, 34%/66% for fumarase and 77%/23% for ATP citrate lyase. In hepatocytes from rats that were induced to synthesize ATP citrate lyase by starvation and re-feeding, the ratio had increased to 95%/5%. The maximum cytosol/intermembrane-space ratio for adenylate kinase was 8%/92%. A procedure is also described for treating commercial digitonin that increases its solubility in water from about 1mg/ml to more than 800mg/ml.     

Copper and zinc ions differentially block asialoglycoprotein receptor-mediated endocytosis in isolated rat hepatocytes.

Asialoglycoprotein receptors on hepatocytes lose endocytic and ligand binding activity when hepatocytes are exposed to iron ions. Here, we report the effects of zinc and copper ions on the endocytic and ligand binding activity of asialoglycoprotein receptors on isolated rat hepatocytes. Treatment of cells at 37 degrees C for 2 h with ZnCl2 (0-220 microM) or CuCl2 (0-225 microM) reversibly blocked sustained endocytosis of 125I-asialoorosomucoid by up to 93% (t1/2 = 62 min) and 99% (t1/2 = 54 min), respectively. Cells remained viable during such treatments. Zinc- and copper-treated cells lost approximately 50% of their surface asialoglycoprotein receptor ligand binding activity; zinc-treated cells accumulated inactive asialoglycoprotein receptors intracellularly, whereas copper-treated cells accumulated inactive receptors on their surfaces. Cells treated at 4 degrees C with metal did not lose surface asialoglycoprotein receptor activity. Exposure of cells to copper ions, but not to zinc ions, blocked internalization of prebound 125I-asialoorosomucoid, but degradation of internalized ligand and pinocytosis of the fluid-phase marker Lucifer Yellow were not blocked by metal treatment. Zinc ions reduced diferric transferrin binding and endocytosis on hepatocytes by approximately 33%; copper ions had no inhibitory effects. These findings are the first demonstration of a specific inhibition of receptor-mediated endocytosis by non-iron transition metals.     

Insulin-like growth factor-II receptors in cultured rat hepatocytes: regulation by cell density

Insulin-like growth factor-II (IGF-II) receptors in primary cultures of adult rat hepatocytes were characterized and their regulation by cell density examined. In hepatocytes cultured at 5 X 10(5) cells per 3.8 cm2 plate [125I]IGF-II bound to specific, high affinity receptors (Ka = 4.4 +/- 0.5 X 10(9) l/mol). Less than 1% cross-reactivity by IGF-I and no cross-reactivity by insulin were observed. IGF-II binding increased when cells were permeabilized with 0.01% digitonin, suggesting the presence of an intracellular receptor pool. Determined by Scatchard analysis and by polyacrylamide gel electrophoresis after affinity labeling, the higher binding was due solely to an increase in binding sites present on 220 kDa type II IGF receptors. In hepatocytes cultured at low densities, the number of cell surface receptors increased markedly, from 10-20,000 receptors per cell at a culture density of 6 X 10(5) cells/well to 70-80,000 receptors per cell at 0.38 X 10(5) cells/well. The increase was not due simply to the exposure of receptors from the intracellular pool, as a density-related increase in receptors was also seen in cells permeabilized with digitonin. There was no evidence that IGF binding proteins, either secreted by hepatocytes or present in fetal calf serum, had any effect on the measurement of receptor concentration or affinity. We conclude that rat hepatocytes in primary culture contain specific IGF-II receptors and that both cell surface and intracellular receptors are regulated by cell density.     

Prohormone processing in the trans-Golgi network: endoproteolytic cleavage of prosomatostatin and formation of nascent secretory vesicles in permeabilized cells

Many peptide hormones are synthesized as larger precursors which undergo endoproteolytic cleavage at paired basic residues to generate a bioactive molecule. Morphological evidence from several laboratories has implicated either the TGN or immature secretory granules as the site of prohormone cleavage. To identify the site where prohormone cleavage is initiated, we have used retrovirally infected rat anterior pituitary GH3 cells which express high levels of prosomatostatin (proSRIF) (Stoller, T. J., and D. Shields. J. Cell Biol. 1988. 107:2087- 2095). By incubating these cells at 20 degrees C, a temperature that prevents exit from the Golgi apparatus, proSRIF accumulated quantitatively in the TGN and no proteolytic processing was evident; processing resumed upon shifting the cells back to 37 degrees C. After the 20 degrees C block, the cells were mechanically permeabilized and pro-SRIF processing determined. Cleavage of proSRIF to the mature hormone was approximately 35-50% efficient, required incubation at 37 degrees C and ATP hydrolysis, but was independent of GTP or cytosol. The in vitro ATP-dependent proSRIF processing was inhibited by inclusion of chloroquine, a weak base, CCCP, a protonophore, or by preincubating the permeabilized cells with low concentrations of N- ethylmaleimide, an inhibitor of vacuolar-type ATP-dependent proton pumps. These data suggest that: (a) proSRIF cleavage is initiated in the TGN, and (b) this reaction requires an acidic pH which is facilitated by a Golgi-associated vacuolar-type ATPase. A characteristic feature of polypeptide hormone-producing cells is their ability to store the mature hormone in dense core secretory granules. To investigate the mechanism of protein sorting to secretory granules, the budding of nascent secretory vesicles from the TGN was determined. No vesicle formation occurred at 20 degrees C; in contrast, at 37 degrees C, the budding of secretory vesicles was approximately 40% efficient and was dependent on ATP, GTP, and cytosolic factors. Vesicle formation was inhibited by GTP gamma S suggesting a role for GTP- binding proteins in this process. Vesicle budding was dependent on cytosolic factors that were tightly membrane associated and could be removed only by treating the permeabilized cells with high salt. After high salt treatment, vesicle formation was dependent on added cytosol or the dialyzed salt extract. The formation of nascent secretory vesicles contrasts with prosomatostatin processing which required only ATP for efficient cleavage. Our results demonstrate that prohormone cleavage which is initiated in the TGN, precedes vesicle formation and that processing can be uncoupled from the generation of nascent secretory vesicles.     

Characterization of an intracellular hyaluronic acid binding site in isolated rat hepatocytes.

125I-HA, prepared by chemical modification at the reducing sugar, specifically binds to rat hepatocytes in suspension or culture. Intact hepatocytes have relatively few surface 125I-HA binding sites and show low specific binding. However, permeabilization of hepatocytes with the nonionic detergent digitonin results in increased specific 125I-HA binding (45-65%) and a very large increase in the number of specific 125I-HA binding sites. Scatchard analysis of equilibrium 125I-HA binding to permeabilized hepatocytes in suspension at 4 degrees C indicates a Kd = 1.8 x 10(-7) M and 1.3 x 10(6) molecules of HA (Mr approximately 30,000) bound per cell at saturation. Hepatocytes in primary culture for 24 h show the same affinity but the total number of HA molecules bound per cell at saturation decreases to approximately 6.2 x 10(5). Increasing the ionic strength above physiologic concentrations decreases 125I-HA binding to permeable cells, whereas decreasing the ionic strength above causes an approximately 4-fold increase. The divalent cation chelator EGTA does not prevent binding nor does it release 125I-HA bound in the presence of 2 mM CaCl2, although higher divalent cation concentrations stimulate 125I-HA binding. Ten millimolar CaCl2 or MnCl2 increases HA binding 3-6-fold compared to EGTA-treated cells. Ten millimolar MgCl2, SrCl2, or BaCl2 increased HA binding by 2-fold. The specific binding of 125I-HA to digitonin-treated hepatocytes at 4 degrees C increased greater than 10-fold at pH 5.0 as compared to pH 7.(ABSTRACT TRUNCATED AT 250 WORDS)     

Endoplasmic reticulum-to-cytosol transport of free polymannose oligosaccharides in permeabilized HepG2 cells.

Free polymannose oligosaccharides have recently been localized to both the vesicular and cytosolic compartments of HepG2 cells. Here we investigated the possibility that free oligosaccharides originating in the lumen of the endoplasmic reticulum (ER) are transported directly into the cystosol. Incubation of permeabilized cells in the absence of ATP at 37 degrees C led to the intravesicular accumulation of free Man9GlcNAc2 which was generated from dolichol-linked oligosaccharide in the ER. This oligosaccharide remained stable within the permeabilized cells unless ATP was added to the incubations at which time the Man9GlcNac2 was partially converted to Man8GlcNAc2, and both these components were released from an intravesicular compartment into the cytosolic compartment of permeabilized cells. In contrast, when permeabilized cells, primed with either free triglucosyl-oligosaccharide or a glycotripeptide, were incubated with ATP both these structures remained associated with the intravesicular compartment. As the conditions in which free oligosaccharides were transported out of the intravesicular compartment into the cytosolic compartment did not permit vesicular transport of glycoproteins from the ER to the Golgi apparatus our data demonstrate the presence of a transport process for the delivery of free polymannose oligosaccharides from the ER to the cytosol.     

Catecholamine secretion from digitonin-treated PC12 cells. Effects of Ca2+, ATP, and protein kinase C activators

PC12 cells, a cloned rat pheochromocytoma cell line, were treated with digitonin to render the plasma membrane permeable to ions and proteins. At a cell density of 2-6 X 10(5) cells/cm2, incubation with 7.5 microM digitonin permitted a Ca2+-dependent release of 25-40% of the catecholamine within 18 min in the presence of 10 microM Ca2+. Half-maximal secretion occurred at 0.5-1 microM Ca2+. PC12 cultures at lower cell densities were more sensitive to digitonin and gave more variable results. Secretion in the presence of digitonin and Ca2+ began after a 2-min lag and continued for up to 30 min. When cells were treated for 3 min in digitonin and then stimulated with Ca2+ in the absence of digitonin, secretion occurred in the same manner but without the initial lag. Optimal secretion from PC12 cells was also dependent upon the presence of Mg2+ and ATP. Permeabilized PC12 cells exhibited a slow time-dependent loss of secretory responsiveness which was correlated with the release of a cytosolic marker, lactate dehydrogenase (134 kDa). This suggests that digitonin permeabilization allows soluble constituents necessary for secretion to leave the cell in addition to allowing Ca2+ and ATP access into the cell interior. Ca2+-dependent secretion was completely inhibited by exposure of digitonin-permeabilized cells to 100 micrograms/ml trypsin (27 kDa), whereas secretion was only slightly inhibited by trypsin exposure prior to digitonin treatment. Thus, an intracellular, trypsin-sensitive protein is probably involved in secretion. The data also indicate that the same population of digitonin-treated cells which responded to Ca2+ was permeable to a 27-kDa protein. 1,2-Dioctanoylglycerol and phorbol esters which activate protein kinase C enhanced the Ca2+-dependent and Ca2+-independent secretion in digitonin-permeabilized PC12 cells. Thus, protein kinase C appears to be involved in the regulation of catecholamine secretion from permeabilized PC12 cells.     

Chromatin recycling of glucocorticoid receptors: implications for multiple roles of heat shock protein 90

Unliganded glucocorticoid receptors (GRs) released from chromatin after hormone withdrawal remain associated with the nucleus within a novel subnuclear compartment that serves as a nuclear export staging area. We set out to examine whether unliganded nuclear receptors cycle between distinct subnuclear compartments or require cytoplasmic transit to regain hormone and chromatin-binding capacity. Hormone-withdrawn rat GrH2 hepatoma cells were permeabilized with digitonin to deplete cytoplasmic factors, and then hormone-binding and chromatin-binding properties of the recycled nuclear GRs were measured. We found that recycled nuclear GRs do not require cytosolic factors or ATP to rebind hormone. Nuclear GRs that rebind hormone in permeabilized cells target to high-affinity chromatin-binding sites at 30 C, but not 0 C, in the presence of ATP. Since geldanamycin, a heat shock protein-90 (hsp90)-binding drug, inhibits hormone binding to recycled nuclear GRs, hsp90 may be required to reassemble the receptor into a form capable of productive interactions with hormone. Geldanamycin also inhibits GR release from chromatin during hormone withdrawal, suggesting that hsp90 chaperone function may play multiple roles to facilitate chromatin recycling of GR.     

Activation of thymocyte glucocorticoid receptors to the steroid binding form. The roles of reduction agents, ATP, and heat-stable factors.

The specific glucocorticoid binding capacity in cytosol preparations of rat thymocytes decays with a half-life of 4 h at 0 degrees C or 20 min at 25 degrees C. Phosphatase inhibitors (molybdate, fluoride, glucose 1-phosphate) added alone do not prevent this inactivation. Dithiothreitol (2 mM) has a large stabilizing effect on the binding capacity at 0 degrees C but only a small effect at 25 degrees C. Addition of 10 mM molybdate plus 2 mM dithiothreitol totally prevents inactivation for at least 8 h at 25 degrees C as well as at 0 degrees C. Fluoride (100 mM) also retards the inactivation if added with dithiothreitol. Addition of dithiothreitol at 25 degrees C to inactivated cytosol receptors results in partial activation of the binding capacity. Addition of dithiothreitol to receptors inactivated at 25 degrees C in the presence of molybdate allows total reactivation of the binding capacity to the maximum zero time value. If binding capacity is inactivated by preincubation of the cytosol at 25 degrees C, addition of ATP with dithiothreitol enhances the activation observed with only dithiothreitol. This ATP stimulated activation is optimal at 1 to 3 mM. ATP (10 mM) is required when molybdate is added to prevent simultaneous inactivation. ADP, GTP, CTP, and UTP have some activating capacity but the effects of all nucleotides are inhibited by the ATP analog, adenyl-5'-yl (beta, gamma-methylene)diphosphonate. ATP-dependent activation can also be prevented with 50 mM EDTA, and addition of magnesium partially overcomes the EDTA inhibition. Dithiothreitol activation of thymocyte glucocorticoid binding capacity can also be enhanced by addition of a heat-stable preparation from thymocytes, L cells, or liver. Sephadex G-25 chromatography, assay of ATP, and inhibition of the activation with adenyl-5'-yl (beta, gamma-methylene)diphosphonate suggest that these preparations contain varying amounts of endogenous reducing equivalents and ATP as well as a larger heat stable factor. Maximum activation is obtained by adding dithiothreitol, ATP, molybdate, and the larger heat-stable factor. These results suggest that stabilization and activation of glucocorticoid binding capacity in thymocytes requires phosphorylation as well as reduction of the receptor itself or of some other component required for the steroid binding reaction.     

The Mouse GalR2 Galanin Receptor: Genomic Organization, cDNA Cloning, and Functional Characterization

Abstract: The diverse physiological actions of galanin are thought to be mediated through activation of galanin receptors (GalRs). We report the genomic and cDNA cloning of a mouse GalR that possesses a genomic structure distinct from that of GalR1 and encodes a functional galanin receptor. The mouse GalR gene consists of two exons separated by a single intron within the protein-coding region. The splicing site for the intron is located at the junction between the third transmembrane domain and the second intracellular loop. The cDNA encodes a 370-amino acid putative G protein-coupled receptor that is markedly different from human GalR1 and rat GalR3 (38 and 57%) but shares high homology with rat GalR2 (94%). In binding studies utilizing membranes from COS-7 cells transfected with mouse GalR2 cDNA, the receptor displayed high affinity ( K _D = 0.47 n M ) and saturable binding with ¹²⁵I-galanin ( B _max = 670 fmol/mg). The radioligand binding can be displaced by galanin and its analogues in a rank order: galanin ⋍ M40 ⋍ M15 ⋍ M35 ⋍ C7 ⋍ galanin (2–29) ⋍ galanin (1–16) ≫ galanin (10–29) ⋍ galanin (3–29), which resembles the pharmacological profile of the rat GalR2. Receptor activation by galanin in COS-7 cells stimulated phosphoinositide metabolism, which was not reversed by pertussis toxin. Thus, the galanin receptor encoded in the cloned mouse GalR gene is the type 2 galanin receptor and is active in both ligand binding and signaling assays.     

ATP-dependent regulation of phospholipase C in permeabilized 3T3 cells

Regulation of phospholipase C (PLC) coupled with a G-protein was studied with Swiss 3T3 cells permeabilized by digitonin. In permeabilized cells, activation of phospholipase C required millimolar concentrations of ATP in addition to a G-protein activator, AlF4- or nonhydrolysable GTP analogues. To determine the mechanism of the action of ATP, we examined the effects of ATP analogues. ATP gamma S directly activated phospholipase C in the presence or absence of AlF4-. On the other hand, neither beta,gamma-methylene ATP nor adenyl-5'-yl imidodiphosphate nor ADP beta S could support the AlF4(-)-dependent activation of phospholipase C. The action of ATP gamma S was not through the substrate supply for phospholipase C, because ATP gamma S did not augment the levels of PIP2 or PIP in permeabilized cells. These results suggested the significance of the gamma-phosphate group of ATP and/or phosphorylation by ATP in the activation of phospholipase C by a putative G-protein.     

Characterization of the in vitro stability of the rat hepatic receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)

The in vitro stability of the Ah receptor from rat hepatic cytosol was evaluated by [3H]TCDD binding studies, gel filtration, and sucrose density gradient ultracentrifugation. Thermal inactivation of unoccupied receptor followed first-order kinetics between 5 and 40 degrees C, with an estimated Ea for inactivation of approximately 35 kcal/mol. Protease inhibitors did not reduce and dilution slightly increased the inactivation rate at 20 degrees C. Recovery and 20 degrees C stability decreased with increasing ionic strength. The TCDD-receptor complex was less susceptible to degradation at 20 degrees C, even in the presence of 0.4 M KCl. Specific binding was markedly pH dependent, with maximum recovery at 7.6. Analysis of the pH curve suggested that cysteine sulfhydryl groups may be involved in TCDD binding. Dithiothreitol (1 mM) maximized recovery and 20 degrees C stability, and addition of the thiol largely reactivated binding sites lost from cytosol prepared without it. Removal of low molecular weight components of cytosol by gel filtration resulted in a rapid 20 degrees C inactivation rate that could not be lessened by dithiothreitol. Glycerol (10% v/v) and EDTA (1.5 mM) maximized recovery of specific binding, but both decreased 20 degrees C stability in a concentration-dependent manner. Calcium chloride (4 mM) increased stability at 20 degrees C by approximately 20%, and retarded the characteristic shift in sedimentation coefficient from approximately 9 to approximately 6 S in high-salt sucrose gradients. The fact that sodium molybdate (20 mM) decreased recovery and 20 degrees C stability when dithiothreitol was present but slightly increased stability in its absence suggested an antagonism between the two compounds. Molybdate mitigated the inactivation induced by 0.4 M KCl, an effect which may be related to the observation of dual peaks in molybdate-containing high-salt sucrose gradients. These data indicate that thermal inactivation of the unoccupied rat hepatic Ah receptor primarily may be due to physical rather than enzymatic processes; (ii) sulfhydryl oxidation, removal of low molecular weight cytosolic components, and high ionic strength result in rapid rates of inactivation at 20 degrees C; and (iii) the large degree of protection conferred by TCDD binding implies a very tight ligand-receptor interaction, and as such accords with TCDDs extraordinary potency and persistence in producing its toxic and biochemical effects.     

Surface and internal galactosyl receptors are heterooligomers and retain this structure after ligand internalization or receptor modulation

We have developed a specific chemical affinity reagent for the hepatic galactosyl receptor (GalR) by derivatizing asialoorosomucoid (ASOR) with the homobifunctional N-hydroxysuccinimide (NHS) ester cross-linker disuccinimidyl suberate [Herzig, M. C. S., & Weigel, P. H. (1989) Biochemistry 28, 600]. NHS-ASOR cross-links with 30-50% efficiency to the three GalR subunits, designated rat hepatic lectins (RHL) 1, 2, and 3. Here, we examined the subunit structure of both surface and internal receptors of two functionally distinct GalR subpopulations, designated state 1 or state 2 GalR. Freshly isolated cells, referred to as state 1 cells, kept at 4 degrees C express only active state 1 GalR on their surface. When these cells are equilibrated at 37 degrees C, they then express both state 1 GalR and state 2 GalR on their surface. These cells are referred to as state 1,2 cells. After incubation at 4 degrees C with NHS-125I-ASOR, surface or internal GalR of state 1 cells or of state 1,2 cells incorporated 125I-ASOR into all three RHL subunits. As analyzed by autoradiography of SDS-PAGE, radiolabeling was identical for all conditions and was in a ratio of 1:1:1 for RHL 1:2:3. Native GalR structure was also examined by first cross-linking nonradiolabeled NHS-ASOR at 4 degrees C to surface or internal receptors of state 1 or state 1,2 hepatocytes. These cells were then washed with EGTA, extracted with Triton X-100, immunoprecipitated with anti-orosomucoid antibody, and subjected to Western blot analysis. Antisera specific for RHL 1 or RHL 2/3 detected cross-linked complexes of Mr congruent to 85K or congruent to 90K-115K, respectively, as well as un-cross-linked native subunits. In all four cases, the ratio of free to cross-linked subunits was greater than or equal to 5:1 for RHL 1 and less than or equal to 0.5:1 for RHL 2/3. Internalized GalR had the same ratio of free to cross-linked subunits as noninternalized GalR. Depletion of ATP either before or after cross-linking GalR to NHS/ASOR also did not alter the ratio of free cross-linked RHL subunits. We conclude that the surface and internal GalR of the two functionally distinct GalR populations have the same heterooligomeric subunit composition and that this GalR structure persists following endocytosis or ATP depletion.     

Diacytosis of 125I-asialoorosomucoid by rat hepatocytes. A non-lysosomal pathway insensitive to inhibition by inhibitors of ligand degradation

Diacytosis of 125I-asialoorosomucoid by rat hepatocytes was studied by preincubating the cells with the labelled ligand at 37 degrees C for 30 min or 18 degrees C for 2 h, washing free of cell surface receptor-bound tracer at 4 degrees C and then reincubating at 37 degrees C. The cells preloaded at 37 degrees C released a maximum of 18% of the total intracellular ligand as undegraded molecules after 1 h of incubation with an apparent first-order rate constant of 0.018 min-1 (t1/2 = 39 min). When the preloaded cells were incubated in the presence of 100 micrograms/ml unlabelled asialoorosomucoid or 5 mM ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, the amount of the released ligand increased to 32 and 37%, respectively, without apparent change in kinetics, indicating that these agents prevented rebinding of the released ligand. In the presence of 5 microM colchicine, 20 microM cytochalasin B, 20 microM chloroquine, 10 mM NH4Cl, 10 microM monensin or 20 microM leupeptin, degradation of the preloaded ligand was inhibited, whereas the release of the ligand was either slightly increased or unchanged. Similar effects of leupeptin, colchicine and asialoorosomucoid were observed with cells preloaded at 18 degrees C. These results indicate that diacytosis of 125I-asialoorosomucoid occurs from a prelysosomal compartment via a route insensitive to inhibition by the inhibitors of ligand degradation.     

Specific intracellular hyaluronic acid binding to isolated rat hepatocytes is membrane-associated

Intact isolated rat hepatocytes show a small amount of specific 125I-labeled hyaluronic acid (HA) binding. However, in the presence of digitonin, a very large increase in the specific binding of 125I-HA is observed. Chondroitin sulfate, heparin and dextran sulfate were as effective as unlabeled HA in competing for 125I-HA binding to permeabilized hepatocytes, indicating that the binding sites may have a general specificity for glycosaminoglycans. After rat hepatocytes had been homogenized in a hypotonic buffer, more than 98% of the 125I-HA binding activity could be pelleted by centrifugation at 100,000 x g for 1 h. Mild alkaline treatment of hepatocyte membranes did not release 125I-HA binding activity, suggesting that the HA binding site is an integral membrane molecule. Furthermore, trypsin treatment of deoxycholate-extracted membranes destroyed the binding activity, as assessed by a dot-blot assay. This suggests that a protein component in the membrane is necessary for 125I-HA binding activity. Rat fibrinogen could be a possible candidate for the HA binding activity because HA binds specifically to human fibrinogen (LeBoeuf et al. (1986) J. Biol. Chem. 261, 12 586). Also, fibrinogen can be found in a quasi-crystalline form in rat hepatocytes and could be pelleted with the membranes. Rat fibrinogen was not responsible for the 125I-HA binding activity, since (1) purified rat fibrinogen did not bind to 125I-HA, and (2) immunoprecipitation of rat fibrinogen from hepatocyte extracts did not decrease the 125I-HA binding of these extracts. We conclude that the internal HA binding sites are membrane- or cytoskeleton-associated proteins and are neither cytosolic proteins nor fibrinogen.     

Nuclear protein import in permeabilized mammalian cells requires soluble cytoplasmic factors

We have developed an in vitro system involving digitonin-permeabilized vertebrate cells to study biochemical events in the transport of macromolecules across the nuclear envelope. While treatment of cultured cells with digitonin permeabilizes the plasma membranes to macromolecules, the nuclear envelopes remain structurally intact and nuclei retain the ability to transport and accumulate proteins containing the SV40 large T antigen nuclear location sequence. Transport requires addition of exogenous cytosol to permeabilized cells, indicating the soluble cytoplasmic factor(s) required for nuclear import are released during digitonin treatment. In this reconstituted import system, a protein containing a nuclear location signal is rapidly accumulated in nuclei, where it reaches a 30-fold concentration compared to the surrounding medium within 30 min. Nuclear import is specific for a functional nuclear location sequence, requires ATP and cytosol, and is temperature dependent. Furthermore, accumulation of the transport substrate within nuclei is completely inhibited by wheat germ agglutinin, which binds to nuclear pore complexes and inhibits transport in vivo. Together, these results indicate that the permeabilized cell system reproduces authentic nuclear protein import. In a preliminary biochemical dissection of the system, we observe that the sulfhydryl alkylating reagent N-ethylmaleimide inactivates both cytosolic factor(s) and also component(s) in the insoluble permeabilized cell fraction required for nuclear protein import. Because this permeabilized cell model is simple, efficient, and works effectively with cells and cytosol fractions prepared from a variety of different vertebrate sources, it will prove powerful for investigating the biochemical pathway of nuclear transport.     

Antiestrogen binding sites in rat liver nuclei

Isolated, intact rat liver nuclei have high-affinity (Kd = 10(-9) M) binding sites that are highly specific for nonsteroidal antiestrogens, especially for compounds of the triphenylethylene series. Nuclear [3H]tamoxifen binding capacity is thermolabile, being most stable at 4 degrees C and rapidly lost at 37 degrees C. More [3H]tamoxifen, however, is specifically bound at incubation temperatures of 25 degrees C and 37 degrees C than at 4 degrees C although prewarming nuclei has no effect, suggesting exchange of [3H]tamoxifen for an unidentified endogeneous ligand. Nuclear antiestrogen binding sites are destroyed by trypsin but not by deoxyribonuclease I or ribonuclease A. The nuclear antiestrogen binding protein is not solubilized by 0.6 M potassium chloride, 2 M sodium chloride, 0.6 M sodium thiocyanate, 3 M urea, 20 mM pyridoxal phosphate, 1% (w/v) digitonin or 2% (w/v) sodium cholate but is extractable by sonication, indicating that it is tightly bound within the nucleus. Rat liver nuclear matrix contains high-affinity (Kd = 10(-9) M) [3H]tamoxifen binding sites present in 5-fold higher concentrations (4.18 pmol/mg DNA) than in intact nuclei (0.78 +/- 0.10 (S.D.) pmol/mg DNA). Low-speed rat liver cytosol (20 000 X g, 30 min) contains high-capacity (955 +/- 405 (S.D.) fmol/mg protein), low-affinity (Kd = 10.9 +/- 4.5 (S.D.) nM) antiestrogen binding sites. In contrast, high-speed cytosol (100 000 X g, 60 min) contains low-capacity (46 +/- 15 (S.D.) fmol/mg protein), high-affinity (Kd = 0.61 +/- 0.20 (S.D.) nM) binding sites. Low-affinity cytosolic sites constitute more than 90% of total liver binding sites, high-affinity cytosolic sites 0.3%-3.2%, and nuclear sites less than 0.5% of total sites.