The endocytic pathway followed by the keratinocyte growth factor receptor

Keratinocyte growth factor (KGF/FGF7) acts specifically on epithelial cells and regulates their proliferation and differentiation. It binds to and activates a receptor tyrosine kinase, the KGF receptor (KGFR), which is a splicing variant of the fibroblast growth factor receptor 2. The endocytic pathway followed by KGF and its receptor was analyzed here using immunofluorescence and confocal microscopy. After 10 min of internalization at 37 degrees C, both KGF and its receptor were localized in early endosomes, and after 30-60 min of endocytosis ligand and receptor were seen to reach perinuclear late endosomes and not the recycling endosomal compartment. Parallel western blot analysis revealed that KGFRs were tyrosine phosphorylated both at early and late steps of internalization, suggesting that KGF and KGFR remain associated in active complexes through the endocytic pathway. Pulse-chase experiments showed that the internalized KGFRs underwent degradation detectable at 1 h of endocytosis at 37 degrees C, indicating that KGFRs are functionally downregulated.     

Binding and processing of (125)I-ACTH by isolated rat splenic lymphocytes

The effect of incubation temperature and ligand competition was tested for (125)I-ACTH binding to isolated rat lymphocytes. AlphaMSH but not Agouti-like peptide was an effective competitive inhibitor for cell surface binding at 4 degrees C. Cells incubated with (125)I-ACTH at 37 degrees C rapidly associated ligand for 10 min and then gradually lost the radioactivity with time. Cells incubated with (125)I-ACTH at 4 degrees C accumulated ligand to only about half the maximal amount when compared to cells incubated at 37 degrees C for 10 min. Temperatures below 20 degrees C and toxins that block lysosomal degradation blocked the loss of cell-associated radioactivity. These results suggest the lymphocyte ACTH receptor is the Melanocortin 5 receptor and the receptor is internalized by endocytosis to deliver ligand to the lysosome.     

Inhibition of sequestration of human B2 bradykinin receptor by phenylarsine oxide or sucrose allows determination of a receptor affinity shift and ligand dissociation in intact cells

Depending on their interaction with intracellular proteins, G protein-coupled receptors (GPCR) often display different affinities for agonists at 37 degrees C. Determining the affinity at that temperature is often difficult in intact cells as most GPCRs are internalized after activation. When sequestration of the B2 bradykinin receptor (B2R) was inhibited by either 0.5 M sucrose or phenylarsine oxide (PAO), a shift in the affinity was detected when the incubation temperature was raised from 4 degrees C to 37 degrees C or lowered from 37 degrees C to 4 degrees C. In contrast, binding of the antagonist [3H]NPC 17731 was temperature-independent. B2R mutants displayed different affinity shifts allowing conclusions on the role of the involved amino acids. By inhibiting receptor sequestration it was possible to determine also dissociation of [3H]BK and of [3H]NPC 17731 from intact cells at 37 degrees C. Surprisingly, both dissociation rates were markedly enhanced by the addition of unlabeled ligand, most likely via prevention of reassociation of dissociated [3H]ligand. This suggests that dissociated [3H]ligand cannot move freely away from the receptor. In summary, our data demonstrate that inhibition of receptor internalization either by PAO or sucrose provides an excellent method to study receptor function and the effects of mutations in intact cells.     

Oxidant-mediated inhibition of ligand uptake by the macrophage mannose receptor

We have investigated the effect of oxidants on ligand recognition and internalization by the macrophage mannose receptor. Rat bone marrow macrophages were treated with increasing concentrations of H2O2 for 30 min at 37 degrees C. Fifty percent inhibition of ligand uptake was observed at 250 microM, with only 10% of control uptake remaining following exposure to 1 mM H2O2 for 30 min. Electron micrographic analysis of macrophages following H2O2 treatment showed no morphological alterations compared to untreated cells. Ligand uptake was also inhibited by the following H2O2 generating systems: menadione, xanthine/xanthine oxidase, glucose/glucose oxidase, and phorbol 12-myristate 13-acetate-stimulated polymorphonuclear leukocytes. Inhibition could be blocked by catalase plus or minus superoxide dismutase. Treatment of macrophages at 4 degrees C with H2O2 had no effect on ligand binding, whereas treatment with H2O2 at 37 degrees C reduced binding to 15% of control levels and decreased the number of surface receptors to one-third of control cells. H2O2 treatment inhibited ligand degradation by macrophages, but did not prevent ligand movement from the surface to the interior of the cell. In addition, ligand delivery to lysosomes was blocked by oxidant treatment. These results suggest that treatment of macrophages with reagent H2O2 or H2O2-generating systems inhibits the normal ligand delivery and receptor recycling process involving the mannose receptor. Potential mechanisms might include receptor oxidation, alterations in ATP levels, or membrane lipid peroxidation.     

Analysis of glucagon-receptor interactions on isolated canine hepatocytes. Formation of reversibly and irreversibly cell-associated hormone

We have investigated the interactions of ligand with the canine hepatic glucagon receptor. Whereas time courses for radiolabeled glucagon binding to receptor and dissociation from receptor revealed fast and slow components at both 30 and 4 degrees C, time courses of ligand dissociation revealed a third component of irreversibly cell-associated (nondissociable) ligand only at the higher temperature. Related experiments identified that (a) the initial rate of formation of nondissociable ligand was slower than that of dissociably bound hormone; (b) the fraction of ligand bound to nondissociable sites achieved a plateau during extended incubations, whereas that bound to dissociable sites was seen to rise and then slowly to fall; (c) the kinetics of formation of a nondissociable ligand was consistent with linked, sequential reactions; (d) dissociable ligand-receptor complexes formed at 4 degrees C were converted to nondissociable complexes during subsequent incubation at 30 degrees C, and (e) nondissociable sites were filled by prior incubation of cells with unlabeled ligand. Analysis of receptor-bound hormone resulting from the incubation of cells with 125I-labeled glucagon and selected concentrations of either glucagon or [[127I]iodo-Tyr10]glucagon at steady state revealed in each case four components of receptor-bound ligand: those corresponding to high and low affinity components of dissociably bound ligand and to high and low affinity components of nondissociably bound ligand. Implications of these findings are considered in terms of mechanisms for the formation of irreversibly bound hormone and for the distribution of hormone among the various components of hepatic glucagon-binding sites.     

Characterization of the asialoglycoprotein receptor in a continuous hepatoma line

The asialoglycoprotein receptor has been identified on a continuous human hepatoma cell line, HepG2. This receptor requires Ca2+ for ligand binding and is specific for asialoglycoprotein. There are approximately 150,000 ligand molecules bound/cell at 4 degrees C. These receptors represent a homogeneous population of high affinity binding sites with Kd = 7 X 10(-9) M. From the rate of 125I-ASOR binding at 4 degrees C, kon was 0.95 X 10(6) M-1 min-1. Uptake of 125I-ASOR at 37 degrees C was approximately 0.02 pmol/min/10(6) cells.     

Temperature-dependent interaction of the bovine hippocampal serotonin(1A) receptor with G-proteins

The ligand binding and G-protein coupling of the bovine hippocampal 5-HT1A receptor as a function of temperature was monitored. There is an almost complete and irreversible loss in agonist binding at 50 degrees C. However, the antagonist binding is reduced only by 50%, and this could be reversed if the temperature is lowered to 25 degrees C. Interestingly, the agonist binding of the 5-HT1A receptor in membranes exposed to 50 degrees C is inhibited to a much lesser extent by GTP-gamma-S, a non-hydrolysable analogue of GTP, indicating uncoupling of the 5-HT1A receptor to G-proteins at 50 degrees C. We propose that high temperature selectively and irreversibly inactivates G-proteins thereby affecting G-protein-receptor interaction and agonist binding of the 5-HT1A receptor.     

Transforming growth factor (TGF)-beta 1 internalization: modulation by ligand interaction with TGF-beta receptors types I and II and a mechanism that is distinct from clathrin-mediated endocytosis

Transforming growth factor-beta (TGF-beta) internalization was studied by monitoring the uptake of (125)I-TGF-beta1 in Mv1Lu cells, which endogenously express TGF-beta receptors types I (RI), II (RII), and III (RIII), and 293 cells transfected with RI and RII. At 37 degrees C internalization occurred rapidly, within 10 min of ligand addition. Internalization was optimal in 293 cells expressing both RI and RII. Internalization was prevented by phenylarsine oxide, a nonspecific inhibitor of receptor internalization, but was not affected by reagents that interfere with clathrin-mediated endocytosis such as monodansylcadaverine, K44A dynamin, and inhibitors of endosomal acidification. Electron microscopic examination of Mv1Lu cells treated with (125)I- TGF-beta1 at 37 degrees C indicated that internalization occurred via a noncoated vesicular mechanism. Internalization was prevented by prebinding cells with TGF-beta1 at 4 degrees C for 2 h prior to switching the cells to 37 degrees C. This was attributed to a loss of receptor binding, as indicated by a rapid decrease in the amount of TGF-beta1 bound to the cell surface at 37 degrees C and by a reduction in the labeling intensities of RI and RII in (125)I-TGF-beta1-cross-linking experiments. Mv1Lu or 293 (RI+RII) cells, prebound with TGF-beta1 at 4 degrees C and subsequently stripped of ligand by an acid wash, nevertheless initiated a signaling response upon transfer to 37 degrees C, suggesting that prebinding promotes formation of stable RI.RII complexes that can signal independently of ligand.     

Receptor-mediated endocytosis of asialoglycoproteins by rat hepatocytes: receptor-positive and receptor-negative endosomes

We have used combinations of subcellular fractionation, specific cytochemical tracers, and quantitative immunoadsorption to determine when, where, and in which intracellular structure internalized asialoglycoproteins (ASGPs) are segregated from their receptor. All membrane vesicles containing the receptor (R+ vesicles) were quantitatively immunoadsorbed from crude microsomes with Staphylococcus aureus cells and affinity-purified anti-ASGP receptor. Using this assay, we varied the time and temperature of exposure of perfused livers to 125I-asialoorosomucoid (125I-ASOR) and followed the movement of ligand from R+ to R- vesicles. After 2.5 min at 37 degrees C, 98% of the internalized ligand could be immunoadsorbed and thus was in R+ vesicles. Over the next 12 min of continuous 37 degrees C perfusion with 125I-ASOR, an increasing fraction of the ligand was not immunoadsorbed and therefore was present in R- vesicles. A maximum of 30% of the ligand could be found in R- vesicles (14-44 min). When livers were maintained at 16 degrees C, ligand was internalized but remained in R+ vesicles. Furthermore, ligand accumulating in R- vesicles at 37 degrees C remained there when livers were cooled to 16 degrees C. R- endosomes could be separated from R+ endosomes by flotation on sucrose density gradients and visualized by the presence of sequestered ASOR-horseradish peroxidase (ASOR-HRP). These structures resembled those labeled by ASOR-HRP in situ: R+ vesicles were relatively dense (1.12 g/cc), frequently tubular or spherical and small (100-nm diam), corresponding to the peripheral and internal tubular endosomes; R- structures were of lower density (1.09 g/cc), large (400-nm diam), and resembled internal multivesicular endosomes (MVEs). Endocytosed ASOR-HRP was found in both the peripheral and internal tubular endosomes in situ under conditions where 95% of the ligand was present in R+ vesicles by immunoadsorption, whereas MVEs containing ASOR-HRP were predominant in situ when ligand was found in R- vesicles and were often in continuity with the tubular internal endosomes. All of these results suggest that complete segregation of ligand and receptor occurs after arrival in the Golgi-lysosome region of the hepatocyte and that MVEs are R- and represent the final prelysosomal compartment.     

Endocytosis and recycling of subunit H1 of the asialoglycoprotein receptor is independent of oligomerization with H2.

The human asialoglycoprotein receptor is composed of two homologous subunits, H1 and H2. By expressing the two subunits in transfected fibroblast cell lines, it has been shown previously that the formation of a hetero-oligomeric complex is necessary for the transport of H2 to the plasma membrane and for high-affinity ligand binding. Here we show that subunit H1, when expressed in the absence of H2, is capable of internalization through coated pits and recycling. The kinetics of these processes are very similar to those of the H1-H2 complex. To study endocytosis in the absence of ligand binding, the cell surface was labeled at 4 degrees C with the 125I-iodinated impermeant reagent sulfosuccinimidyl-3-(4-hydroxyphenyl) propionate, the cells were incubated at 37 degrees C for different times and the amount of internalized receptor was determined by protease digestion of surface proteins and immunoprecipitation. Similarly, recycling of surface-labeled and then internalized receptor protein was studied by monitoring its reappearance on the surface in the presence of exogenous protease. Our results show that subunit H1 contains all the signals necessary for receptor endocytosis and recycling independent of ligand binding.     

Internalization and degradation of receptor-bound interferon-gamma by murine macrophages. Demonstration of receptor recycling

Although the interferon-gamma (IFN-gamma) receptor on murine and human mononuclear phagocytes has been defined and partially characterized, very little data exists which describes the ultimate fate of receptor-bound ligand. The current studies were specifically designed to define the metabolic processes which act on murine recombinant IFN-gamma following its interaction with murine macrophages at physiologic temperatures. Ligand internalization was demonstrated by comparing binding of [125I]IFN-gamma to macrophages at 4 degrees C and 37 degrees C. When binding was carried out at 4 degrees C, 96% of the cell-associated [125I]IFN-gamma remained accessible at the plasma membrane and could be stripped from the cell by exposure to pronase. In contrast, at 37 degrees C, only 35% of the cell-associated radioactivity was pronase strippable. Macrophages degraded [125I]IFN-gamma into trichloroacetic acid-soluble material at 37 degrees C at a constant rate of 7000 molecules/cell/hr over a 12-hr time period. The amount of IFN-gamma degraded correlated with the amount of IFN-gamma bound to the cell surface. The receptor was neither up- nor down-regulated by ligand or by other agents known to regulate macrophage functional activity such as IFN-alpha, IFN-beta, lipopolysaccharide, or phorbol myristate acetate. The constant uptake of IFN-gamma by macrophages was due to the presence of an intracellular receptor pool (62% of the total receptor number) and to a mechanism of receptor recycling. Evidence for the latter was obtained using lysosomotropic agents which blocked degradation but not binding and internalization of ligand and caused the intracellular accumulation of receptor. By comparing the relationship between receptor occupancy and biologic response induction, two activation mechanisms became apparent. Induction of certain functions, such as H2O2 secretion, appeared to require only a single round of receptor occupancy. However, induction of more complex functions such as nonspecific tumoricidal activity appeared to require three to four rounds of receptor occupancy. These results thus support the concept that IFN-gamma internalization and receptor recycling are essential in the induction of nonspecific tumoricidal activity by macrophages.     

Receptor-mediated endocytosis of tissue-type plasminogen activator by the human hepatoma cell line Hep G2

Receptor-mediated endocytosis of tissue-type plasminogen activator (t-PA) was characterized with the human hepatoma cell line Hep G2. At 4 degrees C binding of 125I-t-PA to Hep G2 cells is rapid, specific, saturable, and reflective of a homogeneous population of 76,000 high-affinity surface sites per cell (Kd = 3.7 nM). The kinetics of 125I-t-PA binding to its receptor are characterized by rate constants for association (k1 = 1.2 x 10(6) min-1 M-1) and dissociation (k-1 = 0.001 min-1). A specific glycosylation pattern does not appear to be required for binding. Binding does not appear to be mediated by other recognized hepatic receptor systems. At 37 degrees C a single cohort of bound 125I-t-PA molecules disappears rapidly from the cell surface. Ligand then accumulates intracellularly. Thereafter, the intracellular concentration of ligand declines simultaneously with the release of ligand degradation products into the media. In the continued presence of 125I-t-PA at 37 degrees C the concentration of cell-associated ligand plateaus after 30 min with the concomitant appearance of low molecular weight 125I-labeled fragments in the media. Cumulative degradation then increases linearly with time. Under steady state conditions half-maximal ligand uptake and degradation is 26.6 nM and maximal rate of catabolism is 1.2 pmol/10(6) cells/h. At saturating ligand concentrations uptake and degradation by Hep G2 cells continue linearly for at least 6 h even in the absence of protein synthesis. During this period the cumulative ligand uptake exceeds the total cellular capacity of binding sites, consistent with receptor recycling. We conclude that t-PA clearance in human Hep G2 cells involves ligand binding, uptake, and degradation mediated by a novel high-capacity, high-affinity specific receptor system.     

A method for characterization of endogenous ligands to orphan receptors belonging to the steroid hormone receptor superfamily--isolation of progesterone from pregnancy plasma using progesterone receptor ligand-binding domain.

An analytical method is described whereby progesterone is isolated from pregnancy plasma on the basis of the high affinity and specificity of the progesterone receptor for its ligand. Partially purified progesterone receptor ligand-binding domain, expressed as a protein A fusion protein in Escherichia coli, is incubated with a neutral steroid fraction obtained by extraction and ion-exchange chromatography of human late-pregnancy plasma. The incubated sample is passed through two Lipidex 1000 (lipophilic gel) beds. The first, at 4 degrees C, separates the specific ligand-fusion protein complex from nonspecifically bound and unbound compounds, and the second, at 40 degrees C, separates the specific ligand from the protein. Elution of the second bed with methanol yields a fraction containing specific ligand that can be characterized by gas chromatography-mass spectrometry. This methodology may be valuable for identification of endogenous ligands to orphan receptors of the steroid hormone receptor superfamily.     

Characterisation of receptors for 1,25-dihydroxyvitamin D3 in the human testis

Specific high affinity receptors for 1,25-dihydroxyvitamin D3 have been demonstrated in the human testes. The mean binding affinity (Kd +/- SD) of the receptor for 1,25-dihydroxyvitamin D3 was 1.75 +/- 0.32 x 10(-10) M but the binding capacity was low (mean Nmax +/- SD = 0.53 +/- 0.18 fmol/mg protein). Binding was time- and temperature-dependent, with a maximum binding achieved after 1 h at 25 degrees C. Although binding also took place at 4 and 37 degrees C, higher and more rapid binding was found at 25 degrees C. Furthermore, the binding between the ligand and the receptor was specific since only unlabelled 1,25-dihydroxyvitamin D3 competed with the labelled ligand. Binding of 1,25-dihydroxyvitamin D3 was abolished by trypsin and heat. Sucrose density gradient centrifugation revealed a sedimentation coefficient of 3.6S.     

The large intracellular pool of asialoglycoprotein receptors functions during the endocytosis of asialoglycoproteins by isolated rat hepatocytes

The function of intracellular asialoglycoprotein receptors during the endocytosis of asialo-orosomucoid in isolated hepatocytes was assessed by following changes in the occupancy of intracellular receptors. Unoccupied total cellular (inside and surface) or surface receptors were quantified at 0 degrees C by the binding of 125I-asialo-orosomucoid in the presence or absence, respectively, of digitonin. Freshly isolated cells had about 17% of their total receptors on the surface. After incubation at 37 degrees C, the receptor distribution changed to 25 to 50% on the cell surface and 50 to 75% inside the cell. At 37 degrees C, the average total number of receptors/cell was 4.5 x 10(5). Dissociation constants, determined from equilibrium binding studies in the presence or absence of digitonin to assess total or surface receptors, were identical (5.4 +/- 1.4 and 5.6 +/- 1.1 x 10(-9) M, respectively). In the presence of asialo-orosomucoid at 37 degrees C, there was both a time- and a concentration-dependent decrease in surface and intracellular receptor activity. This receptor activity decrease was reversed by removing asialo-orosomucoid from the medium or by washing the digitonin-permeabilized cells with ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid prior to quantification of receptor activity. Within 1 to 2 h in the presence of excess asialo-orosomucoid, a steady state was attained in which approximately 70% of the intracellular receptors were occupied. The kinetics of receptor activity recovery on the cell surface after internalization of a pulse of ligand is different than the rate of recovery of internal receptor activity. The results suggest that all of the internal asialoglycoprotein receptors are functional and participate during endocytosis. Internal receptors may be functionally equivalent to those on the surface or they may serve a reservoir or routing function for internalized ligand.     

Estrogen receptor in chicken oviduct: receptor dissociation kinetics and transformation

Cytosolic and nuclear estrogen receptor forms of chicken oviduct have been studied by (1) measuring hormone dissociation kinetics and by (2) sucrose density gradient analysis on high salt gradients. Estradiol dissociates from the receptor in chicken oviduct cytosol at 22 degrees C following a two-phase exponential process. The fraction of receptor with a fast dissociation rate (k = 120 X 10(-3) min-1) decreases as a function of the pre-incubation at 22 degrees C; after prolonged pre-incubation only the slowly dissociating (k = 12.3 X 10(-3) min-1) form remains. Dissociation of moxestrol, a synthetic estrogen with a higher affinity, from the cytosol receptor at 30 degrees C is similar, showing a transition of a fast dissociating form (k = 120 X 10(-3) min-1) to a slowly dissociating form (k = 7.6 X 10(-3) min-1) as a result of pre-incubation at 30 degrees C. A concomitant temperature-dependent shift of the estrogen receptor from a 4.8 S to a 6.1 S form was observed with moxestrol but not with estradiol as a ligand. Sodium molybdate (20 mM) and NaSCN (400 mM) inhibit the temperature-dependent increase in sedimentation coefficient, but molybdate allows the formation of a receptor form which shows intermediary dissociation kinetics. Estrogen receptor, precipitated with ammonium sulfate (0-35%) shows monophasic dissociation kinetics of estradiol (k = 39.5 X 10(-3) min-1) and for moxestrol (k = 10.8 X 10(-3) min-1), suggesting full receptor activation only with moxestrol as a ligand. Moxestrol-receptor complexes obtained by ammonium sulfate precipitation sediment at 0 degree C at 4.8 S. Only after subsequent incubation at 30 degrees C a shift from 4.8 S to 5.9 S is observed, suggesting that the formation of the slowly dissociating form of the receptor may precede the formation of a stable transformed receptor complex. The nuclear estrogen receptor with estradiol as a ligand shows biphasic dissociation kinetics at 22 degrees C (k = 70 X 10(-3) min-1; k = 14.0 X 10(-3) min-1). The ratio of both components (1:1) does not change after preincubation of the nuclear receptor extract at 22 degrees C. Moxestrol dissociates from the nuclear receptor at 30 degrees C monophasically with a slow rate (k = 6.1 X 10(-3) min-1), suggesting that it is extracted as an activated hormone-receptor complex.(ABSTRACT TRUNCATED AT 400 WORDS)     

Lysosomal degradation of receptor-bound urokinase-type plasminogen activator is enhanced by its inhibitors in human trophoblastic choriocarcinoma cells.

We have studied the effect of plasminogen activator inhibitors PAI-1 and PAI-2 on the binding of urokinase-type plasminogen activator (u-PA) to its receptor in the human choriocarcinoma cell line JAR. With 125I-labeled ligands in whole-cell binding assays, both uncomplexed u-PA and u-PA-inhibitor complexes bound to the receptor with a Kd of approximately 100 pM at 4 degrees C. Transferring the cells to 37 degrees C led to degradation to amino acids of up to 50% of the cell-bound u-PA-inhibitor complexes, whereas the degradation of uncomplexed u-PA was 15%; the remaining ligand was recovered in an apparently intact form in the medium or was still cell associated. The degradation could be inhibited by inhibitors of vesicle transport and lysosomal hydrolases. By electron microscopic autoradiography, both 125I-u-PA and 125I-u-PA-inhibitor complexes were located over the cell membrane at 4 degrees C, with the highest density of grains over the membrane at cell-cell interphases, but, after incubation at 37 degrees C, 17 and 27% of the grains for u-PA and u-PA-PAI-1 complexes, respectively, appeared over lysosomal-like bodies. These findings suggest that the u-PA receptor possesses a clearance function for the removal of u-PA after its complex formation with a specific inhibitor. The data suggest a novel mechanism by which receptor-mediated endocytosis is initiated by the binding of a secondary ligand.     

Protein kinase C-mediated gonadotropin-releasing hormone receptor sequestration is associated with uncoupling of phosphoinositide hydrolysis

Gonadotropin-releasing hormone (GnRH) regulates pituitary gonadotropin release by a Ca2+-dependent mechanism involving receptor-mediated phosphoinositide hydrolysis. Previous studies indicate that activation of pituitary protein kinase C (PKC), while not required for acute gonadotropin release in response to GnRH, is likely involved in the chronic regulation of gonadotrope responsiveness. Studies from our laboratory have shown that activation of PKC by phorbol esters produces both the uncoupling of GnRH-stimulated phosphoinositide hydrolysis and the selective enhancement of GnRH agonist binding in pituitary cell cultures. In the present work, we have examined the possibility that these processes are related in mechanism. Dissociation of bound agonist radioligand at 23 degrees C was found to be reduced in the presence of phorbol esters, and ligand bound in the presence of phorbol ester was resistant to displacement by competing ligands at 4 degrees C. However, agonist bound in the presence of phorbol ester was dissociable by subsequently washing cells at pH 3. Receptor photoaffinity labeling studies confirmed that agonist association with membrane component(s) identified as the GnRH receptor was increased in the presence of phorbol ester. These results suggest that, in the presence of a phorbol ester PKC activator, agonist-occupied GnRH receptors remain at the cell surface, but are sequestered in some manner. In other experiments, cell preloaded with [3H]inositol were treated with GnRH agonist ligand and phorbol ester at 4 degrees C to form a pool of sequestered, agonist-occupied receptors, and then displaceable (nonsequestered) agonist was removed by incubation with antagonist ligand. After addition of LiCl and warming to 37 degrees C, [3H]inositol phosphate production (an index of phosphoinositide hydrolysis) in phorbol ester-treated cells was reduced to 67% of vehicle control, although residual specific agonist binding had been increased to greater than 300% of control. The appearance of sequestered receptors and inhibition of [3H]inositol phosphate production had similar phorbol ester concentration dependencies. These results suggest that the same agonist-occupied GnRH receptors sequestered as a result of PKC activation also are preferentially uncoupled from phosphoinositide hydrolysis.     

Recycling of the asialoglycoprotein receptor and the effect of lysosomotropic amines in hepatoma cells

Receptor-mediated uptake and degradation of 125I-asialoorosomucoid (ASOR) in human hepatoma HepG2 cells is inhibited by the lysosomotropic amines chloroquine and primaquine. In the absence of added ligand at 37 degrees C, these amines induce a rapid (t1/2 5.5-6 min) and reversible loss of cell surface 125I-ASOR binding sites as well as a rapid decrease in 125I-ASOR uptake and degradation. There is no effect of these amines on the binding of 125I-ASOR to the cell surface at 4 degrees C or on the rate of internalization of prebound 125I-ASOR. The loss of 125I-ASOR surface binding at 37 degrees C is not attributable to altered affinity of ligand-receptor binding. In the presence of added ligand at 37 degrees C, there is a more rapid (t1/2 2.5-3 min) loss of hepatoma cell surface receptors. In addition, the amines inhibit the rapid return of the internalized receptor to the cell surface. We examined the nature of this loss of 125I-ASOR surface binding sites by following the fate of receptor molecules after biosynthetic labeling and after cell surface iodination. At 37 degrees C, chloroquine and primaquine induce a loss of asialoglycoprotein receptor molecules from the hepatoma cell surface to an internal pool.     

Quaternary structure of the Mr 46,000 mannose 6-phosphate specific receptor: effect of ligand, pH, and receptor concentration on the equilibrium between dimeric and tetrameric receptor forms

The Mr 46,000 mannose 6-phosphate specific receptor exists in solution as a mixture of noncovalently associated dimeric and tetrameric forms. The two quaternary forms were separated by sucrose density centrifugation, and their composition was assessed by cross-linking with bifunctional reagents followed by SDS-polyacrylamide gel electrophoresis. The dependence of equilibrium between the dimeric and tetrameric forms on pH, receptor concentration, and presence of mannose 6-phosphate was studied. The formation of tetrameric forms is favored by pH values around 7, high receptor concentration, and presence of mannose 6-phosphate ligand. Tetrameric forms bind stronger at pH 7 to phosphomannan-Sepharose 4B than dimeric forms. Both quaternary forms dissociate at the same pH from a mannose 6-phosphate affinity matrix. When starting with dimeric or tetrameric forms, the equilibrium between dimeric and tetrameric forms is reached at pH 7.5 and 4 degrees C after 6-8 days. The presence of 5 mM mannose 6-phosphate shifts the equilibrium toward tetrameric forms. At pH 4.5 and 4 degrees C, the association of dimeric to tetrameric forms is negligible, while tetrameric forms dissociate to dimeric forms within 12 h. The results demonstrate that oligomerization is an intrinsic property of MPR-46 that is affected by ligand binding, pH, and receptor concentration.