Biomedical subjects

B I Posner

Publications and source records attributed to B I Posner.

At least 19 recordsLinked to original sources

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

Decrease in beta-subunit phosphotyrosine correlates with internalization and activation of the endosomal insulin receptor kinase.

In a previous study, we showed that the rat hepatic insulin receptor (IR) kinase of endosomes (ENs) was transiently activated to levels exceeding those of plasma membrane (PM) receptors following insulin injection. Phosphatase treatment of EN receptors abolished IR kinase activation implicating beta-subunit autophosphorylation as a mediator of the activation process (Khan, M. N., Baquiran, G., Brule, C., Burgess, J., Foster, B., Bergeron, J. J. M., and Posner, B. I. (1989) J. Biol. Chem. 264, 12931-12940). In the present study, the phosphotyrosine (PY) content of the IR beta-subunit in PM and ENs was estimated by two different methods. In one method, direct in vivo labeling with 32Pi followed by receptor immunoprecipitation was carried out. In the second method, immunoblotting with antibodies against the submembrane domain of the IR beta-subunit, encompassing residue 960 (alpha 960), and with antibodies against PY (alpha PY) was used to determine the content of PY/beta-subunit in PM and ENs following injection of insulin. By both methods, it was found that the PY content of PM IR was significantly greater than that of IR in ENs. With doses of 1.5 micrograms of insulin/100 g body weight (50% receptor occupancy) or 15 micrograms/100 g body weight (receptor saturation), the PY/beta-subunit of PM IR attained a level 2.0 to 2.5-fold of that observed for the IR of ENs. Surprisingly, the IR of ENs incorporated 3 to 5 times more PY/beta-subunit than those of PM consequent to autophosphorylation. Exogenous IR kinase activity (poly(Glu:Tyr)) in PM changed only slightly with insulin dose. In contrast, EN receptors exhibited a dose-dependent increase in kinase activity coincident with the decrease in PY/beta-subunit levels. A comparison of the proportion of receptor and kinase activity immunoprecipitated by alpha PY both before and after autophosphorylation indicated that ENs but not PM contained a small population of lightly phosphorylated but highly activated receptors. Since Thr12-Lys (IR kinase residues 1142-1153) efficiently inhibited IR autophosphorylation of both PM and EN receptors, Tris phosphorylation of beta-subunit regulatory tyrosines was unlikely. These results may be explicable by a dephosphorylation-dependent activation of IR kinase, as seen with the src family of tyrosine kinases.

Amino Acid Sequence↗

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↗

PubMed1992

Prolactin enhancement of its own uptake at the choroid plexus.

The choroid plexus contains PRL receptors that function in part to transport PRL from the blood into the cerebrospinal fluid (CSF). The blood PRL concentration of female rats was altered by 1) three daily injections of haloperidol (chronic hyperprolactinemia) with or without bromocriptine administration 4 h before death, 2) bromocriptine alone for 4 h (acute hypoprolactinemia), and 3) a single vascular injection of ovine PRL (acute hyperprolactinemia). Changes in the uptake of PRL by the choroid plexus was assessed by quantitative in vivo autoradiography after the injection of radiolabeled PRL. Correlation of changes in PRL uptake at the choroid plexus with changes in PRL transport from blood to CSF was evaluated by subjecting CSF samples to sodium dodecyl sulfate-polyacrylamide gel electrophoresis after vascular injection of radiolabeled PRL. Autoradiography revealed that both chronic and acute hyperprolactinemia resulted in a significant increase in the uptake of radiolabeled PRL by the choroid plexus compared to that in untreated control animals. In contrast, bromocriptine had no effect on PRL uptake at the choroid plexus relative to that in control (untreated) animals. Chronic hyperprolactinemia, but not acute hyperprolactinemia, resulted in a significant increase in the transport of radiolabeled PRL from the blood to the CSF compared to that in untreated controls. The results are consistent with the up-regulation of PRL receptors in the choroid plexus by circulating PRL and the consequent augmentation of transport of PRL from blood to CSF.

Animals↗

PubMed1992

Characterization of the structure and glycosylation properties of intracellular and cell surface rat hepatic prolactin receptors.

In this study we examined the structure of the PRL receptor of rat liver. We used immunoblotting with monoclonal antibodies to binding and nonbinding site epitopes of the PRL receptor to assess receptor subtypes in different hepatic subcellular fractions. Analysis of frozen-thawed cell fractions revealed 40- and 42-kilodalton (kDa) species. Digestion with neuraminidase indicated that both species were terminally sialylated. Freshly isolated membranes exhibited a single 42-kDa species in all subcellular fractions, whereas freeze-thawing generated the 40-kDa species. The carbohydrate linkages present in the PRL receptor were examined using enzymes to deglycosylate iodinated purified receptors. These studies indicated that oligosaccharides comprise about 7 kDa of receptor mass and that they are exclusively N-linked, consisting of tri- and/or tetra-antennary complex glycans. Monoclonal antibodies to the receptor recognized the deglycosylated receptor. Maximally deglycosylated receptors retained about 85% of their binding capacity. After in vivo tunicamycin treatment of rats, total PRL receptors (as determined by immunoblot analysis and binding activity) disappeared with a half-time of about 25 min. In this circumstance, no aglycosylated receptor species were recognized by monoclonal antibodies. Since deglycosylation of mature receptors did not markedly reduce binding capacity, we infer that mature receptors do not accumulate during the blockade of glycosylation by tunicamycin. Thus, glycosylation appears to be required for the acquisition of a mature receptor status, but is not necessary for the maintenance of that status.

Animals↗

PubMed1991

A protein-tyrosine phosphatase with sequence similarity to the SH2 domain of the protein-tyrosine kinases.

The phosphorylation of proteins at tyrosine residues is critical in cellular signal transduction, neoplastic transformation and control of the mitotic cycle. These mechanisms are regulated by the activities of both protein-tyrosine kinases (PTKs) and protein-tyrosine phosphatases (PTPases). As in the PTKs, there are two classes of PTPases: membrane associated, receptor-like enzymes and soluble proteins. Here we report the isolation of a complementary DNA clone encoding a new form of soluble PTPase, PTP1C. The enzyme possesses a large noncatalytic region at the N terminus which unexpectedly contains two adjacent copies of the Src homology region 2 (the SH2 domain) found in various nonreceptor PTKs and other cytoplasmic signalling proteins. As with other SH2 sequences, the SH2 domains of PTP1C formed high-affinity complexes with the activated epidermal growth factor receptor and other phosphotyrosine-containing proteins. These results suggest that the SH2 regions in PTP1C may interact with other cellular components to modulate its own phosphatase activity against interacting substrates. PTPase activity may thus directly link growth factor receptors and other signalling proteins through protein-tyrosine phosphorylation.

Amino Acid Sequence↗

PubMed1991

The differential regulation by glucagon and growth hormone of insulin-like growth factor (IGF)-I and IGF binding proteins in cultured rat hepatocytes.

The liver is a major site of production of insulin-like growth factor-I (IGF-I) and IGF binding proteins (IGF-BPs). GH decisively influences IGF-I production. To study the role of GH and glucagon in the regulation of IGF-I and IGF-BP production, we examined IGF-I and IGF-BPs secreted by primary rat hepatocytes cultured in a serum-free medium. Glucagon (1 x 10(-8) M) stimulated IGF-I secretion and IGF-BP secretion. Bovine GH (bGH, 300 ng/ml) stimulated IGF-I secretion but suppressed IGF-BP secretion. Combining bGH and glucagon significantly augmented IGF-I secretion above the level seen with each individual agent. The inhibitory effect of bGH on IGF-BP secretion was reversed by glucagon. The major species of IGF-BPs secreted by hepatocytes were found, on Western ligand blotting, to be 24K and 30-34K. All species of secreted IGF-BPs appeared to be comparably affected by glucagon, bGH, and their combination. Northern analysis of IGF-I mRNA revealed three transcripts of 0.7-1.1 kilobases (kb), 1.8 kb, and 7.0 kb. Glucagon stimulated IGF-I mRNA levels 1.8- to 2.0-fold, whereas bGH stimulated IGF-I mRNA levels 2.0- to 2.5-fold. When hepatocytes were incubated with glucagon and bGH for 6 h, IGF-I mRNA levels were augmented 10-fold. Glucagon, in the presence of 50 ng/ml bGH, had a dose-dependent effect on IGF-I mRNA accumulation from a 6-fold level of stimulation at 50 ng/ml of glucagon to a 9-fold level of stimulation at 1000 ng/ml glucagon to a 9-fold level of stimulation at 1000 ng/ml glucagon. This study has demonstrated that glucagon, as well as GH, has significant effects on the production of both IGF-I and IGF-BPs. Of particular interest was the marked augmentation of hepatic IGF-I messenger RNA levels and the reversal of the low levels of IGF-BP production seen on adding glucagon to bGH.

Animals↗

PubMed1990

The distribution of lactogen receptors in the mammalian hypothalamus: an in vitro autoradiographic analysis of the rabbit and rat.

The hypothalamus contains a high concentration of lactogen receptors as detected with in vitro radioreceptor assay techniques. In an effort to define the location of the lactogen receptors relative to specific hypothalamic nuclei, an in vitro autoradiography technique was applied to frozen sections of rat and rabbit brains. Three lactogenic hormones, i.e. human growth hormone (hGH), ovine prolactin (oPRL), and rat prolactin (rPRL), were radiolabeled with iodine-125. Competition for observed binding sites was assessed with unlabeled hGH, oPRL, and bovine growth hormone (bGH). Analysis of the autoradiographs with a microcomputer-based densitometry system revealed that the rabbit hypothalamus contains specific lactogen binding sites within the supraoptic, paraventricular, suprachiasmatic, ventromedial, arcuate, and dorsomedial nuclei and the medial preoptic area. Unlabeled bGH was effective in competing for binding sites in all areas when hGH but not oPRL was used as the radiolabeled ligand, suggesting the presence of growth hormone receptors in the rabbit hypothalamus with a distribution similar to that of the lactogen binding sites. In contrast to the rabbit, no lactogen binding sites were detected in the rat hypothalamus regardless of the ligand used in the assay. All of the ligands were successful, however, in detecting lactogen receptors within the rat choroid plexus and liver. The results from the rabbits indicate that the influences of prolactin on hypothalamic activity are mediated via lactogen receptors that are widely distributed throughout the various pertinent hypothalamic nuclei. The broad distribution of lactogen receptors in the rabbit hypothalamus attests to the extensive influence of prolactin on hypothalamic regulatory systems. The results from the rat raise questions as to the nature of rat brain prolactin receptors in comparison to prolactin receptors in rat peripheral tissues.

Animals↗

PubMed1990

Determination of insulin and insulin-like growth factors in the ovarian circulation.

Recent in vitro studies implicate the ovary as an extra-hepatic source of insulin-like growth factors (IGFs) with production regulated by gonadotropins and local steroids. Because previous studies have failed to show any significant variations in IGF levels in peripheral blood during the menstrual cycle, we measured the concentrations of IGF-I, IGF-II, and insulin in ovarian and peripheral venous blood samples obtained simultaneously from nine women undergoing abdominal hysterectomy to obtain more detailed data on the ovarian contribution. A significant decreased ovarian gradient was found for IGF-II but not for IGF-I or insulin. Although there was no significant ovarian vein insulin gradient, insulin levels were higher in follicular than in luteal phase ovarian samples. These data suggest that IGF-II may be locally regulated by the ovary. Both insulin and IGFs may regulate ovarian function in vivo.

Adult↗

PubMed1990

Selective degradation of insulin within rat liver endosomes.

To characterize the role of the endosome in the degradation of insulin in liver, we employed a cell-free system in which the degradation of internalized 125I-insulin within isolated intact endosomes was evaluated. Incubation of endosomes containing internalized 125I-insulin in the cell-free system resulted in a rapid generation of TCA soluble radiolabeled products (t1/2, 6 min). Sephadex G-50 chromatography of radioactivity extracted from endosomes during the incubation showed a time dependent increase in material eluting as radioiodotyrosine. The apparent Vmax of the insulin degrading activity was 4 ng insulin degraded.min-1.mg cell fraction protein-1 and the apparent Km was 60 ng insulin.mg cell fraction protein-1. The endosomal protease(s) was insulin-specific since neither internalized 125I-epidermal growth factor (EGF) nor 125I-prolactin was degraded within isolated endosomes as assessed by TCA precipitation and Sephadex G-50 chromatography. Significant inhibition of degradation was observed after inclusion of p-chloromercuribenzoic acid (PCMB), 1,10-phenanthroline, bacitracin, or 0.1% Triton X-100 into the system. Maximal insulin degradation required the addition of ATP to the cell-free system that resulted in acidification as measured by acridine orange accumulation. Endosomal insulin degradation was inhibited markedly in the presence of pH dissipating agents such as nigericin, monensin, and chloroquine or the proton translocase inhibitors N-ethylmaleimide (NEM) and dicyclohexylcarbodiimide (DCCD). Polyethylene glycol (PEG) precipitation of insulin-receptor complexes revealed that endosomal degradation augmented the dissociation of insulin from its receptor and that dissociated insulin was serving as substrate to the endosomal protease(s). The results suggest that as insulin is internalized it rapidly but incompletely dissociates from its receptor. Dissociated insulin is then degraded by an insulin specific protease(s) leading to further dissociation and degradation.

Animals↗

PubMed1990

Hepatic protein phosphotyrosine phosphatase. Dephosphorylation of insulin and epidermal growth factor receptors in normal and alloxan diabetic rats.

Polypeptide hormone signal transmission by receptor tyrosine kinases requires the rapid reversal of tyrosine phosphorylation by protein phosphotyrosine phosphatases (PPTPases). We studied hepatic PPTPases in the rat with emphasis on acute and chronic regulation by insulin. PPTPase activity with artificial substrates ([32P]Tyr-reduced, carboxyamidomethylated, and maleylated lysozyme and [32P]Tyr-poly[glutamic acid:tyrosine] 4:1) was present in distinct membrane, cytoskeletal, and cytosolic fractions. These PPTPase activities were unaffected by alloxan diabetes. Acute administration of insulin to normal animals also did not change PPTPase activity in liver plasma membranes or endosomal membranes. Although alloxan diabetes did not affect PPTPase activity measured with artificial substrates or with epidermal growth factor receptors, a decrease in insulin receptor dephosphorylation was noted. Dephosphorylation of hepatic receptors from normal and diabetic rats by membrane PPTPase from control rats was similar. These results indicate that alloxan diabetes does not lead to a generalized effect on hepatic PPTPase activity, although a substrate-specific decrease in activity with the insulin receptor may occur.

Animals↗

PubMed1990

Effects of mannose-6-phosphate on receptor-mediated endocytosis of insulin-like growth factor-II.

The insulin-like growth factor-II (IGF-II) and lysosomal enzymes containing a mannose-6-phosphate (M6P) recognition site bind to different sites of the same receptor molecule. We have observed that M6P increases the receptor-mediated uptake of IGF-II into IM-9 cells. We now confirm this phenomenon in a different line, the H-35 rat hepatoma cells, and present additional characterization of the underlying mechanisms. When incubated in the presence of radiolabeled IGF-II, H-35 cells accumulated, in a time-dependent fashion, radioactivity that was resistant to removal by trypsin digestion at 15 C, indicating that it was endocytosed. In the presence of 3 mM M6P, endocytosed counts were approximately 2-fold higher after 5 min of incubation, an enhancement that peaked at 10 min, then declined, but was still evident after 40 min (1.5-fold). The rate of release of cell-associated IGF-II, degraded or intact, as measured in a chase experiment, was not affected by M6P. These observations indicate that M6P increased accumulation of IGF-II by accelerating its rate of endocytosis rather than by interfering with IGF-II degradation or with the recycling of intact hormone-receptor complexes to the cell surface. Electrophoresis after affinity cross-linking of labeled cells demonstrated that the enhancement in radioactivity could be located at a molecular size of approximately 250 kDa, corresponding to IGF-II-receptor complexes. Preincubation with M6P did not significantly alter the specific binding of IGF-II to the cell surface of H-35 cells, as measured by a binding assay at 4 C. Finally, pretreatment with cycloheximide for up to 8 h, to remove all newly synthesized lysosomal enzymes bound to the M6P/IGF-II receptor, did not affect IGF-II endocytosis beyond what could be accounted for by some loss of receptor, suggesting that the observed effect of M6P is due to the binding of M6P itself to the receptor and not to displacement of lysosomal enzymes. We conclude that simultaneous occupancy of the M6P/IGF-II receptor by ligands on both binding sites enhances its rate of endocytosis.

Animals↗

PubMed1990

Prolactin receptors in the primate choroid plexus.

The presence of prolactin receptors in the choroid plexus of primates has until now been assumed to be based on observations made on lower vertebrate animal models. An in vitro autoradiographic technique employing the principles of competitive binding was used to demonstrate the presence of specific prolactin binding sites in the choroid plexus of monkeys. Thus the primate choroid plexus resembles that of lower vertebrate mammals in that it possesses the prerequisite receptor for a prolactin blood-to-CSF transport mechanism.

Animals↗

PubMed1989

Pervanadate [peroxide(s) of vanadate] mimics insulin action in rat adipocytes via activation of the insulin receptor tyrosine kinase.

Both vanadate and hydrogen peroxide (H2O2) are known to have insulin-mimetic effects. We previously reported that the mixture of vanadate plus H2O2 results in the generation of a peroxide(s) of vanadate, which strongly enhances IGF-II binding to rat adipocytes (Kadota et al., 1987b). We now report that pervanadate mimics insulin in isolated rat adipocytes to (1) stimulate lipogenesis, (2) inhibit epinephrine-stimulated lipolysis, and (3) stimulate protein synthesis. The efficacy of pervanadate is comparable to that of insulin. However, it is 10(2)-10(3) times more potent than vanadate alone. Exposure of intact rat adipocytes to pervanadate was found to activate the WGA-purified insulin receptor tyrosine kinase assayed with the exogenous substrate poly(Glu80/Tyr20) in a dose-dependent manner to a maximum of 1464% of control at 10(-3) M compared with a maximum insulin effect of 1046% at 10(-6) M. In contrast, in vitro assayed autophosphorylation of the WGA-purified extract was increased 3-fold after exposure of intact cells to insulin but not significantly increased after pervanadate. Furthermore, high concentrations of pervanadate (10(-5) M) inhibited subsequent in vitro added insulin-stimulated autophosphorylation. In vitro addition of pervanadate to WGA-purified receptors could not stimulate autophosphorylation or exogenous tyrosine kinase activity and did not inhibit insulin-stimulated autophosphorylation. Labeling of intact adipocytes with [32P]orthophosphate followed by exposure to 10(-4) M pervanadate increased insulin receptor beta-subunit phosphorylation (7.9 +/- 3.0)-fold, while 10(-7) M insulin and 10(-4) vanadate increased labeling (5.3 +/- 1.8)- and (1.1 +/- 0.2)-fold, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Adipose Tissue↗

PubMed1989

Internalization and activation of the rat liver insulin receptor kinase in vivo.

The preparation of clearly delineated plasmalemma (PM) and endosomal subcellular fractions from rat liver has allowed us to compare insulin receptor (IR) kinase activity at the cell surface and in hepatic endosomes (ENs) as a function of dose and time after injected insulin. Tyrosine kinase activity in PM and ENs was measured, after solubilization and partial purification by wheat germ agglutinin chromatography (lectin-purified), using poly(Glu:Tyr) as substrate. Following the injection of a subsaturating dose of insulin (1.5 micrograms/100 g body weight), lectin-purified receptor showed peak activation at 30 s in PM and at 2 min in ENs. As observed previously (Khan, M. N., Savoie, S., Bergeron, J. J. M., and Posner, B. I. (1986) J. Biol. Chem. 261, 8462-8472) autophosphorylation activity was also augmented following insulin injection. In a pattern virtually identical to that of exogenous kinase activity, autophosphorylation attained peak activity at 30 s in PM and at 2 min in ENs. The time course of IR autophosphorylation in intact membranes was very similar to that observed for lectin purified receptors and was seen with an injected insulin dose as low as 150 ng/100 g body weight. Phosphatase treatment of the solubilized endosomal receptor abolished its enhanced activity. Hence, insulin treatment led to in vivo receptor phosphorylation which was reflected in the enhancement of both tyrosine kinase and autophosphorylation activities. Significant differences in the phosphorylation activities of PM and ENs were observed. Phosphoamino acid analyses revealed that the activated IR of intact PM was autophosphorylated in vitro, at both serine (55%) and tyrosine (45%) residues; whereas the activated IR of intact ENs was phosphorylated in vitro exclusively on tyrosine autophosphorylation specific activity for the activated IR of ENs was 3- to 4-fold that of the IR of PM. This was observed for the lectin purified IRs as well as for IRs of intact cell fractions. The reduced level of IR autophosphorylation in PM was not due to occlusion of tyrosine acceptor sites by prior in vivo phosphorylation. The rapidity with which activated IR accumulates in ENs as well as the sensitivity of endosomal IR kinase to activation by injected insulin are consistent with the endosomal apparatus serving a physiologically significant site for the regulation of transmembrane signaling.

Animals↗

PubMed1989

Homologous induction of growth hormone receptors in cultured rat hepatocytes.

The administration of growth hormone (GH) to animals has been reported to induce GH receptors in liver and adipose tissue. However, GH addition to cultured fibroblasts and lymphoblasts downregulated GH receptors, suggesting an indirect mechanism for GH upregulation of its receptors in vivo. We evaluated the direct role of GH by adding it to rat hepatocytes cultured in serum-free medium supplemented as previously described (Barash et al. (1988) Endocrinology 122, 1151-1158). After 3 days in culture the initial 125I-bGH specifically bound (0.18 ng per mg protein) had declined 5-fold. Binding continued to decrease thereafter to 0.008 ng by day 9 of culture. When added after 3 days in culture both hGH and bGH induced GH receptors. The maximum level (0.1 ng/mg protein) was attained 2 days later (day 5 of culture) and remained at this plateau through day 9 of culture. Induction occurred with 10 ng/ml hGH and was maximal (4- to 12-fold control) at 250 ng/ml. At a supramaximal dose of 1000 ng/ml hGH downregulated GH receptor. GH receptor induction was equally seen with hGH, bGH and rGH and did not occur on incubation with oPRL or ACTH. Thyroxine (1 X 10(-5) M) augmented 125I-bGH binding to levels 3-fold those of control but did not further augment the inductive effect of GH alone. We conclude that hepatic GH receptors are upregulated by GH acting through its own receptor. The induction occurs rapidly without a lag phase. The failure to restore fully receptor levels to those seen in freshly prepared hepatocytes implies a role for other modulating factor(s).

Animals↗

PubMed1989

Ligand-mediated internalization, recycling, and downregulation of the epidermal growth factor receptor in vivo.

EGF receptor internalization, recycling,a nd downregulation were evaluated in liver parenchyma as a function of increasing doses of injected EGF. The effect of ligand occupancy in vivo on the kinetics and extent of internalization was studied with changes in the receptor content of isolated plasmalemma and endosome fractions evaluated by direct binding, Scatchard analysis, and Western blotting. For all doses of injected EGF, receptor was lost from the plasmalemma and accumulated in endosomes in a time- and dose-dependent fashion. However, at doses of injected EGF equivalent to less than or equal to 50% surface receptor occupancy (i.e., less than or equal to 1 microgram/100 g body weight), receptor levels returned by 120 min to initial values. This return was resistant to cycloheximide and therefore did not represent newly synthesized receptor. Neither was the return due to replenishment by an intracellular pool of low-affinity receptors as such a pool could not be detected by Scatchard analysis or Western blotting. Therefore, receptor return was due to the recycling of previously internalized receptor. At doses of injected EGF greater than 50% receptor occupancy, net receptor loss-i.e., downregulation-was observed by evaluating the receptor content of total particulate fractions of liver homogenates. At the higher saturating doses of injected EGF (5 and 10 micrograms/100 g body weight), the majority of surface receptor content was lost by 15 min and remained low for at least an additional 105 min. As the kinetics of ligand clearance from the circulation and liver parenchyma were similar for all doses of EGF injected, then the ligand-mediated regulation of surface receptor content and downregulation were not a result of a prolonged temporal interaction of ligand with receptor. Rather, the phenomena must be a consequence of the absolute concentrations of EGF interacting with receptor at the cell surface and/or in endosomes.

Animals↗

PubMed1989

Ligand-mediated autophosphorylation activity of the epidermal growth factor receptor during internalization.

The association of EGF with its receptor in endosomes isolated from rat liver homogenates was assessed biochemically by polyethylene glycol precipitation and morphologically by electron microscope radioautography. The proportion of receptor-bound ligand in endosomes at 15 min after the injection of doses of 0.1 and 1 microgram EGF/100 g body weight was 57%. This value increased to 77% for the dose of 10 micrograms EGF injected. Quantitative electron microscope radioautography carried out on endosomes isolated at 15 min after the injection of 10 micrograms 125I-EGF demonstrated that most radiolabel was over the endosomal periphery thereby indicating that ligand-receptor complexes were in the bounding membrane but not in intraluminal vesicles of the content. EGF receptor autophosphorylation activity during internalization was evaluated in plasmalemma and endosome fractions. This activity was markedly but transiently reduced on the cell surface shortly after the administration of saturating doses of EGF. The same activity, however, was augmented and prolonged in endosomes for up to 30 min after EGF injection. The transient desensitization of cell surface activity was not due to prior in vivo phosphorylation since receptor dephosphorylation in vitro failed to restore autophosphorylation activity. Transient desensitization of cell surface autophosphorylation activity coincided with a diminished capacity for endocytosis of 125I-EGF with endocytosis returning to normal after the restoration of cell surface autophosphorylation activity. The inhibition of cell surface autophosphorylation activity and the activation of endosomal autophosphorylation activity coincident with downregulation suggest that EGF receptor traffic is governed by ligand-regulated phosphorylation activity.

Animals↗