Search PubMed⌕ Search

Biomedical subjects

L Jarett

Publications and source records attributed to L Jarett.

At least 37 records · Page 2Linked to original sources
PubMed1994

Demonstration by two-color flow cytometry that tyrosine kinase activity is required for down-modulation of the oncogenic neu receptor.

Expression of rat oncogenic neu receptor, p185T-neu (a growth factor receptor with constitutive tyrosine kinase activity), causes cells to become transformed. Treatment with anti-neu receptor monoclonal antibodies reverts the transformed phenotype by down-modulation of p185T-neu. Monoclonal antibody treatment of cells expressing normal neu receptor, p185C-neu (which lacks constitutive tyrosine kinase activity), does not result in down-modulation of p185C-neu. To understand further the role the biochemical activity of p185T-neu plays in transformation and endocytosis, we created a series of mutations in p185T-neu. We found that fibroblasts expressing the tyrosine kinase-defective mutants cannot form foci in culture, colonies in soft agar, or tumors in immunocompromised mice. To follow the antibody-induced endocytosis of neu receptors expressed in these transfectants, we developed a novel two-color flow cytometric assay and confirmed receptor localization by electron microscopy. Cells were treated with mAb7.16.4 over time. After 4 hr of antibody treatment, less than 50% of full-length p185T-neu and of mutant T691 remained on the cell surface, whereas internal expression of the neu receptors within these cells initially increased and then decreased to the original internal receptor level. In contrast, the level of kinase-deficient mutated neu receptors remaining on the cell surface initially decreased by 35%, but, after 4 hr of antibody treatment, the cell surface expression level returned to approximately the original level. Concurrently, fluctuations in expression levels were seen internally over time as well. These cell lines were also treated with gold-conjugated mAb7.16.4. Using electron microscopy, we consistently found the gold particles within multivesicular bodies of cell lines expressing full-length or mutated neu receptor. These data strongly suggest that the fate of the neu receptor, once internalized, is directed by its tyrosine kinase activity. When the kinase activity of the neu receptor is disrupted, the receptor is internalized but recycled to the cell surface, whereas neu receptors which have constitutive kinase activity are internalized and presumably degraded when engaged with anti-neu receptor mAb. Understanding the regulation of receptor endocytosis, degradation, and recycling will contribute to the development of novel therapeutic protocols to combat human malignancies, particularly those associated with the overexpression of the human homologue of the neu receptor, c-erbB2.

Animals↗
PubMed1994

1,10-Phenanthroline increases nuclear accumulation of insulin in response to inhibiting insulin degradation but has a biphasic effect on insulin's ability to increase mRNA levels.

Previous reports demonstrated that insulin is translocated through the cytoplasm to the nucleus of H35 hepatoma cells and suggested that nuclear insulin may be involved in stimulating transcription of immediate-early genes. In a recent study, inhibition of insulin-degrading enzyme with 1,10-phenanthroline, a Zn2+ chelator, caused a significant increase in the nuclear accumulation of insulin. The present study characterized the effects of 1,10-phenanthroline and its nonchelating isomer, 1,7-phenanthroline, on insulin degradation, nuclear accumulation, and stimulation of immediate-early gene expression. 1,10- but not 1,7-phenanthroline inhibited insulin degradation and increased nuclear accumulation of insulin in a dose-dependent manner. 1,7-phenanthroline caused a dose-dependent decrease in the expression of insulin-stimulated immediate-early genes, but had no significant effect on alpha-tubulin mRNA levels. In the presence of insulin, Northern analysis revealed that 1,10-phenanthroline at all concentrations tested increased alpha-tubulin mRNA levels, but had a biphasic effect on insulin-stimulated immediate-early gene expression. At low concentrations (5-200 microM), 1,10-phenanthroline increased the expression of insulin-stimulated g33, c-fos, and Egr-1 mRNA. At concentrations greater than 1 mM, insulin-stimulated immediate-early gene expression was decreased similar to the effect seen with 1,7-phenanthroline. Nuclear run-on analysis demonstrated that high concentrations of 1,10-phenanthroline decreased insulin-stimulated immediate-early gene transcription but had no effect on transcription of alpha-tubulin. However, low concentrations of 1,10-phenanthroline did not increase transcription of any genes.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

A subgroup of murine monoclonal anti-deoxyribonucleic acid antibodies traverse the cytoplasm and enter the nucleus in a time-and temperature- dependent manner.

BACKGROUND: The capacity of lupus autoantibodies to enter living cells and bind to molecules for which they have intrinsic affinity is not well appreciated. In previous studies, we identified a subgroup of three murine monoclonal IgG anti-DNA antibodies, derived from lupus-prone MRL-lpr/lpr mice, that localized within nuclei of cells in multiple organs and induced functional perturbations, in vivo, after passive transfer to normal mice. To examine the mechanisms of this phenomenon, we now extend these observations, using the same monoclonal anti-DNA antibodies and cultured cell lines. EXPERIMENTAL DESIGN: Multiple experimental approaches were utilized to track nuclear localization of anti-DNA antibodies, including direct immunofluorescence, confocal microscopy and immunoelectron microscopy. The requirements for nuclear localization were further evaluated quantitatively, in nuclei isolated from co-cultures of cells and 125I-Ig, under varying experimental conditions. RESULTS: Nuclear localization was observed with the same subset of anti-DNA antibodies that localized within nuclei in vivo; it was dependent on the antigen-binding region of the molecule; and it was not found with other anti-DNA antibodies. At progressive intervals, the Ig were observed: at the cell surface, within the cytoplasm, clustered at the nuclear pore, and within the nucleus. Nuclear localization of Ig was found to be a time- and temperature- dependent process, specific for a subset of anti-DNA antibodies and dependent on the antigen binding region of the Ig. CONCLUSIONS: This is the first demonstration that monoclonal autoantibodies can traverse both the cell and nuclear membranes to localize within the nuclei of cultured cells. Furthermore, nuclear localization of Ig was regulated in a manner analogous to that of other large cytoplasmic proteins that enter the nucleus. This confirms and extends our results using the same antibodies in whole animals, and it provides the basis to further examine the underlying mechanisms and consequences of this phenomenon.

Animals↗
PubMed1993

Genistein inhibits insulin-stimulated glucose transport and decreases immunocytochemical labeling of GLUT4 carboxyl-terminus without affecting translocation of GLUT4 in isolated rat adipocytes: additional evidence of GLUT4 activation by insulin.

A recent study from this laboratory (Abler et al., J. Biol. Chem. 267, 18172-18179, 1992) showed genistein blocked insulin-stimulated glucose oxidation without affecting receptor autophosphorylation or tyrosine kinase activity. The mechanism by which genistein inhibited insulin-stimulated glucose metabolism was investigated in the present study. Insulin caused a approximately 12-fold increase in 3-O-methyl-D-glucose (3OMG) uptake compared to that of control cells. Basal and insulin-stimulated 3OMG transport was inhibited 40-60% by genistein in a concentration-dependent manner (10-100 micrograms/ml). Genistein had no effect on insulin-stimulated GLUT4 translocation from low density microsomes to plasma membranes as determined by Western blotting. These results suggested that genistein inhibited glucose transport in adipocytes by decreasing the intrinsic activity, rather than the number, of the plasma membrane-associated glucose transporters. We also previously reported that insulin treatment of adipocytes resulted in the immunocytochemically visualized unmasking of the carboxyl-terminus of plasma membrane-associated GLUT4 and suggested the unmasking might be related to an insulin-induced increase in the intrinsic activity of the glucose transporter (Smith et al., Proc. Natl. Acad. Sci. USA 88, 6893-6897, 1991). In the present study, genistein decreased immunocytochemical labeling of plasma membrane-associated GLUT4 by approximately 50% in control and insulin-treated adipocytes by carboxyl-terminus antibodies but had no effect on labeling observed in an amino-terminus antibody. Since genistein did not affect the number of plasma membrane-associated GLUT4 transporters, this result supports the hypothesis that conformational changes in the glucose transporter, reflected by the ability of anti-carboxyl-terminus antibodies to bind to the transporter, may be an indication of the intrinsic activity of the plasma membrane-associated transporter. We therefore conclude that conformational changes in and activation of glucose transporters, in addition to insulin-stimulated GLUT4 translocation, play an important role in insulin-regulated glucose transport in adipocytes.

3-O-Methylglucose↗
PubMed1993

Insulin induces an unmasking of the carboxyl terminus of G(i) proteins in rat adipocytes.

Several groups have shown a relationship between the insulin receptor and inhibitory G proteins, G(i). An antisera, 8729, to a peptide sequence (KNNLKDCGLF) corresponding to the carboxyl termini of G(i)alpha subunits was used to investigate this relationship by immunoelectron microscopy. Rat adipocytes were incubated in the absence or presence of 100 ng/ml insulin for 1 h and fixed for immunoelectron microscopy. Insulin-treated adipocytes stained with 8729 were labeled at the cell surface at a much higher density than control adipocytes. Subcellular fractionation of insulin-treated and control cells was followed by PAGE and Western blots of the plasma membrane and low-density microsomes with 8729. The density of the bands did not change in response to insulin treatment. Antibodies to noncarboxyl terminus sequences of the alpha subunit were used for immunoelectron microscopy and no difference was noted between insulin-treated and control adipocytes. These results indicated that 8729 was detecting a conformational change in the structure of G(i)alpha subunit in the plasma membrane in response to insulin. This unmasking of the carboxyl terminus was also seen in response to treatment with phenylisopropyladenosine and prostaglandin E2. Pertussis toxin-catalyzed ADP ribosylation also unmasked the carboxyl terminus. In contrast, isoproterenol, an agonist of stimulatory G proteins (Gs), did not induce an unmasking of the carboxyl terminus. These results support the hypothesis that some of insulin's effects are mediated through G(i) proteins in adipocytes.

Adipose Tissue↗
PubMed1993

Activation and inhibition of insulin receptor autophosphorylation by trypsin treatment of intact H35 cells.

1. Treatment of intact cultured H35 cells with trypsin (1 mg/ml) for 15 min at low temperature (4 degrees C) or for 30 sec at 37 degrees C causes activation of the insulin receptor subsequently isolated from the cells. 2. Receptor activation was assessed by increased phosphotyrosine content of the beta-subunit of the receptor, and increased autophosphorylation using [32P]-ATP. 3. Treatment of the cells for 15 min at 37 degrees C however completely abolished insulin binding and all insulin receptor kinase activity. 4. These data demonstrate that proteolytic damage of the extracellular domain of the insulin receptor can render the receptor kinase inactive and lead to a cell which is unresponsive to insulin.

Adenosine Triphosphate↗
PubMed1993

Mouse preimplantation embryos exhibit receptor-mediated binding and transcytosis of maternal insulin-like growth factor I.

High-resolution microscopy in conjunction with colloidal gold-labeled insulin-like growth factor I (IGF-I) has been used to provide evidence that the IGF-I receptor is first detected in 8-cell-stage mouse embryos, confirming the results of previous reverse transcriptase polymerase chain reaction (RT-PCR) studies. Specificity for the IGF-I receptor was demonstrated by displacement with unlabeled IGF-I and dual-labeling experiments with colloidal gold-labeled or unlabeled insulin. Labeled IGF-I ligand is internalized by means of receptor-mediated endocytosis following its concentration in coated pits, and it can be visualized within cytoplasmic organelles. Immunocytochemical analyses at the blastocyst stage, using gold-labeled antibodies to the receptor, confirmed the expression of IGF-I receptors on all cells of the embryo. Similar studies with antibodies directed against the ligand demonstrated that IGF-I internalized by the embryo in vivo is maternally derived. Approximately 40% of blastocysts showed apical plasma membrane binding of gold-labeled ligand ("responders"), while approximately 60% did not demonstrate binding ("nonresponders"); however, both classes of embryo expressed receptors on basolateral membranes of trophectoderm cells and on the surface of inner masses. Functional studies show that incubating embryos in physiological levels of IGF-I (40 ng/ml) results in increased numbers of cells in the inner cell mass (p < 0.05), but not the trophectoderm, as compared to controls.

Animals↗
PubMed1993

A truncated human insulin receptor missing the COOH-terminal 365 amino acid residues does not undergo insulin-mediated receptor migration or aggregation.

A previous study of tyrosine kinase-defective insulin receptors demonstrated that receptor autophosphorylation or tyrosine kinase activity was required for concentrating insulin receptors in coated pits, but not for their migration or aggregation on the cell surface. Furthermore, receptor migration and aggregation on the cell surface were not sufficient to cause internalization of the occupied receptors in coated pits. In the present study, biochemical and ultrastructural techniques were used to compare insulin receptor mobility and internalization in Rat 1 fibroblasts expressing wild-type human insulin receptors (HIRc) with those in cells expressing receptors truncated at residues 978 (HIR delta 978) or 1301 of the carboxyl-terminus (HIR delta CT). There were no significant differences in the mobility or internalization of insulin receptors on HIR delta CT cells compared to those of insulin receptors on HIRc cells. Ultrastructural analysis revealed that truncated insulin receptors on HIR delta 978 cells failed to migrate from their initial location on the microvilli, move to the plasma membrane, and aggregate in coated pits. Receptor-mediated insulin internalization in HIR delta 978 cells was markedly decreased due entirely to a decrease in ATP-dependent, coated pit-mediated internalization. ATP-independent endocytosis in non-coated pinocytotic invaginations was not affected by receptor truncations. These results provide evidence of the roles that regions of the beta-subunit play in the processing of occupied insulin receptors. 1) The carboxyl-terminus of the insulin receptor is not involved in the events leading to receptor internalization, i.e. migration, aggregation, and concentration in coated pits. 2) Internalization of insulin receptors by the ATP-independent noncoated invagination pathway is not regulated by residues in the insulin receptor beta-subunit distal to 978. 3) Sequences in the beta-subunit between 978-1300, but not the autophosphorylation and kinase domains, are involved in insulin-induced receptor migration and aggregation.

Amino Acid Sequence↗
PubMed1993

Inhibition of insulin-degrading enzyme increases translocation of insulin to the nucleus in H35 rat hepatoma cells: evidence of a cytosolic pathway.

We previously demonstrated the translocation of insulin to the nucleus in several cell types and partially characterized the uptake mechanisms and pathways in H35 rat hepatoma cells. Nuclear accumulation of insulin was energy independent, time and temperature dependent, and apparently was not saturable at insulin concentrations which resulted in full receptor occupancy. We also have shown insulin could be internalized by both receptor-mediated and fluid-phase endocytosis. This study investigated subsequent steps involved in the nuclear accumulation of insulin following internalization. We examined the effects of inhibiting insulin degrading enzyme (IDE) with 1,10-phenanthroline on the nuclear accumulation of insulin in H35 cells. 1,10-phenanthroline (2 mM) which markedly inhibited insulin degradation, significantly increased nuclear accumulation of insulin without having any effects on total cell-associated and intracellular insulin. This reagent increased 125I-insulin on the cellular membrane and decreased 125iodine (125I-insulin and 125I-insulin degradation products) in the cytosolic fractions. Chemical extraction and Sephadex G-50 chromatography revealed the insulin associated with the nucleus in 1,10-phenanthroline-treated cells formed the same complex(es) with the nuclear matrix as in control cells. These results suggested that inhibition of cytosolic IDE activity resulted in increased insulin translocation from the cytosol to the nucleus. Furthermore, when IDE activity was inhibited by high cytosolic insulin concentrations, the amount of 125I-insulin in the nucleus was significantly increased. Our study suggests internalized insulin is probably released from endosomes into the cytosol where modulation of IDE activity could have significant effects on the accumulation of insulin, or insulin-cytoplasmic protein complexes, in nuclei. The IDE regulatory mechanism, by controlling the translocation of insulin to the cell nucleus, could play a crucial role in insulin's regulation of gene expression and cell proliferation.

Animals↗
PubMed1992

Direct stimulation of immediate-early genes by intranuclear insulin in trypsin-treated H35 hepatoma cells.

H35 hepatoma cells were treated with trypsin to abolish insulin binding and insulin-stimulated receptor kinase activity. Insulin was, however, internalized by fluid-phase endocytosis in trypsin-treated cells. Furthermore, nuclear accumulation of insulin was similar in control and trypsin-treated hepatoma cells. Northern blot analysis revealed insulin increased g33 and c-fos mRNA concentrations identically in control and trypsin-treated cells but had no effect on beta 2-microglobulin mRNA. Actinomycin D treatment prior to or after insulin addition demonstrated that insulin increased gene transcription and had no effect on mRNA degradation. These studies suggest that the accumulation of intact insulin in cell nuclei may be directly involved in the increased transcription of immediate-early genes.

Cell Cycle↗
PubMed1992

Genistein differentially inhibits postreceptor effects of insulin in rat adipocytes without inhibiting the insulin receptor kinase.

Genistein, an isoflavone putative tyrosine kinase inhibitor, was used to investigate the coupling of insulin receptor tyrosine kinase activation to four metabolic effects of insulin in the isolated rat adipocyte. Genistein inhibited insulin-stimulated glucose oxidation in a concentration-dependent manner with an ID50 of 25 micrograms/ml and complete inhibition at 100 micrograms/ml. Genistein also prevented insulin's (10(-9) M) inhibition of isoproterenol-stimulated lipolysis with an ID50 of 15 micrograms/ml and a complete effect at 50 micrograms/ml. The effect of genistein (25 micrograms/ml) was not reversed by supraphysiological (10(-7) M) insulin levels. In contrast, genistein up to 100 micrograms/ml had no effect on insulin's (10(-9) M) stimulation of either pyruvate dehydrogenase or glycogen synthase activity. We determined whether genistein influenced insulin receptor beta-subunit autophosphorylation or tyrosine kinase substrate phosphorylation either in vivo or in vitro by anti-phosphotyrosine immunoblotting. Genistein at 100 micrograms/ml did not inhibit insulin's (10(-7) M) stimulation of insulin receptor tyrosine autophosphorylation or tyrosine phosphorylation of the cellular substrates pp185 and pp60. Also, genistein did not prevent insulin-stimulated autophosphorylation of partially purified human insulin receptors from NIH 3T3/HIR 3.5 cells or the phosphorylation of histones by the activated receptor tyrosine kinase. In control experiments using either NIH 3T3 fibroblasts or partially purified membranes from these cells, genistein did inhibit platelet-derived growth factor's stimulation of its receptor autophosphorylation. These findings indicate the following: (a) Genistein can inhibit certain responses to insulin without blocking insulin's stimulation of its receptor tyrosine autophosphorylation or of the receptor kinase substrate tyrosine phosphorylation. (b) In adipocytes genistein must block the stimulation of glucose oxidation and the antilipolytic effects of insulin at site(s) downstream from the insulin receptor tyrosine kinase. (c) The inhibitory effects of genistein on hormonal signal transduction cannot necessarily be attributed to inhibition of tyrosine kinase activity, unless specifically demonstrated.

Adipose Tissue↗
PubMed1992

Nonreceptor mediated nuclear accumulation of insulin in H35 rat hepatoma cells.

We previously demonstrated that insulin accumulated in the nucleus in several cell types and partially characterized the uptake mechanisms and pathways in H35 rat hepatoma cells. Nuclear accumulation of insulin was energy independent, time, temperature, and insulin concentration dependent, but apparently nonsaturable. This study investigated further the initial endocytotic pathways that contribute to the nuclear accumulation of insulin using trypsin treatment of the cells to prevent insulin binding to its plasma membrane receptor. Total cell-associated, intracellular, and nuclear insulin were compared in control and trypsin-treated H35 hepatoma cells. Trypsin treatment markedly decreased total cell-associated and intracellular insulin as well as the nuclear accumulation of insulin when cells were incubated with 2.8 ng/ml insulin. When the cells were incubated with 100 ng/ml insulin, trypsin treatment totally inhibited insulin binding to the plasma membrane for at least 90 min. However, intracellular accumulation of insulin was reduced by only 50% at 60 min, and trypsin treatment failed to inhibit the nuclear accumulation of insulin. Chemical extraction and Sephadex G-50 chromatography revealed nuclear associated insulin in trypsin-treated cells was identical to that in control cells incubated with either 2.8 or 100 ng/ml insulin. These results suggest that a nonreceptor mediated uptake pathway, i.e., fluid-phase endocytosis, contributed significantly to the nuclear accumulation of insulin at high insulin concentrations, but at lower insulin concentrations the receptor-mediated pathway predominated. No matter which initial endocytotic route was used to internalize insulin, the insulin apparently associated with the same nuclear matrix proteins. This association of insulin with the nuclear matrix may be involved in regulation of nuclear events such as cell growth and differentiation or gene transcription.

Animals↗
PubMed1992

The effect of orthovanadate on phosphoinositide metabolism in NIH 3T3 fibroblasts.

Orthovanadate is an agent known to stimulate cell growth and mimic insulin action. The effects of this compound on phosphoinositides in NIH 3T3 cells were examined. Both 100 and 1000 microM orthovanadate were found to increase the cellular content of inositol phosphate secondary to the activation of phosphatidylinositol-specific phospholipase C (PtdIns-PLC). The time course, dependence on orthovanadate concentration, and sensitivity to the isoflavone genistein were similar for orthovanadate-induced accumulation of inositol phosphate and protein tyrosine phosphate, indicating that there is a correlation between cellular protein tyrosine phosphate levels and PtdIns-PLC activity. Increased phosphatidylinositol phosphate (PtdInsP) content also occurred when cells were incubated with orthovanadate and appeared to result from the activation of PtdIns kinase. This effect was not correlated with cellular protein tyrosine phosphate content. Hence, orthovanadate is shown to affect phosphoinositide metabolism at a minimum of two sites by both tyrosine phosphate-dependent and -independent mechanisms.

Animals↗
PubMed1992

Developmental expression and cellular localization of glucose transporter molecules during mouse preimplantation development.

Two general mechanisms mediate glucose transport, one is a sodium-coupled glucose transporter found in the apical border of intestinal and kidney epithelia, while the other is a sodium-independent transport system. Of the latter, several facilitated transporters have been identified, including GLUT1 (erythrocyte/brain), GLUT2 (liver) and GLUT4 (adipose/muscle) isoforms. In this study, we used Western-blot analysis and high resolution immunoelectron microscopy (IEM) to investigate the stage-related expression and cellular localization of GLUT1, 2 and 4. The Western blot results demonstrate that GLUT1 is detectable in the oocyte and throughout preimplantation development. GLUT2 isoforms were not detectable until the blastocyst stage, while the GLUT4 isoform was undetectable in the oocyte through blastocyst stages. The present findings confirm previous studies at the molecular level which demonstrated that mRNAs encoding the same GLUT isoforms are detectable at corresponding developmental stages. GLUT1 and GLUT2 display different cellular distributions at the blastocyst stage as shown by IEM studies. GLUT1 has a widespread distribution in both trophectoderm and inner cell mass cells, while GLUT2 is located on trophectoderm membranes facing the blastocyst cavity. This observation suggests a different functional significance for these isoforms during mouse preimplantation development.

Animals↗
PubMed1992

Insulin stimulates accumulation and efflux of macromolecules in isolated nuclei from H35 hepatoma cells.

This study used 10-nm gold particles with 5-7 insulin molecules attached (Au10-Ins) to investigate the site of interaction of insulin with the nuclear envelope during insulin uptake into intact isolated nuclei. Despite its size, and in the absence of ATP, Au10-Ins entered nuclei through the nuclear pore and associated with the heterochromatin. Because Au10-Ins is essentially gold-bovine serum albumin (Au-BSA) with a few insulin molecules attached, the effect of insulin and other growth factors on the nuclear accumulation of BSA coupled to 10-, 15-, and 24-nm-diam colloidal gold particles (Au10-BSA, Au15-BSA, and Au24-BSA) was determined. The Au-BSA complexes were excluded from nuclei in the absence of insulin. Insulin (0.5-100 ng/ml) caused a dose-dependent accumulation of Au10-BSA in the nucleus. The nuclear membrane was shown to be intact by several criteria, therefore, accumulation of Au-BSA occurred via the nuclear pore and was not due to leakage across or through the membrane. Uptake of 15- and 24-nm Au-BSA molecules was not affected by insulin, suggesting the hormone had a limited effect in increasing the functional diameter of the nuclear pores. Glucagon, epidermal growth factor, platelet-derived growth factor, insulinlike growth factor I, and insulin A or B chains did not stimulate the accumulation of Au10-BSA. The insulin-stimulated accumulation of Au10-BSA was blocked by concanavalin A, mimicked by wheat-germ agglutinin, and did not require ATP. The Au10-BSA in the nucleus was associated with heterochromatin, suggesting it bound to a nuclear element.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1992

Insulin and IGF1 receptors in a model renal epithelium: receptor localization and characterization.

Insulin and IGF1 stimulate transepithelial Na+ transport in the urinary bladder of the toad Bufo marinus, a model renal epithelium. The signal transduction mechanisms for the natriferic action are unknown. Ultrastructural techniques were used to localize both receptors and ligands in the epithelium. Electron microscopy using gold-labelled anti-insulin or anti-IGF1 receptor antibodies demonstrated the majority of the insulin receptors were associated with the basolateral membrane while IGF1 receptors were found on basolateral and apical membranes. Both insulin and IGF1 receptors were found in endosomes and on the membranes surrounding subapical granules. In intact tissues incubated with iodinated IGF1 or insulin, both ligands were associated with the basolateral membrane. IGF1 was internalized to a greater extent than insulin and only IGF1 accumulated in cell nuclei.

Animals↗
PubMed1991

Tyrosine kinase-defective insulin receptors undergo insulin-induced microaggregation but do not concentrate in coated pits.

Biologically active colloid-gold complexes were used to compare ligand-induced microaggregation, redistribution, and internalization of insulin receptors on Rat 1 fibroblasts expressing wild type (HIRc) or tyrosine kinase-defective (HIR A/K1018) human insulin receptors. Insulin-like growth factor I (IGF I) and alpha 2-macroglobulin receptors also were compared. On both cell types, all four unoccupied receptor types occurred predominantly as single receptors. Ligand binding caused receptor microaggregation. Microaggregation of wild type or kinase-defective insulin receptors or IGF I receptors was not different. alpha 2-Macroglobulin receptors formed larger microaggregates. Compared to wild type insulin or IGF I receptors, accumulation of kinase-defective insulin receptor microaggregates in endocytic structures was decreased, and the size of microaggregates in coated pits was significantly smaller. As a result, receptor-mediated internalization of gold-insulin by HIR A/K1018 cells was less than 6% of the cell-associated particles compared to approximately 60% of the particles in HIRc cells. On HIR A/K1018 cells, alpha 2-macroglobulin and IGF I were internalized via coated pits demonstrating that those structures were functional. These results suggest that: 1) ATP binding, receptor autophosphorylation, and activation of receptor kinase activity are not required for receptor microaggregation; 2) receptor microaggregation per se is not sufficient to cause ligand-induced receptor-mediated internalization or the biological effects of insulin; and 3) autophosphorylation of the beta-subunit or activation of the receptor kinase activity is required for the insulin-induced concentration of occupied receptors in coated pits.

Animals↗