Biomedical subjects

D A McClain

Publications and source records attributed to D A McClain.

At least 19 recordsLinked to original sources

PubMed1992

Glucose and glucosamine regulate growth factor gene expression in vascular smooth muscle cells.

We have investigated the regulation of the expression of two growth factors found in vascular smooth muscle, transforming growth factor alpha (TGF alpha) and basic fibroblast growth factor (bFGF). Cells cultured in medium containing 30 mM glucose exhibited a 2-fold increase in TGF alpha mRNA and a 3-fold increase in bFGF mRNA compared with cells grown in normal (5.5 mM) glucose. Glucosamine was more potent than glucose, leading to a 6-fold increase in TGF alpha mRNA. TGF alpha protein levels were also increased by glucosamine treatment, and the predominant species present was the membrane-bound precursor form of TGF alpha. To examine further the regulation of growth factors by sugars, cultured rat aortic smooth muscle cells were transfected with a plasmid construct consisting of a 1.2-kilobase-pair fragment of the TGF alpha promoter linked to a luciferase reporter gene. Increasing the concentration of glucose in the culture medium from 5.5 mM to 30 mM led to a rapid, 1.7-fold increase in the activity of the TGF alpha promoter. Glucosamine was much more potent than glucose in this stimulation, with 2 mM glucosamine causing a 12-fold increase in TGF alpha promoter activity. Insulin had no effect on luciferase activity in either the presence or the absence of added sugars. The glucose response element of the TGF alpha gene maps to a 130-base-pair segment that includes three potential binding sites for the transcription factor Sp1. We conclude that high glucose concentrations such as are reached in diabetes mellitus can stimulate the transcription of the genes for growth factors in vascular smooth muscle cells. This signaling pathway apparently involves the metabolism of glucose to glucosamine. This effect could be representative of nutritional regulation of a family of genes and could contribute to the toxicity of hyperglycemia and the vascular complications of diabetes.

Animals↗

PubMed1992

Mechanism and role of insulin receptor endocytosis.

Like many other cell surface receptors for nutrients and polypeptide hormones, the insulin receptor undergoes a complex endocytotic itinerary. Upon insulin binding, the receptor is activated as a tyrosine-specific protein kinase and autophosphorylates. This autophosphorylation is necessary for the receptor to internalize. After endocytosis, the ligand (insulin) and its receptor are dissociated. Most of the insulin is degraded, whereas the receptors are largely recycled to the cell surface. The signals in the receptor that control and specify its endocytotic pathway are beginning to be understood. Through the techniques of in vitro mutagenesis, noninternalizing receptors have been engineered and their structural and functional properties have been analyzed. For example, the immediate submembranous domain of the insulin receptor has been found to contain sequences (Gly-Pro-Leu-Tyr and, to a lesser extent, Asn-Pro-Gln-Tyr) that are necessary for normal endocytosis. Receptors deleted or mutated in these sequences retain tyrosine kinase activity but fail to undergo endocytosis. Unlike the better understood low density lipoprotein and transferrin receptors, however, these sequences are not sufficient for endocytosis. An insulin receptor with only these sequences exposed in the cytoplasm does not internalize. Tyrosine kinase activity is thought to be needed to lead to autophosphorylation and a conformational change that exposes the otherwise buried endocytosis sequences in the normally dimerized insulin receptor. Non-internalizing mutants of the insulin receptor have been used to examine the role of endocytosis in insulin action. It was found that an endocytosis-defective receptor could induce a short-term metabolic action of insulin (glycogen synthetase stimulation) as well as longer-term mitogenic effects of insulin. Furthermore, insulin action deactivated after the hormone was removed from the noninternalizing receptors. Apparently, endocytosis is not necessary for insulin action, but probably is important for removing the insulin from the cell so the target cell for insulin responds in a time-limited fashion to the hormone.

Amino Acid Sequence↗

PubMed1992

Tyrosine kinase-defective insulin receptors undergo decreased endocytosis but do not affect internalization of normal endogenous insulin receptors.

To characterize tyrosine kinase activity in signaling ligand/receptor internalization, metabolic labeling and surface radioligand binding were used to follow the processing of both normal and tyrosine kinase-deficient human insulin receptors. The mutant receptor (A/K1018) has an alanine substituted for lysine 1018 in the ATP-binding domain. Rat 1 fibroblasts, expressing either normal human insulin receptors (HIRc) or A/K1018 receptors, were assayed to determine the insulin receptor half-life as well as internalization and down-regulation. Our results show that insulin greatly reduces the half-life of normal insulin receptors (from 9.9 to 5.7 h). The A/K1018 receptor had a much longer half-life (24 h), which was not reduced by the presence of saturating insulin concentrations. The A/K1018 receptor does not undergo down-regulation after long term insulin exposure, while HIRc cells showed a 34% decrease in insulin receptor number. This down-regulation is accounted for by the accelerated turnover rate of normal receptors in the presence of insulin. To confirm that the kinase activity is necessary for normal endocytosis, we also show that ATP depletion in HIRc cells resulted in significant decreases in receptor internalization and that tyrosine kinase-defective receptors also fail to internalize in a different cell type (rat Fao hepatocytes). Lastly, the complement of normal rat insulin receptors in cells expressing the kinase-defective receptors endocytose normally. We conclude that the defect in endocytosis observed in kinase-defective receptors is intrinsic to this receptor and not due to a dominant inhibitory effect on cellular endocytotic machinery.

Adenosine Triphosphate↗

PubMed1991

Amino acid sequences Gly-Pro-Leu-Tyr and Asn-Pro-Glu-Tyr in the submembranous domain of the insulin receptor are required for normal endocytosis.

We have recently shown that the immediately submembranous domain of the human insulin receptor (hIR) is required for rapid ligand-dependent internalization (Thies, R. S., Webster, N. J., and McClain, D. A. (1990) J. Biol. Chem. 265, 10132-10137). This region contains one copy of an NPXY sequence that is required for endocytosis of the low density lipoprotein receptor. In order to dissect and analyze the specific sequences involved in endocytosis of the insulin receptor, we have mutated the NPXY sequence from NPEY (residues 957-960) to APEA (NPEY/APEA). In addition, we have mutated a similar sequence in the same region, changing GPLY (residues 950-953) to APLA (GPLY/APLA). The cDNAs encoding the normal hIR and these mutant receptors were transfected into Rat 1 fibroblasts. The expressed receptors bound insulin with high affinity and retained insulin-stimulated tyrosine kinase activity. Despite the ability of these mutant receptors to bind insulin and undergo autophosphorylation, the GPLY/APLA receptor internalized insulin at only 32% of the rate of normal hIR at low receptor occupancy. On the other hand, the NPEY/APEA receptor internalized insulin at 87% of the normal rate. These results were confirmed by measuring internalization of photoaffinity-labeled insulin receptors. Another receptor with both the NPEY/APEA and GPLY/APLA mutations internalized to a lesser degree than the GPLY/APLA receptor and at a rate equivalent to that seen for a receptor with the entire submembranous domain deleted. A receptor with the complete normal submembranous domain but with the tyrosine kinase and C-terminal region of the hIR deleted exhibited only a basal internalization rate. We conclude that the information contained in the GPLY and, to a lesser extent, the NPEY sequences are necessary but not sufficient for signaling internalization of the insulin receptor.

Amino Acid Sequence↗

PubMed1991

Transdominant inhibition of tyrosine kinase activity in mutant insulin/insulin-like growth factor I hybrid receptors.

Classical insulin and insulin-like growth factor I (IGF-I) receptors exist as well defined alpha 2 beta 2 heterotetrameric complexes that are assembled from two identical alpha beta heterodimeric half-receptor precursors. Recent evidence suggests that insulin and IGF-I half-receptors can heterologously assemble to form alpha 2 beta 2 insulin/IGF-I hybrid receptor complexes in vivo and in vitro. We have utilized hybrid receptor complexes to examine ligand-stimulated transmembrane signaling of wild-type insulin (alpha beta INS.WT) or IGF-I (alpha beta IGF.WT) half-receptors assembled with a kinase-defective insulin half-receptor mutant (alpha beta INS.A/K). In vitro assembly of either (alpha beta)IGF.WT/(alpha beta)INS.A/K or (alpha beta)INS.WT/(alpha beta)INS.A/K hybrid receptors resulted in decreased substrate protein kinase activity. The degree of protein kinase inactivation directly correlated with the amount of immunologically cross-reactive hybrid receptors formed. In contrast to substrate kinase activity, insulin-stimulated autophosphorylation of the (alpha beta)INS.WT/(alpha beta)INS.A/K hybrid receptor complex was completely unaffected in comparison to the wild-type (alpha beta)INS.WT/(alpha beta)INS.WT receptor. To assess a molecular basis for this difference, autophosphorylation of a hybrid receptor composed of a truncated beta-subunit insulin half-receptor with the kinase-defective half-receptor, (alpha beta)INS. delta CT/(alpha beta)INS.A/K, demonstrated the exclusive autophosphorylation of the (alpha beta)INS.A/K half-receptor beta subunit. These results demonstrate that ligand-dependent substrate phosphorylation by insulin and IGF-I holoreceptors requires interactions between two functional beta subunits within the alpha 2 beta 2 heterotetrameric complex and occurs through an intramolecular trans-phosphorylation reaction.

Cell Membrane↗

PubMed1991

Different ligand affinities of the two human insulin receptor splice variants are reflected in parallel changes in sensitivity for insulin action.

The human insulin receptor (hIR) is expressed in two variant forms that are generated by tissue-specific alternative splicing of the 11th exon of the IR gene. This leads to receptors that differ in their affinities for insulin based on the absence (hIR-A) or presence (hIR-B) of a 12-amino acid insert near the C-terminus of the alpha-subunit. To explore further the functional significance of the difference in these receptor subtypes, the properties of hIR-A(exon 11-) and hIR-B(exon 11+) receptors have been compared in parallel. Despite their different affinities for insulin, the receptor variants retain equivalent acid sensitivity for insulin binding and bind proinsulin with the same relative affinity. Both hIR-A and hIR-B are able to signal a variety of insulin's actions, but the insulin dose-response curves for receptor autophosphorylation and for mitogenesis and glycogen synthase stimulation in cells are shifted to the right for hIR-B receptors compared to hIR-A receptors. The magnitude of these rightward shifts, 1.5- to 3-fold in the assays listed above, are similar to and presumably accounted for by the 2-fold difference in insulin affinity exhibited by the receptor variants. Occupied hIR-A and hIR-B receptors undergo indistinguishable endocytotic itineraries after insulin binding. Both lead to insulin degradation that is quantitatively and kinetically similar, and both down-regulate when exposed to saturating insulin for 24 h. Thus, the functional consequences of the alternative splicing of IRs are limited to those related to the variants' differing affinities for insulin.

Animals↗

PubMed1991

Insulin-receptor cDNA sequence in NIDDM patient homozygous for insulin-receptor gene RFLP.

Resistance to insulin action is a well-established feature of non-insulin-dependent diabetes mellitus (NIDDM) and is believed to contribute to the etiology of this condition. A strong genetic contribution to the etiology of NIDDM exists, and we previously identified an insulin-receptor gene restriction-fragment-length polymorphism (RFLP) associated with the NIDDM phenotype. In an attempt to elucidate whether structural defects in the insulin receptor could be a primary cause of insulin resistance in NIDDM, we analyzed the insulin-receptor cDNA sequence in a subject with NIDDM who is also homozygous for this RFLP. The insulin-receptor cDNA was sequenced with the polymerase chain reaction (PCR). mRNA from transformed lymphocytes was reverse transcribed and amplified with five overlapping sets of primers that span the coding sequence of both alpha- and beta-subunits. No difference was found in the predicted amino acid sequence of the subject's insulin receptor compared with the normal insulin receptor. At nucleotide positions 831 and 2247, the subject is heterozygous for silent nucleotide polymorphisms that do not affect the amino acid sequence. Exon 11 encodes a 12-amino acid insert in the alpha-subunit, which, due to alternate splicing, is not expressed in lymphocyte insulin-receptor mRNA. Consequently, exon 11 was amplified from genomic DNA by PCR; the sequence of exon 11 was found to be normal. In addition, when this patient's transformed lymphocytes were maintained in culture, no abnormalities in insulin binding were observed. We conclude that the insulin resistance seen in this NIDDM subject is not due to a structural alteration in the insulin receptor itself.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1990

Endocytosis of insulin receptors is not required for activation or deactivation of the hormone response.

To examine the role of endocytosis in insulin action, hormone responsiveness was studied in transfected Rat 1 fibroblasts stably expressing a noninternalizing insulin receptor. The latter receptor (hIR delta ex16) was engineered by deleting the immediately submembranous 22 amino acids encoded by the 16th exon of the human insulin receptor and has previously been shown not to internalize despite having normal insulin-stimulated tyrosine kinase activity. It is shown in the present study that hIR delta ex16 receptors do mediate insulin action. Insulin dose-response curves both for activation of glycogen synthetase and for mitogenic stimulation demonstrate greater insulin sensitivity in hIR delta ex16 cells compared with untransfected Rat 1 cells. In addition, increases in the absolute levels of glycogen synthetase activity are seen in the hIR delta ex16 cells. Species-specific agonistic antibodies to the insulin receptor also stimulate hIR delta ex16 cells, confirming the activity of the mutant receptor. The non-internalizing receptors are rapidly dephosphorylated after removal of insulin, and the activation of glycogen synthetase decays no more slowly in hIR delta ex16 cells than in cells expressing wild-type receptors. The results demonstrate that receptor endocytosis is not necessary for activation or deactivation of the insulin response.

Animals↗

PubMed1990

The ligand binding characteristics of a kinase-defective A/K1018 human insulin receptor expressed in Rat 1 fibroblasts.

Expression of the cDNA encoding a human insulin receptor with replacement of alanine for lysine at residue 1018 in the ATP binding domain of the beta subunit results in a receptor that is not only kinase-defective, but also biologically inactive. Interestingly, this mutated receptor shows a decreased insulin binding affinity when expressed at high level. We, therefore, studied the binding property of this mutant receptor expressed in Rat 1 fibroblasts. The association rate (Ka) of insulin to the mutant receptor was comparable to normal, but the dissociation rate (Kd) was twice as fast. Furthermore, the Kd of the mutant receptor was also more sensitive to changes in pH, accelerating more rapidly with pH changes than did the Kd of normal receptors. Despite this difference, the mutant receptor still exhibited negative cooperativity. These results indicate that the loss of tyrosine kinase activity of the beta subunit of the insulin receptor leads to alteration of the ligand binding affinity of the alpha subunit.

Animals↗

PubMed1990

A domain of the insulin receptor required for endocytosis in rat fibroblasts.

To study the mechanism and role of ligand-dependent endocytosis, we have engineered a mutant insulin receptor that retains its insulin binding and insulin-stimulated tyrosine kinase activities but does not exhibit ligand-induced internalization. The mutant has a deletion of the 16th exon which encodes 22 amino acids (residues 944-965) on the cytoplasmic side of the transmembrane region of the receptor beta-subunit. When the cDNA is transfected in Rat 1 cells, the mutant receptor (HIR delta ex16) is processed to a glycosylated alpha 2 beta 2 heterotetramer and expressed at the cell surface. HIR delta ex16 receptors bind insulin with lower affinity than normal receptors (ED50 for insulin competition = 1.1 nM compared with 0.2 nM for normal receptors), but binding is normal in detergent solution. The mutant HIR delta ex16 receptor undergoes insulin-dependent autophosphorylation and activation as a tyrosine kinase toward exogenous substrates in vitro. In vivo, the receptor is also enzymatically active, as assessed 1) by the ability of antiphosphotyrosine antibodies to precipitate equivalent proportions (58-60%) of occupied wild type or mutant receptors and 2) by immunoblotting extracts of insulin-stimulated cells using antiphosphotyrosine antibodies. In the latter experiment, cells expressing HIR delta ex16 receptors exhibit tyrosine phosphorylation of insulin receptor beta-subunits as well as of pp 185, a putative substrate of the receptor. Despite the ability to bind insulin and activate as a tyrosine kinase, HIR delta ex16 receptors do not internalize in Rat 1 cells. Whereas normal surface receptors covalently labeled with the photoaffinity reagent 125I-NAPA-DP insulin are 36% intracellular after 1 h at 37 degrees C, only background levels of internalization are seen when HIR delta ex16 receptors are labeled. The HIR delta ex16 receptors mediate no internalization or degradation of 125I-insulin compared with control untransfected Rat 1 cells, and they do not down-regulate after long exposure to saturating concentrations of insulin. We conclude that the 16th exon encodes a domain necessary for ligand-dependent endocytosis.

Base Sequence↗

PubMed1990

Characterization of an insulin receptor mutant lacking the subunit processing site.

An insulin receptor mutant was constructed utilizing site-directed mutagenesis to delete the Arg-Lys-Arg-Arg basic amino acid cleavage site (positions 720-723) from the cDNA encoding the human insulin proreceptor. This mutant was transfected into Chinese hamster ovary cells. Immunoprecipitation of metabolically labeled cells revealed a 205-kDa proreceptor which bound to wheat germ agglutinin. Processed 130-kDa alpha and 95-kDa beta subunits were also observed and contained approximately 20% as much protein as the proreceptor on a molar basis. Trypsin digestion of intact metabolically labeled cells decreased the proreceptor band by 80%. Pulse-chase studies revealed a half-life of 28 h for the proreceptor. When cells were photolabeled with 125I-B2(2-nitro-4-azidophenylacetyl)-des-PheB1 (NAPA)-insulin, the proreceptor incorporated 10% as much label as the 130-kDa alpha subunit in spite of a 5-fold molar excess. Incubation of NAPA-labeled cells at 37 degrees C for 20 min resulted in 60% of the labeled subunits, but little labeled proreceptor, becoming resistant to trypsin degradation. Immunoprecipitation of NAPA-insulin-stimulated cells with anti-phosphotyrosine antibodies revealed that 62% of the processed labeled receptors, but very little proreceptor, contained phosphotyrosine. Thus, this mutant receptor is synthesized, glycosylated, and expressed on the cell surface as uncleaved proreceptor, although some processing to alpha and beta subunits still occurs. It exhibits a markedly decreased affinity for insulin, and when insulin is bound to, demonstrates defective internalization, down-regulation, and autophosphorylation. These data suggest that cleavage of the mutant proreceptor into subunits is required not only for the development of high affinity binding sites, but also for normal transduction of the signal which activates the beta subunit tyrosine kinase.

Affinity Labels↗

PubMed1990

Dissection of the growth versus metabolic effects of insulin and insulin-like growth factor-I in transfected cells expressing kinase-defective human insulin receptors.

We have recently reported that the expression of an in vitro mutated, kinase-defective insulin receptor (A/K1018) leads to cellular insulin resistance when expressed in Rat 1 fibroblasts. That is, despite the presence of normal numbers of activatable native insulin receptors in the host cell, the A/K1018 receptors prevent the normal receptors from phosphorylating endogenous substrates and from signalling insulin action, perhaps by competing for limiting amounts of these substrates. We report here that insulin-like growth factor I-stimulated phosphorylation of two endogenous substrate proteins, pp220 and pp170, is also inhibited in cells expressing A/K1018 receptors. Because insulin-like growth factor I stimulation of glucose uptake is not inhibited in cells with A/K1018 receptors while pp220 and pp170 phosphorylation is inhibited, it is unlikely that either pp220 or pp170 are involved in mediating the stimulation of glucose transport. In contrast, insulin-like growth factor I-mediated stimulation of mitogenesis is inhibited in cells with A/K1018 receptors. Thus, pp170 or pp220 could be involved in mitogenic signalling. We also report that both H2O2 and tetradecanoylphorbolacetate stimulate glucose transport normally in cells with A/K1018 receptors. Phorbol esters also lead to the phosphorylation of both normal and A/K1018 receptors on serine and/or threonine. This argues that phorbol esters or H2O2 bypass the normal proximal steps in signalling insulin action.

Animals↗

PubMed1990

Functionally distinct insulin receptors generated by tissue-specific alternative splicing.

Cloning of the insulin receptor cDNA has earlier revealed the existence of two alternative forms of the receptor differing by the presence or absence of 12 amino acids near the C-terminus of the receptor alpha-subunit. This insert has been shown by others to be encoded by a discrete exon, and alternative splicing of this exon leads to tissue-specific expression of two receptor isoforms. We have studied the functional significance of the receptor isoforms and have confirmed that they are generated by alternative splicing. When cDNAs encoding the two forms of the insulin receptors are expressed in Rat 1 cells, the receptor lacking the insert (HIR-A) has a significantly higher affinity for insulin than the receptor with the insert (HIR-B). This difference in affinity is maintained when insulin binding activity is assayed in solution using detergent solubilized, partially purified receptors. These data, combined with the tissue specificity of HIR-A and HIR-B expression, suggest that alternative splicing may result in the modulation of insulin metabolism or responsiveness by different tissues.

Amino Acid Sequence↗

PubMed1990

Insulin action in cells expressing truncated or kinase-defective insulin receptors. Dissection of multiple hormone-signaling pathways.

An attempt has been made to dissect insulin-signaling pathways by means of in vitro mutagenesis of the insulin receptor. Studies of two mutants are reviewed: a COOH-truncated receptor and a mutant of the ATP-binding site that is tyrosine kinase defective. The COOH-truncated receptor has normal insulin binding and normal endocytosis and undergoes autophosphorylation and activation as a kinase in vitro and in vivo. Loss of the COOH-terminal 43 amino acids has, however, impaired the ability of the receptor to signal metabolic events while augmenting its ability to signal mitogenesis. Thus, domains have been partially identified that are separately involved in metabolic and mitogenic signaling, facilitating future studies of these pathways. The kinase-defective receptor is not only biologically inactive, but has a dominant inhibitory effect on the endogenous insulin receptors in the cell. That is, transfected Rat-1 cells carrying defective human receptors still have normally active rat insulin receptors but are incapable of signaling insulin action. The fact that metabolic signaling by insulinlike growth factor I (IGF-I) is intact in these cells allows definition of separate insulin and IGF-I pathways, leading to stimulation of glucose transport. Analysis of dose responsiveness for mitogenesis implies that mitogenic signaling pathways are shared by insulin and IGF-I, however. The utility of these cell lines in defining these pathways is discussed.

Animals↗

PubMed1989

Augmented mitogenesis and impaired metabolic signaling mediated by a truncated insulin receptor.

Recently, we have described a COOH-terminal deletion mutation of the human insulin receptor (HIR delta CT) that exhibits normal insulin-mediated kinase activity and endocytosis, but is inefficient in stimulating glucose transport and glycogen synthase (McClain, D. A., Maegawa, H., Levy, J., Huecksteadt, T., Dull, T. J., Lee, J., Ullrich, A., and Olefsky, J.M. (1988) J. Biol. Chem. 263, 8904-8911; Maegawa, H., McClain, D. A., Freidenberg, G., Olefsky, J. M., Napier, M., Lipari, T., Dull, T. J., Lee, J., and Ullrich, A. (1988) J. Biol. Chem. 263, 8912-8917). In this paper, we report that despite this defect in metabolic signaling, the truncated receptor exhibits augmented mitogenic activity compared to normal receptors. These results were verified in three independently isolated clones of Rat 1 fibroblasts transfected with the HIR delta CT cDNA. The increase in insulin sensitivity of mitogenic stimulation was proportional to the number of HIR delta CT receptors expressed on the cells. By contrast, only the cells with normal receptors and none of the HIR delta CT clones exhibit increased sensitivity for a metabolic action of insulin, the stimulation of glucose uptake. Stimulation of cells by other mitogens and autoradiographic analysis confirm that the enhanced mitogenic effects seen in HIR delta CT cells are attributable only to the presence of the truncated insulin receptors. These receptors mediate the tyrosine phosphorylation of a number of cellular proteins, and the pattern of these phosphorylations differs quantitatively from that seen in cells with normal receptors. We conclude: 1) The COOH terminus plays a role in signaling metabolic actions of insulin, perhaps through its recognition of substrates for the receptor kinase. 2) By contrast, the COOH terminus is an inhibitory regulator of mitogenesis, and removal of the terminal 43 amino acids converts the receptor from a moderately active growth signaler to a very active one. 3) The changes seen in biologic activities of the HIR delta CT receptor are associated with quantitative changes in substrate phosphorylation by the receptor kinase.

Animals↗

PubMed1989

Effects of growth and insulin treatment on the levels of insulin receptors and their mRNA in Hep G2 cells.

We have studied the variations in the number of insulin receptor and insulin receptor mRNA levels in (Hep G2) cells in response to growth and insulin treatment. The levels of insulin receptors are relatively low in growing cells. After approximately 5 days in culture, if cells are not refed they cease to divide and the number of receptors/cell increases, reaching 4 times the initial values by the 9th day. Refeeding the cells completely prevented both growth arrest and the increase in insulin receptor number. Insulin added daily to cells at 0.33 microM caused receptor down-regulation but did not prevent a 3-fold increase in binding with growth arrest. Pulse-chase studies of metabolically labeled ([35S]methionine) cells showed that the receptor degradation rate (apparent t 1/2, 18-20 h) was comparable in rapidly growing versus growth-arrested cells. The increased receptor level in non-refed cells is not due to generation of a soluble factor by confluent cells, nor is it caused by depletion of insulin, glucose, or insulin-like growth factor I from the culture medium. The levels of insulin receptor mRNA measured on Northern blots increased in growth-arrested cells in parallel to the increase in receptor number. The mRNA value begins to increase from the 3rd day in culture and by the 9th day reaches a level 6.0 times that on the 3rd day. Chronic insulin-induced receptor down-regulation did not alter insulin receptor mRNA levels at any time point studied. These data demonstrate that the increase in insulin receptor number/cell in growth-arrested cells is paralleled by an increase in insulin receptor mRNA content with no change in the receptor degradation rates. This suggests that the increase in the number of insulin receptors is due to enhanced receptor synthesis due to increased receptor mRNA content. Conversely, down-regulation of the insulin receptor does not affect the level of insulin receptor mRNA and thus must be due to increased receptor degradation.

Blotting, Northern↗

PubMed1988

Insulin receptors with defective tyrosine kinase inhibit normal receptor function at the level of substrate phosphorylation.

Rat 1 fibroblasts have been transfected with the cDNA encoding a kinase-defective mutant human insulin receptor (A/K1018). Expression of this cDNA results in a receptor that is not only biologically inactive but also inhibits normal insulin action through the normal endogenous rat receptors in this fibroblast line (McClain, D. A., Maegawa, H., Lee, J., Dull, T. J., Ullrich, A., and Olefsky, J. M. (1987) J. Biol. Chem. 262, 14663-14671). We have investigated the mechanism of this inhibition and show that: 1) rat receptors are expressed at normal to increased levels in two cell lines which also express A/K1018 receptors at low (A/K1018-A, 5700 total receptors) or high (A/K1018-B, 2.2 x 10(5) total receptors) levels. 2) The rat receptors in the A/K1018 lines can be normally autophosphorylated under the control of insulin in vitro. 3) A/K1018 receptors do not inhibit the kinase activity of normal receptors when mixed together in vitro. 4) In intact A/K1018-B cells, the ability of insulin to stimulate autophosphorylation of the rat receptor is unimpaired; furthermore, the autophosphorylated rat receptor becomes normally activated as a tyrosine kinase. 5) The expression of receptors for insulin-like growth factor I and stimulation of hexose uptake mediated by this receptor are unaffected in cells expressing inhibitory A/K1018 receptors. 6) Expression of the A/K1018 receptor inhibits insulin-stimulated phosphorylation of two endogenous protein substrates (pp220 and pp170) by the native rat receptors. We conclude that the inhibition of insulin action seen in the A/K1018 cells is not mediated at the levels of native receptor expression or activation, nor is the effector (hexose uptake) mechanism affected by the A/K1018 receptors. The expression of this kinase-defective receptor does, however, inhibit the phosphorylation of substrate molecules by the normally activated endogenous rat receptors.

Animals↗

PubMed1988

Properties of a human insulin receptor with a COOH-terminal truncation. I. Insulin binding, autophosphorylation, and endocytosis.

In order to test the contribution of the insulin receptor COOH terminus to insulin action, a truncation of 43 COOH-terminal amino acids was engineered by cDNA-based deletion mutagenesis. This cDNA (HIR delta CT), as well as cDNA encoding the complete receptor (HIRc) was transfected into Rat 1 fibroblasts. Cells expressing 6.4 X 10(3) and 1.25 X 10(6) normal receptors and 2.5 X 10(5) HIR delta CT receptors, as well as control Rat 1 fibroblasts were selected for further analysis. All cell lines exhibited insulin binding of similar affinity. Partial tryptic digestion and immunoprecipitation by region-specific antibodies verified that the HIR delta CT receptors were truncated at the COOH terminus. Purified HIRc and HIR delta CT receptors underwent autophosphorylation with similar insulin and ATP sensitivity, although the HIR delta CT receptors were slightly more active in the absence of insulin. Transfected HIRc and HIR delta CT receptors undergo endocytosis in a normal fashion. Insulin internalization and degradation in both HIRc and HIR delta CT cells is increased in proportion to receptor number. Intracellular insulin processing, degradation, and release were qualitatively comparable among the transfected cell lines. Complete and truncated receptors internalize, recycle, and down-regulate normally. We conclude the following: 1) the COOH-terminal portion of the insulin receptor is not necessary for partial autophosphorylation or endocytosis; 2) following internalization the intracellular itinerary of the receptor and ligand appear normal with the truncated receptor; and 3) truncation of the COOH terminus does not impair recycling of the receptor or retroendocytosis of internalized ligand.

Animals↗