Cell Biology International (2007) 31, 815-824 - Akm A. Sattar and others - Human insulin receptor juxtamembrane domain independent insulin signaling

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Cell Biology International (2007) 31, 815–824 (Printed in Great Britain)

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Human insulin receptor juxtamembrane domain independent insulin signaling

Akm A. Sattar^*, Chali Berhanu, Surafel Gebreselassie and Paulos Berhanu

Division of Endocrinology, Department of Internal Medicine, Wayne State University School of Medicine, 421 E. Canfield Avenue, Detroit, MI 48201-1928, USA

Abstract

The exon 16-encoded juxtamembrane (JM) domain of human insulin receptor (hIR) harbors the NPEY motif which couples the insulin-activated hIR kinase to downstream signal transduction molecules. We sought to determine if signal transduction requires the entire exon 16-encoded 22-amino acid JM domain. Transfected CHO cells were generated stably expressing either the wild-type hIR (hIR-WT) or two mutant hIRs (hIRΔEx16 in which the JM domain was deleted, and hIRrosJM in which the deleted segment was replaced by the corresponding domain of v-ros protein). The mutant hIRΔEx16 and hIRrosJM exhibited similar insulin-binding as the hIRWT. Insulin internalization and insulin dose-response experiments toward activation of downstream signal transduction molecules demonstrated that: i) the presence of intact hIR-JM domain which harbors the NPEY motif is essential for Shc phosphorylation but not for IRS-1 phosphorylation; ii) insulin signal transduction can occur independent of the JM domain of hIR and without participation of the NPEY motif; iii) engagement of this putative alternative downstream signal transduction is Shc independent and is dependent on insulin concentration; and iv) insulin internalization does not necessarily require the hIR specific aa sequence of the JM domain which can be partially substituted by the JM domain of the v-ros tyrosine kinase.

Keywords: Akt, Insulin signaling, Insulin receptor, Insulin receptor substrate 1, MAP kinase, Tyrosine phosphokinase.

^*Corresponding author. Tel.: +1 313 577 9405; fax: +1 313 577 8615.

1 Introduction

The human insulin receptor (hIR), a transmembrane glycoprotein composed of α and β subunits in a tetrameric α2β2 stoichiometry, mediates the diverse biological actions of insulin (Ebina et al., 1985; Brunetti and Goldfine, 1995; Shao et al., 2000; Dunaif et al., 2001; Bartucci et al., 2001; Reiter and Gardner, 2003). The functional organization of the hIR molecule indicates that it is a modular protein encoded by the 22 exons of the IR gene where exon 16 encodes the juxtamembrane domain of the β-subunit. Insulin binding to the α sub-unit of the IR induces rapid phosphorylation of tyrosine residues in the β-subunit TPK regulatory domain. This event leads to conformational changes in the receptor, activates it as a tyrosine kinase, and facilitates its interaction with down stream signal transduction molecules. The initiation and propagation of this transmembrane signaling cascade plays an essential role in mediating the multiple biological effects of insulin (Liu et al., 2001; Bevan, 2001; Pirola et al., 2004; Zick, 2004; De Meyts, 2005; Taniguchi et al., 2006).

Based on various mutational analysis of hIR β-subunit, there is a general consensus that its immediate cytoplasmic juxtamembrane domain that is encoded by exon 16 is important for endocytic function of the receptor (Berhanu et al., 1991; Kaburagi et al., 1993; Berhanu et al., 1995; Schranz et al., 1996; Najjar et al., 1998). This domain contains two tyrosine residues that exist as GPLY and NPEY motifs (Berhanu et al., 1991, 1995). We have previously shown that the tetrameric amino acid sequence Asn-Pro-Glu-Tyr (NPEY) contained in the JM domain is important but not absolutely essential for coupling of hIR kinase to insulin receptor substrate 1 and p85 or for mediating insulin metabolic and mitogenic effects (Berhanu et al., 1997). But the role of the JM domain apart from the GPLY and NPEY motifs (Berhanu et al., 1991, 1995, 1997) remains undetermined especially for the B (exon 11+) isoform of the IR molecule. The B isoform with the 12 amino acids insertion in the C-terminal of the α-subunit is the predominant form expressed in the major insulin target tissues responsible for glucose homeostasis, i.e. fat, muscle and liver (Kosaki et al., 1995). It is also reported to signal more efficiently in response to insulin binding as compared to the A (exon 11-deleted) isoform (Kosaki et al., 1995).

The amino acid sequence of the hIR tyrosine kinase domain resembles that of tyrosine kinases of epidermal growth factor, insulin-like growth factor, and platelet-derived growth factor as well as member of the tyrosine kinase family of oncogene products such as the avian sarcoma virus UR2 transforming protein p68^gag-ros (v-ros) (Neckameyer and Wang, 1985). Among the known tyrosine kinase family v-ros shares the greatest homology with the insulin receptor. Although, the juxtamembrane domain of the v-ros shares some organizational and functional similarity with that of the hIR-JM with respect to transmembrane signaling it lacks the key tyrosine residues that upon phosphorylation are known to mediate the interaction of the activated hIR kinase with the downstream signal transduction molecules. In the present study we have comparatively examined insulin signal transduction pathways in transfected CHO cells stably expressing the wild type (WT) receptor or mutant receptors where the whole 22-a.a segment of the JM domain is deleted or replaced by the corresponding domain of v-ros sequences (Neckameyer and Wang, 1985) to determine the role of the JM domain in insulin-induced internalization and insulin signal transduction. Our data indicate that the presence of intact hIR-JM domain, which harbors tyrosine motif(s), is essential for insulin-induced Shc phosphorylation but not essential for IRS-1 phosphorylation; insulin signal transduction can occur independent of the JM domain of hIR and without the participation of the NPEY motif. The mechanism underlying of this putative alternative downstream signal transduction is Shc independent and is dependent on insulin concentration. In this study we also show that insulin internalization does not necessarily require the hIR specific sequence of the JM domain, which can be partially substituted by the JM domain of the similar yet heterogeneous v-ros tyrosine kinase.

2 Materials and methods

2.1 Materials

Minimum essential medium, Ham's F-12 medium and Geneticin (G418) were purchased from GIBCO BRL Life Technologies, Inc. Fetal bovine serum (FBS) was purchased from Hyclones (Logan, UT, USA) and insulin-free bovine serum albumin (BSA) purchased from Fluka. Tissue culture laboratory ware was purchased from Falcon. Human biosynthetic insulin was kindly supplied by Eli Lilly and Co. Antibodies to the active Thr202/Tyr204 phosphorylated pp42/pp44 MAPK, and to the Ser473 phosphorylated Akt were purchased from New England BioLabs. Anti phosphotyrosine (αPY20) antibody was purchased from Upstate Biotechnology Inc. Electrophoretic reagents were obtained from Bio-Rad. The chemiluminescence detection reagent kit, [¹²⁵I]insulin (human), monoiodinated at tyrosine A-14 position (2000Ci/mmol), and Na¹²⁵I were from Amersham (Arlington heights, IL, USA). The plasmid pET, which contains the entire hIR coding sequence and the mammalian expression vector pECE, were as described previously (Ellis et al., 1987). All other chemicals were reagent grade and purchased from Sigma.

2.2 Construction of hIR mutant lacking the exon 16 domain and replacement of the JM domain by corresponding domain of the oncogen product v-ros

First, the entire coding region of hIR was excised from the bacterial propagation plasmid pET and ligated into the mammalian expression vector pECE as described previously (Berhanu et al., 1990). The resultant plasmid, pECE-hIR, codes for wild type hIR under the transcriptional control of the SV40 early promoter (Berhanu et al., 1991). Deletion of the nucleotides encoding the exon 16 domain was performed by oligonucleotide directed mutagenesis using the Altered Sites Mutagenesis System (Promega) and standard molecular biology techniques. Briefly, the hIR cDNA fragment was cloned into pSelect ampicillin-sensitive phagemid vector (Promega) and transfected into E. coli JM109 cells. Subsequently, these cells were infected with a helper phage, R408, to generate a single-stranded DNA template. A 30nt mutagenic oligonucleotide (5′-AGAGCATGGAAACACCCTCTTTCTCAGGAA-3′) composed of the complimentary 15 nucleotides from both side of the exon 16 was used to prime the synthesis of the opposite strand of the single-stranded template with DNA polymerase. Subsequent replication of the mutant strand according to the Altered Sites protocol resulted in looping out and deletion of the exon 16 coding amino acids as described previously (Berhanu et al., 1995). The newly constructed plasmid lacking the exon 16 region, hIRΔExon16, was confirmed by restriction digestion and sequence analysis. The entire coding region was excised from pSelect and ligated back into pECE.

To replace the exon 16 sequence of wild-type hIR with the corresponding v-ros sequence, we used the hIRΔExon16 in pECE as starting template. The following sense (5′-AGGACAGATTGTTGCTTGTGAAGGAGGATAAGGAGCTTGCTCAGGTGTTTCCATGCTCTGTGTA-3′) and antisense (5′-TCACAAGCACAATCTGTCCTGTAGAAGCCGGCTTT CTAGACTTCCTCTTTCTCAGGAATAGAT-3′) primers, each having 43 bases of v-ros JM sequence and 20 bases complementary to hIRΔExon16 plasmid, were used to generate a DNA fragment containing the 22 amino acids of v-rosJM replacing exon 16 of hIR. This was achieved by carrying out two sequential PCR reactions utilizing additional sense (5′-GACGTCCCGTCAAATATTGC-3′ and antisense (5′-GATCTTCTCTCGAGACACCT-3′) amplimers containing SspI and XhoI sites (underlined) coding for sequences located just upstream and downstream of exon 16, respectively. The final PCR product was digested with SspI and XhoI and religated between the SspI and XhoI sites of the hIRΔExon16 in pECE. The correct sequence of the resultant plasmid, designated hIRrosJM, was verified by appropriate restriction enzyme digestion and dideoxy sequence analysis.

2.3 Transfection and stable expression of wild type and mutant receptors

The generation and characterization of the stably transformed Chinese hamster ovary cell lines expressing wild type and mutant hIR were performed according to previously described procedures (Ellis et al., 1987). Each of these cell lines stably express >10⁵ IRs/cell in comparison with the untransformed CHO cells, which express 2800 rodent IRs/Cell. Briefly, CHO cells (&007E;10⁶cells/100-mm plate) maintained in F-12/10% fetal calf serum culture medium were cotransfected as previously described (Berhanu et al., 1990) with 2μg of pSV2 neo plasmid DNA and either 10μg of pECE-hIR or pECE-hIR Δ exon 16 or hybrid hIR-rosJM plasmid DNA by the calcium phosphate precipitation method with the addition of a glycerol shock step (20% glycerol in F-12 medium) after 4h. After 36h, the cells were split 1:20 and allowed to grow in the presence of 400μg/ml of the neomycin analog G418. Ten to 14days later, individual G418-resistant colonies were harvested with trypsin/EDTA and replated in triplicate in 24-well culture plates. After confluence, cells were analyzed for insulin binding, and the cell populations giving high levels of binding were subcloned by limiting dilution to obtain several pure cell lines each with a similar high level (>10⁵ IRs/cell) expression of the wild-type and mutant hIR species. Cells transfected with pSV2 neo alone were also processed in parallel as controls.

2.4 Cell culture

Cell lines were maintained in Ham's F12 media supplemented with 10% (v/v) FCS, 2mM glutamine, and 400μg/ml G418. Cells were split every five days and all experiments were conducted with cells under passage 15.

2.5 Insulin binding assay

Insulin binding to whole cells was quantified using A-14 monoiodinated 125I-insulin as described previously (Berhanu et al., 1991). 2×10⁶cells in 35×10mm dishes expressing wild-type or mutant hIR were exposed to 0.02pM 125I-insulin in 1ml binding buffer containing MEM, 10mM Hepes, and 10mg/ml BSA, pH 7.4 for 2h at 4°C with gentle shaking. The cells were rinsed with ice-cold PBS for 3 times and then lysed with 1ml of 1% SDS and specific binding of 125I-insulin was determined by gamma counting and expressed as the percent of the initial 125I-insulin added.

2.6 [¹²⁵I]Insulin internalization

To assess synchronous internalization of insulin prebound to cell surface IR, cell monolayers of the WT and mutant (hIRΔEx16 and hIRrosJM) receptors in 35×10mm dishes were first incubated with 0.02nM 125I-insulin for 2h at 4°C. Replicate dishes of cells were then washed and solubilized to measure initial surface bound insulin. To the remaining dishes of washed cells, binding buffer prewarmed to 37°C was added and the cells further incubated at 37°C. At selected time points (0–120min), the media were removed and the cell monolayers were then incubated in ice-cold pH 3.0 buffer to remove 125I-insulin that was still surface bound. The acid-resistant (internalized) pool of 125I-insulin was then quantified following solubilization of the cells and measurement of the associated radioactivity (Berhanu et al., 1991). 125I-insulin internalization velocity was measured at 2min intervals for 10min and the specific internalization rate constant (Ke) was determined by using a previously described initial rate determination method (Lund et al., 1990).

2.7 Serum-starvation and insulin stimulation

The CHO cell lines described were cultured in 100mm tissue culture dishes until 70% confluency, and then were washed twice with ESPG (150mM NaCl, 0.5mM dextrose and 20mM KPO4). Cells were then incubated in serum-free F12 media containing 0.1% insulin-free BSA for 48h with a serum-free media change 16h prior to insulin treatment. This step was necessary to reduce basal IRS-1, MAPK, Akt phosphorylations. For dose response studies, insulin, in serum-free media, was added to the cells for 5min at 37°C, at final concentrations of 0.05, 0.1, 1, 5, 10 or 100nM. Media lacking insulin (0nM) served as a vehicle control. Cells were then placed on ice and quickly washed twice with ice-cold phosphate buffer saline, pH 7.5 (PBS). The cells were then lysed in 4°C solubilizing buffer containing 1% Triton X-100, 50mM HEPES (pH 7.6), 150mM NaCl, 1mM EDTA, 1mM NaF, 10μg/ml aprotinin, 1mM NaVO4, 10% glycerol. Lysates were centrifuged at 5000×g for 2min and clarified supernatants were stored at −20°C until determination of protein concentration and western immunoblot analysis.

2.8 Immunodetection of phosphorylated Shc

The supernatant (200μg of total protein equivalent) collected from the control and insulin-stimulated cells expressing the hIRWT, hIRrosJM or hIRΔEx16 receptors (and control CHONeo cells) were incubated separately with 5μg anti-Shc antibody for 2h at 4°C, followed by addition of 20μl of protein A agarose suspension, and further incubated for overnight at 4°C. The immunoabsorbed complexes were collected by centrifugation at 3000×g for 3min, and the proteins in the pellet were released by heating in SDS-polyacrylamide gel electrophoresis sample buffer and resolved in 10% SDS-polyacrylamide gel. The proteins were transferred to nitrocellulose membrane and immunoblotted with anti-phosphotyrosine antibody (PY20). Labeled bands were detected using anti-mouse IgG conjugated with horseradish peroxidase and the enhanced chemiluminescence kit according to manufacturer's (Amersham Corp.) instructions. All blots were developed and exposed to film under identical conditions. Autoradiograms were scanned and densitometric data obtained using a Kodak Digital Science ID. Immunoreactivity of the phosphorylated Shc detected in Western blots were expressed and compared as relative band intensity of that from the resting vs. insulin stimulated CHO cells.

2.9 Immunodetection of pIRS-1, pp42/pp44 MAPK and pAkt

Samples from control and insulin-treated cells were diluted with 5x sample buffer (0.5M Tris–HCl, pH 6.8, 100mM DTT, 8.5% SDS, 27.5% sucrose and 0.03% bromophenol blue), heated to 100°C for 3min and equal amounts of protein (20μg) resolved by 10% SDS-PAGE. Proteins were transferred to nitrocellulose membrane and the phosphorylated, active forms of p42/p44 MAPK (p-p42/p-p44 MAPK or Ser 473-phosphorylated Akt (p-Akt) immunodetected using the specific antibodies described previously. All primary antibodies were diluted 1:1000. The secondary antibody (goat anti-rabbit IgG, horseradish peroxidase-conjugated) was diluted 1:10,000. The phosphorylated IRS-1 (pIRS-1) was detected by matching apparent molecular weights and subsequently using anti-phosphotyrosine antibody (PY20) and anti-mouse IgG conjugated with horseradish peroxidase. The same blot was also immunoblotted with anti-IRS-1 antibody to confirm the presence of IRS-1 at the same electrophoretic sites as p-IRS-1. The immunoblots were treated with chemiluminescence reagents and exposed to Amersham's hyper-film. All blots were developed and exposed to film under identical conditions. Autoradiograms were scanned and densitometric data obtained using an AlphaImager. Immunoreactivity of the protein of interest detected in Western blots were expressed and compared as relative band intensity of that from the resting vs. insulin stimulated CHO cells.

2.10 Statistical analysis

Data are expressed as means±SEM. The p-values were calculated by Student's t-tests and a p-value of <0.05 was considered statistically significant.

3 Results

3.1 Insulin binding and internalization

Earlier the hIR lacking juxtamembranous NPEY sequence (hIRΔNPEY) was shown to mediate insulin signaling through an alternate mitogenic signaling pathway that is independent of Shc phosphorylation (Berhanu et al., 1997), we were interested in determining what role, if any, the entire JM domain had in the intact insulin receptor where multiple sorting signals are present. In this study, we utilized transfected CHO cell lines stably expressing either the wild-type (hIRWT) or two mutant hIRs: one in which the JM domain 22 aa were deleted (hIRΔEx16), and the other in which the exon 16 domain was replaced by the corresponding domain of v-ros (hIRrosJM), an oncogen product having significant structural and functional similarities with the hIR kinase, but whose JM contains neither the NPEY motif nor any tyrosine residues (Fig. 1A). Our first goal was to investigate the possible gross misfolding of the hIR due to deletion or chimeric mutation of the JM domain of hIR. Thus, we determined insulin binding capability of the cells harboring the mutant constructs and compared to that with the wild-type. Cells expressing either wild-type hIRWT receptor or the chimeric receptor hIRrosJM or the deletion mutant receptor hIRΔEx16 similarly bound &007E;60–65% of the initial added insulin (Fig. 1B).

Fig. 1

CHO cells expressing wild-type and JM domain mutant hIR constructs bind insulin with apparent similarity. (A) Schematic diagram shows the location of the structural/functional domains of the β-subunit segment and the amino acid sequence of the JM domain of the wild-type human insulin receptor (hIR.WT), exon 16 deletion (-JM) mutant (hIRΔEx16), and the JM chimeric receptor (hIR.RosJM). The tyrosine residues in exon 16 of hIRWT are shown in bold-type and please note that no corresponding tyrosine residues in vRosJM domain. (B) 125I-insulin binding in hIRWT, hIRΔEx16, and hIRRosJM expressing CHO cells at 4°C in the presence of 0.02pM insulin. The data is expressed as the percent bound of initial added 125I-insulin. Values are the mean±SEM from three experiments.

Internalization of a single cohort of prebound 125I-insulin molecules were next compared in cells expressing hIRWT, hIRrosJM and hIRΔEx16 (Fig. 2). The hIRΔEx16 and hIRrosJM receptors exhibited &007E;80% and 50% inhibition of insulin internalization, respectively, as assessed by measurement of single cohort internalization of the prebound insulin at 37°C (Fig. 2A) and by determination of the specific internalization rate constant (Fig. 2B) using the initial rate method (Lund et al., 1990).

Fig. 2

Internalization of cell -surface bound insulin. (A) CHO cells expressing either wild-type or mutant insulin receptors were first incubated for 2h at 4°C with 0.02nM of 125I-insulin to allow the binding equilibration on the cell surface. After this, groups of cells were washed and immediately solubilized for measurement of the amount of initial surface-bound insulin. The remainder of the cells were washed and incubated at 37°C, and at the times shown the accumulation of internalized (acid non-dissociable) insulin in the cells were measured and expressed as the percent of the initial surface bound insulin. Internalized 125I-insulin is plotted as a function of time for CHO cells expressing either wild-type (WT), chimeric (RosJM), or the exon 16 deleted mutant (ΔEx 16) insulin receptors. The data represent the mean±SEM from three experiments. (B) Rate constant for insulin receptor internalization (ke) in each cell line was calculated as described under Section 2. Values are the mean±SEM from three experiments. * P<0.05 and **P<0.005 compared with control (WT).

3.2 Insulin stimulation of IRS-1 phosphorylation

We examined the effects of the deletion of the entire Exon16 that harbors NPEY⁹⁷² motif and the chimeric hIRrosJM (lacking NPEY motif or tyrosine residues) on insulin stimulated IRS-1 phosphorylation. The amount of IRS-1 phosphorylation increased in an insulin dose-dependent manner as shown by immunoblotting (Fig. 3A) and quantified by densitometric scanning (Fig. 3B). Insulin stimulated p185 (IRS-1) phosphorylation occurred to similar extent in hIRWT and hIRΔEx16 mutant. Apparently, there was more p185 (IRS-1) phosphorylation in the chimeric hIRrosJM than the hIRWT cells. However, there was also more of the basal level p185 (IRS-1) phosphorylation in hIRrosJM than that of the hIRWT. This relatively higher IRS-1 phosphorylation trend in chimeric hIRrosJM was still obvious even after normalization with that of the hIRWT (data not shown). Thus, endogenous substrate p185 (IRS-1) phosphorylation in response to insulin stimulation was similar in magnitude in both the hIRWT and the mutant hIREx16 cells, except the chimeric hIRros cells. The control Neo cells exhibited minimal insulin stimulation of IRS-1 phosphorylation. Both the JM deletion mutant hIREx16 and the JM chimeric mutant hIRrosJM were as effective as hIRWT receptor in IRS-1 phosphorylation may suggest that the hIR JM domain specific amino acid sequence that harbors tyrosine motif(s) may not play a critical role for IRS-1 phosphorylation.

Fig. 3

Insulin-stimulated phosphorylation of IRS-1. (A) Dose dependent insulin stimulated phosphorylation of IRS-1 from CHO cells expressing wild-type or mutant insulin receptors. The cells were serum-starved and incubated with the indicated concentrations of insulin for 5min at 37°C. The cells were then solubilized and the extracts were subjected to SDS-PAGE followed by electroblotting onto a nitrocellulose membrane and were analyzed by immunoblotting with anti-phosphotyrosine antibody and by stripping and reimmunoblotting with anti-IRS-1 antibody. The arrowhead indicates phosphorylated IRS-1 band. The Western blot shown is representative of three similar experiments. (B) Immunoreactive p-IRS-1 detected in the Western blots were quantitated by densitometric scanning and were expressed and compared as relative band intensities as a function of indicated concentrations of insulin used to stimulate in each cell type. Values are the mean±SEM from three experiments at each insulin dose.

3.3 Insulin stimulation of Shc phosphorylation

Having found that the juxtamembrane domain of human insulin receptor not playing a critical role for IRS-1 phosphorylation, we investigated the effects of the hIR JM deletion and chimeric mutation on Shc phosphorylation, as shown by immunoblotting (Fig. 4A) and quantifying by densitometric scanning (Fig. 4B) insulin stimulated phosphorylation of the 52-kDa Shc isoform in cells expressing hIRWT. Phosphorylation of 52-kDa Shc in cells expressing hIRWT was also insulin concentration dependent (data not shown). Neither the hIREx16 mutant nor the hIRrosJM mutant showed insulin-stimulated Shc phosphorylation and exhibited essentially similar pattern like the control cells harboring Neo (Fig. 4). Both the JM deletion mutant hIREx16 and the JM chimeric mutant hIR.rosJM were defective in Shc phosphorylation may suggest that the hIR JM domain specific amino acid sequence that harbors tyrosine motif(s) play a role for Shc phosphorylation.

Fig. 4

Insulin-stimulated tyrosine phosphorylation of Shc. (A) CHO cells expressing either wild-type or mutant insulin receptors were serum-starved and incubated without (−) or with (+) 100nM insulin for 5min at 37°C. The cells were then solublized and the lysates were subjected to immunoprecipitation with anti-Shc antibody followed by electrophoresis and Western blot analysis using anti-phosphotyrosine antibody. (B) Immunoreactive p-Shc bands detected in the Western blots were scanned and compared as relative band intensities of that from the resting (−) vs. insulin stimulated (+) CHO cells. Values are the mean±SEM of three experiments.

3.4 Insulin stimulation of Akt phosphorylation

Our observation that hIR JM domain specific amino acid sequence that harbors tyrosine motif(s) apparently does not play a critical role for IRS-1 phosphorylation, led us to investigate further if the hIR JM domain deletion mutant and chimeric mutant would have the similar effects on insulin-stimulated Akt phosphorylation. Akt phosphorylation increased in an insulin dose-dependent manner as shown by immunoblotting (Fig. 5A) and quantified by densitometric scanning (Fig. 5B). At higher concentrations of insulin ≥5nM, phosphorylation of Akt was similar in both the WT and the mutant receptors. On the contrary, at lower concentrations of insulin <5nM, the extent of Akt phosphorylation was different in the WT and the mutant receptors. As shown in Fig. 5, besides, the negative control Neo, both the JM deletion mutant hIRΔEx16 and the JM chimeric mutant hIRRosJM were less responsive than the hIRWT to the insulin (<5nM) -stimulation of Akt phosphorylation. Although, the least responsive mutant hIRΔEx16 still exhibited 50–60% of the insulin (1nM) -stimulated Akt phosphorylation compared to that of the hIRWT. These results demonstrate insulin concentration dependent activation of the downstream Akt signaling molecule in the absence of hIRJM domain specific sequence that harbors tyrosine motif(s).

Fig. 5

Insulin-stimulated phosphorylation of Akt. (A) Dose dependent insulin stimulated phosphorylation of Akt from CHO cells expressing wild-type or mutant insulin receptors. The cells were serum-starved and incubated with the indicated concentrations of insulin for 5min at 37°C. The cells were then solubilized and the extracts were subjected to SDS-PAGE followed by electroblotting onto a nitrocellulose membrane and were analyzed by immunoblotting with an anti Ser473-p-Akt antibody. The arrowheads indicate phosphorylated Akt bands. The Western blot shown is representative of three similar experiments. (B) Immunoreactive p-Akt bands detected in the Western blots were scanned for each cell type and compared as relative band intensities as a function of indicated insulin concentrations used to stimulate the cells. Values are the mean±SEM from three experiments at each insulin dose.

3.5 Insulin stimulation of MAP kinase

We investigated to find out how the hIR JM domain deletion mutant and chimeric mutant would have displayed their effects on insulin-stimulated MAPK phosphorylation (Fig. 6). MAPK phosphorylation increased in an insulin dose-dependent manner as shown by immunoblotting (Fig. 6A) and quantified by densitometric scanning for p-p44 MAPK (Fig. 6B) and for p-p42 MAPK (Fig. 6C), respectively. At higher concentrations of insulin ≥5nM, the pattern and the extent of phosphorylation of MAPK (p-p44) were similar in both the WT and the mutant receptors. On the contrary, at lower concentrations of insulin <5nM, the extent of MAPK phosphorylation was different in the WT and the mutant hIRΔEx16 receptor. As shown in Fig. 6, besides, the negative control Neo, both the deletion mutant hIRΔEx16 and the chimeric mutant hIRRosJM were less responsive than the hIRWT to the insulin (<0.5nM) -stimulation of MAPK phosphorylation. Although, the least responsive mutant hIRΔEx16 still exhibited 30–40% of the insulin (1nM) -stimulated MAPK phosphorylation compared to that of the hIRWT. However, the JM deletion mutant hIREx16 was more defective than the JM chimeric mutant hIRrosJM in MAPK phosphorylation might suggest that the hIR JM domain specific amino acid sequence that harbors tyrosine motif(s) could partly contribute to MAPK activation possibly through Shc dependent pathway. Nevertheless, this study demonstrates insulin dose dependent activation of the downstream MAPK in the absence of hIR JM domain specific sequence.

Fig. 6

Insulin-stimulated activation of MAP kinase. (A) Dose dependent insulin stimulated phosphorylation (activation) of MAP kinases (p44 and p42) from CHO cells expressing wild-type or mutant insulin receptors. The cells were serum-starved and incubated with the indicated concentrations of insulin for 5min at 37°C. The cells were then solubilized and the extracts were subjected to SDS-PAGE followed by electroblotting onto a nitrocellulose membrane and were analyzed by immunoblotting with an anti Thr202-p-p42/Tyr204-p-p44 MAPK antibody. The arrowhead indicates either phosphorylated P44 (**) or phosphorylated P42 (*) band. The Western blot shown is representative of three similar experiments. (B) Quantitative profile of immunoreactive p-p44. The immunoblotted p-44 bands were scanned and the relative band intensities were plotted as a function of the insulin concentrations used to stimulate the cells. Values are the mean±SEM from three experiments at each insulin dose. (C) Quantitative profile of immunoreactive p-p42. Values are the mean±SEM from three experiments at each insulin dose.

4 Discussion

Insulin action on target tissues requires insulin receptor. With the exception of the tyrosine regions, functional domains of the insulin receptor are less well characterized, and in particular the regions of the receptor responsible for signaling are incompletely understood. To better understand the role of endocytosis in insulin action, it is necessary to identify the IR domain mediating internalization. Various mutational analysis of the hIR-β subunit by us (Berhanu et al., 1991, 1995, 1997) and by other investigators (Backer et al., 1990, 1992; Kaburagi et al., 1993; Prager et al., 1994; Eck et al., 1996; Najjar et al., 1998) have established that its immediate cytoplasmic juxtamembrane domain is necessary for endocytic signaling. Since tyrosine based motifs have been suggested to provide internalization signals in other membrane proteins (Chen et al., 1990; Bohm et al., 1997; Denzer et al., 1997; Cassard et al., 1998; Banbury et al., 2003), two such motifs in hIR, GPLY⁹⁶⁵ and NPEY⁹⁷² have been evaluated as candidates for insulin internalization and signal transduction (Berhanu et al., 1991, 1995).

In the present study we further elucidated the role of the whole JM domain in insulin internalization and signal transduction by constructing and utilizing two mutant receptors, one in which the JM domain (22 aa) was deleted and the other in which this domain was replaced by the corresponding domain of v-ros, as v-ros among the known tyrosine kinase family shares the greatest homology with the insulin receptor. Although, the juxtamembrane domain of the v-ros shares some organizational and functional similarity with that of the hIR-JM with respect to transmembrane signaling it lacks the key tyrosine residues that upon phosphorylation known to mediate the interaction of the activated hIR kinase with the downstream signal transduction molecules. The WT and the mutant receptors were each stably expressed in CHO cells as α2β2 heterotetramers and bound insulin with similar affinity. The endocytic functions of both the mutant receptors were assessed in comparison to that of the wild-type hIR by determining the single cohort internalization of the prebound insulin and measuring the specific internalization rate constant, Ke, for each cell line using a previously described initial rate determination method (Lund et al., 1990).

The JM deletion mutant ΔEx 16 and the JM chimeric mutant hIRrosJM showed 80 and 50% reduction in endocytic function, respectively. The result demonstrates that the intact exon 16 encoded JM domain is required for hIR's normal endocytic function. This finding correlates with the studies done by Thies S.R (Thies et al., 1990) who used hIR ΔExon 16 constructed from the A isoform of hIR and showed that despite the ability to bind insulin, the hIR Δexon 16 receptors did not internalize in Rat 1 cells (Thies et al., 1990). In addition, we have shown that partial restoration of endocytic function can occur in the hIR ros JM cells where the JM domain is replaced by v-ros oncogene which lacks the NPEY motif. As reported by Chen et al. (1990), there are receptors of the tyrosine kinase family that do not have NPXY sequence (e.g. the receptor for platelet-derived growth factor or colony stimulating factor I) thus the requirement for a sub-membranous NPXY sequence is not universal for all receptors.

The hIRΔEx16 from the A isoform was unable to internalize in Rat 1 cells despite its ability to bind insulin and activate tyrosine kinase (Thies et al., 1990). Although, our B isoform hIRΔEx16 and chimeric hIR rosJM are partially defective in their ability to internalize insulin, both the mutant receptors bound insulin with similar affinity as that of the wild-type, suggesting that the mutant receptors are not grossly misfolded. Therefore, these B isoform mutants provided a reasonably valid system for comparative analysis of the role of the juxtamembrane domain of insulin receptor in insulin signal transduction.

The early step in transmembrane insulin signaling involves rapid autophosphorylation of the receptor β-subunit on specific tyrosine residues, a process that activates the hIR kinase and initiates a further phosphorylation cascade involving downstream signal transduction molecules. Since IRS-1 is the key target molecule for hIR kinase to propagate downstream insulin signaling, we determined the effects of the deletion of the entire Exon16 JM domain that harbors NPEY⁹⁷² motif of hIR and the chimeric hIRrosJM lacking NPEY motif or tyrosine on insulin-stimulated IRS-1 phosphorylation. The IRS-1 phosphorylation increased in an insulin dose-dependent manner. Insulin-stimulated p185(IRS-1) phosphorylation occurred to similar extent in hIRWT and hIRΔExon 16 mutant. Noticeably, there was more of the basal as well as insulin-stimulated p185 (IRS-1) phosphorylation in the chimeric hIRrosJM than the hIRWT cells. Apparently, higher IRS-1 phosphorylation potency in hIRrosJM possibly due to the chimeric nature of the receptor as there is a lack of major amino acid sequence identity between the JM domains of v-ros and hIR.

We speculate that amino acid sequence of the JM domain of v-ros may favor the interaction of the alternative domain of the hIR β-subunit in hIRrosJM for mediating IRS-1 phosphorylation. On the other hand, endogenous substrate p185 (IRS-1) phosphorylation in response to insulin stimulation was similar in magnitude in both the WT and the hIRΔExon 16 mutant cells except the negative control Neo cells, which exhibited minimal insulin stimulation of tyrosine phosphorylation of the endogenous substrate p185 (IRS-1). Both the JM deletion mutant hIREx16 and the JM chimeric mutant hIRrosJM are as effective as hIRWT receptor in IRS-1 phosphorylation. Thus, this finding suggest that the hIR JM domain specific amino acid sequence that harbors tyrosine motif(s) may not play a critical role for IRS-1 phosphorylation. It is known that IRS-1 utilizes its phosphotyrosine binding (PTB) domain to bind to the tyrosine phosphorylated NPEY972 motif to couple the activated IR kinase to downstream signaling molecules (Berhanu et al., 1997). The phosphorylation of IRS-1 in the absence of the JM domain of hIR suggest the presence of NPEY sequence in hIR is not essential and the involvement of other conserved tyrosine outside of the JM domain of hIR in interaction with IRS-1 and its phosphorylation may not be ruled out.

Insulin activation of the IR kinase leads to phosphorylation of Shc, an Src homology 2 (SH2)-domain containing protein, and this process has been implicated in mitogenic signaling (Chen et al., 1990; Cheatham and Kahn, 1995; Saltiel, 1996; Bohm et al., 1997; Denzer et al., 1997). The phosphorylated Shc binds to the JM domain of the IR-β subunit that contains Tyr⁹⁷² (Sasaoka et al., 1994a,b; Kaburagi et al., 1995; Gustafson et al., 1995). Accordingly, we examined the effect of the deletion of the entire Exon16 that harbors NPEY⁹⁷² motif and on chimeric hIRrosJM lacking NPEY motif or tyrosine on insulin stimulation of Shc phosphorylation. Insulin stimulated phosphorylation of the 52-kDa Shc isoform in cells expressing hIRWT was observed. Insulin stimulation of the 52-kDa Shc phosphorylation in hIRWT-expressing cells is insulin concentration dependent (data not shown). In contrast, the hIREx16 and the hIRrosJM mutant showed little or no insulin-stimulated Shc phosphorylation. The cells expressing the hIREx16 and the hIRrosJM receptors exhibited essentially similar pattern like the control cells harboring Neo with respect to insulin stimulated Shc phosphorylation. Diminished Shc phosphorylation in both the JM deletion mutant hIREx16 and the JM chimeric mutant hIRrosJM suggest that the hIR JM domain specific amino acid sequence and its motif(s) containing the tyrosine residue(s) is important for Shc phosphorylation. This finding is in agreement with the earlier study showing that deletion of NPEY⁹⁷² motif from hIRWT abolish insulin-stimulated Shc phosphorylation (Berhanu et al., 1997).

The finding that the hIR JM domain specific amino acid sequence that harbors tyrosine motif(s) apparently does not play a critical role for IRS-1 phosphorylation, led us to investigate further if the hIR JM domain deletion mutant and chimeric mutant would have similar effects on insulin-stimulated Akt phosphorylation, a downstream serine/threonine kinase that is linked to metabolic functions like glucose transport and glycogen synthesis (Kohn et al., 1996; Moule et al., 1997) and is activated by PI3 kinase docked in phosphorylated IRS-1. Insulin dose-dependent Akt phosphorylation was observed. However, at higher concentrations of insulin ≥5nM, phosphorylation of Akt was similar in both the WT and the mutant receptors. The mutants were indistinguishable from the wild-type with respect to insulin-stimulated Akt phosphorylation. On the contrary, at lower concentrations of insulin <5nM, the extent of Akt phosphorylation was different in the WT and the mutant receptors. Besides, the control Neo, both the hIR JM deletion mutant hIRΔEx16 and the chimeric mutant hIRrosJM were less responsive than the hIRWT to the insulin-stimulation (<5nM) of Akt phosphorylation. Although, among the mutants, hIRΔEx16 was the least responsive to insulin stimulation of Akt phosphorylation, still it exhibited 50–60% of the insulin-stimulated (1nM) Akt phosphorylation compared to that of the hIRWT. This study demonstrates that insulin dose dependent activation of the downstream Akt signaling molecule can occur in the absence of hIR JM domain specific sequence that harbors tyrosine motif(s).

Having found that the hIR JM domain specific amino acid sequence and its motif(s) containing the tyrosine residue(s) plays differential roles for IRS-1 and Shc phosphorylations, we investigated a step further to find out whether hIR JM domain deletion or chimeric mutation would affect insulin-stimulated MAPK phosphorylation, a downstream kinase that is linked to gene expression/mitogenesis. Both phosphorylated IRS-1 and Shc provide docking sites for Grb2, a key molecule that is known to activate a sequentially linked downstream signaling proteins, Ras-Raf-MEK in MAP kinase pathway (Cheatham and Kahn, 1995; Saltiel, 1996; Myers and White, 1996). Insulin dose-dependent MAPK phosphorylation (p-p44 and p-p42) was observed. However, at higher concentrations of insulin ≥5nM, the pattern and the extent of phosphorylation of MAPK (p-p44) were similar in both the WT and the mutant receptors; the mutants were indistinguishable from the wild-type. On the contrary, at lower concentrations of insulin <5nM, the extent of MAPK phosphorylation was different in the WT and the mutant hIRΔEx16 receptor; and at concentrations of insulin <0.5nM, the extent of MAPK phosphorylation were different for both hIRΔEx16 and hIRrosJM mutants compared to that of the hIRWT. Although, among the mutants, hIRΔEx16 was the least responsive to insulin stimulation of MAPK phosphorylation, still it exhibited 30–40% of the insulin (1nM) -stimulated MAPK phosphorylation compared to that of the hIRWT. The JM deletion mutant hIREx16 was more defective than the JM chimeric mutant hIRrosJM in MAPK phosphorylation. This suggests that the hIR JM domain specific amino acid sequence that harbors tyrosine motif(s) may partly contribute to MAPK activation possibly through Shc dependent pathway. This study also demonstrates that insulin dose dependent activation of the downstream MAPK signaling molecules can occur even in the absence of hIR JM domain specific sequence that harbors tyrosine motif(s).

Taken together, our study demonstrates that in the absence of the JM domain of the hIR there is little or no insulin-stimulated tyrosine phosphorylation of Shc; by contrast, in the absence of the JM domain of the hIR, tyrosine phosphorylation of IRS-1 and activation of the down stream signaling molecules such as Akt and MAPK are dependent on insulin dose. Certain similarities and differences are apparent between the current and previous studies reported by other investigators. Our results are somehow in agreement with that of Thies et al. (1990) who reported no impairment of p185 phosphorylation upon deletion of the entire exon 16 encoded JM region containing the NPEY sequence. McClain (1990) also reported a modest decrease in insulin sensitivity without appreciable alteration in maximal response on the Δexon 16 mutant. But both studies that examined the role of the JM domain in signaling utilized the A-isoform of the hIR (White et al., 1988; Backer et al., 1991; Thies et al., 1990; McClain, 1990; Kaburagi et al., 1993). In the present study we have used the B-isoform for expression of WT and mutant IRs. The B isoform has been reported to signal more efficiently in response to insulin binding despite having a two fold lower affinity for insulin (Kosaki et al., 1995). Divergent isoform dependent signaling mechanisms have been demonstrated in the pancreas β cells with different activation of different PI3 Kinase and protein isoforms in response to insulin (Leibiger et al., 2001).

In summary, in this work we provide the following evidence: (i) the presence of intact hIR-JM domain which harbors tyrosine motif(s) is essential for insulin-induced Shc phosphorylation but not essential for IRS-1 phosphorylation; (ii) insulin signal transduction can occur independent of the JM domain of hIR and without the participation of the NPEY motif; (iii) engagement of this putative alternative downstream signal transduction is Shc independent and is dependent on insulin concentration; and (iv) insulin internalization does not necessarily require the hIR specific sequence of the JM domain which can be partially substituted by the JM domain of the similar yet heterogeneous v-ros tyrosine kinase. This leads to the conclusion that the insulin receptor contains the information necessary to engage multiple signaling pathways and maintain redundancy for signal transduction that can be differentially activated.

Acknowledgements

This work was supported by research grant DK54475 from NIH/NIDDK (P.B.) and in part by funds from Wayne State University Department of Internal Medicine (A.A.S.).

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Received 27 November 2006/5 January 2007; accepted 18 January 2007

doi:10.1016/j.cellbi.2007.01.033

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