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Cell Biology International (2007) 31, 815824 (Printed in Great Britain)
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
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.
The human insulin receptor (hIR), a transmembrane glycoprotein composed of α and β subunits in a tetrameric α
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 p68gag-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
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 (2000
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 30
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;106
2.4 Cell culture
Cell lines were maintained in Ham's F12 media supplemented with 10% (v/v) FCS, 2
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
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
2.7 Serum-starvation and insulin stimulation
The CHO cell lines described were cultured in 100
2.8 Immunodetection of phosphorylated Shc
The supernatant (200
2.9 Immunodetection of pIRS-1, pp42/pp44 MAPK and pAkt
Samples from control and insulin-treated cells were diluted with 5x sample buffer (0.5
2.10 Statistical analysis
Data are expressed as means
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).
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
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 Fig. 2 Internalization of cell -surface bound insulin. (A) CHO cells expressing either wild-type or mutant insulin receptors were first incubated for 2
Internalization of cell -surface bound insulin. (A) CHO cells expressing either wild-type or mutant insulin receptors were first incubated for 2
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.
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 5
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.
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 (+) 100
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 ≥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 5
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 ≥5
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 5
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 α
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 NPEY
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 ≥5
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 ≥5
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.
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 2007doi:10.1016/j.cellbi.2007.01.033