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Endocrinology Vol. 139, No. 8 3578-3584
Copyright © 1998 by The Endocrine Society

ARTICLES

Comparison of the Signaling Abilities of the Cytoplasmic Domains of the Insulin Receptor and the Insulin Receptor-Related Receptor in 3T3-L1 Adipocytes1

Ajai A. Dandekar2, Brenda J. Wallach2, Andreas Barthel and Richard A. Roth

Department of Molecular Pharmacology, Stanford University School of Medicine, Stanford, California 94305

Address all correspondence and requests for reprints to: Dr. Richard A. Roth, Department of Molecular Pharmacology, Stanford Medical Center, Stanford, California 94305. E-mail: roth{at}cmgm.stanford.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In the present work a chimeric receptor containing the intracellular domain of the insulin receptor-related receptor (IRR) and the extracellular domain of the colony stimulating factor-1 (CSF-1) receptor was expressed in 3T3-L1 adipocytes and compared with the parallel chimeric receptor containing the cytoplasmic domain of the insulin receptor (IR). Both chimeric receptors exhibited CSF-stimulated tyrosine kinase activity when assayed in vitro after in vivo activation comparable to that of the endogenous IR present in these cells. No cross-activation of the expressed chimeric and endogenous receptors was observed. The cytoplasmic domain of the IRR was found to 1) mediate activation of the Ser/Thr kinase Akt/PKB, 2) stimulate glucose uptake, 3) inhibit lipolysis, and 4) stimulate glycogen synthase, all with a potency comparable to those of the expressed CSF-1R/IR chimera and the endogenous insulin receptors. These results indicate that despite the extensive differences in sequence between the cytoplasmic domains of the IRR and IR, the elements required for insulin-specific responses have been conserved in this distinct member of the insulin receptor family.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
BINDING of insulin to its receptor (IR) induces a wide variety of distinct biological responses, most of which appear to be mediated via activation of the receptor’s intrinsic tyrosine-specific phosphotransferase activity (1, 2, 3, 4). Extensive studies have documented the role of specific endogenous substrates such as insulin-receptor substrate-1 in mediating the subsequent biological responses (5). In adipocytes, the IR appears unique among the various receptors with tyrosine kinase activity in its ability to stimulate various biological responses, including glucose uptake, activation of the glycogen synthase, and inhibition of lipolysis (1, 2, 3, 4). The IR is a member of a family of receptors that includes the insulin-like growth factor I (IGF-I) receptor and the insulin receptor-related receptor (IRR) (6). Both differences and similarities in the signaling abilities of these three receptors have been noted. For example, some studies have indicated that the IGF-I receptor is more potent at stimulating cellular proliferation, whereas the insulin receptor is better at stimulating metabolic responses (7, 8, 9). The cytoplasmic domain of the IGF-I receptor exhibits a greater degree of sequence identity with the IR than the IRR (10, 11); the juxtamembrane, kinase domain, and carboxy-tail of the IGF-I receptor are 61%, 84%, and 44% identical, respectively, to the sequences of the corresponding regions of the IR, whereas the same domains of the IRR are 53%, 79%, and 19% identical to the comparable regions of the IR. In addition, the carboxy-tail of IRR is 69 or 76 amino acids shorter than that of the IR or IGF-I receptor, respectively (11). This carboxy-tail region of the IR and IGF-I receptors that is missing in IRR includes both tyrosine and serine/threonine phosphorylation sites, which have been proposed to play a role in cellular signaling (12, 13). Finally, whereas both the IR and IGF-I receptors exhibit widespread tissue distributions, the IRR is expressed in only a few limited cell types, including dorsal root and trigeminal neurons, neuroendocrine cells in gastric glands of the fundic mucosa, and non-A intercalated cells in the kidney (14, 15, 16, 17, 18).

In the present studies we therefore sought to compare the ability of the cytoplasmic domain of the IRR with that of the IR in stimulating insulin-specific biological responses in adipocytes. Recent studies by Chaika et al. and Kalloo-Hosein et al. have demonstrated that chimeric receptors containing the extracellular domains of either the neurotropin 3 or colony-stimulating factor-1 (CSF-1) receptors and the cytoplasmic domains of the IR can be expressed in 3T3-L1 cells and specifically stimulated by their appropriate ligands (19, 20). This approach thereby allows one to determine the relative abilities of different cytoplasmic domains and/or specific mutants to mediate various adipocyte-specific responses. Indeed, Kalloo-Hosein et al. used this system to demonstrate a differential signaling ability of the intracellular domain of the IGF-I receptor in comparison to that of the IR (19). In particular, they reported that in 3T3-L1 fibroblasts, the IGF-I receptor cytoplasmic domain was poorer than that of the IR in stimulating glycogen synthesis. In the present studies we examined the ability of the intracellular domain of the more distantly related IRR to mediate various insulin-specific biological responses in 3T3-L1 adipocytes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
The following were purchased: restriction enzymes from New England Biolabs (Beverly, MA) and Life Technologies (Gaithersburg, MD), oligonucleotides from Operon Technologies (Alameda, CA), T4 DNA ligase from New England Biolabs, deoxy-NTPs and protein G-Sepharose beads from Pharmacia (Uppsala, Sweden), protein A-agarose beads from RepliGen (Cambridge, MA), G418 from Sigma (St. Louis, MO), antibodies to the extracellular domain of the CSF-1R from Zymed (South San Francisco, CA), and the GSK-3 peptide (GRPRTSSFAEG) from the Beckman PAN facility (Stanford, CA). The antibodies to the IR (2G7) and Akt were prepared as described previously (21, 22). The following were gifts: the Phoenix retroviral packaging cell line and the pWZLneo retroviral vector from Dr. Garry P. Nolan (Stanford University), CSF-1 from the Genetics Institute (Cambridge, MA), and the CSF-1R/IR and CSF-1R/K1030M IR plasmids from Drs. O. Chaika and R. Lewis (University of Nebraska, Omaha, NE) (20).

Construction of the CSF-1R chimerae
The CSF-1R/IRR chimera was constructed using a PCR site-overlap-extension strategy (see schematic below). The template for the CSF-1R extracellular domain was CSF-1R/IR in pcDNA3 (20). The template for the IRR intracellular domain was IR/IRR in pECE (23). The upstream primer (primer A) for the CSF-1R extracellular domain was for a section of pcDNA3 and contained an introduced EcoRI site. The downstream primer (primer B) corresponded to the final 25 bases of CSF-1R before the transmembrane (TM) domain and contained a 18-base overhang corresponding to the first 18 bases from the noncoding strand of the TM domain of IR. The upstream primer (primer C) for IRR intracellular domain likewise corresponded to the first 25 coding bases of the IR TM domain and also had an 18-base identity with the 3'-end of the coding strand of the CSF-1R extracellular domain. The downstream primer (primer D) was based on a sequence from pECE immediately following the end of IRR and contained an introduced XhoI site. The CSF-1R and IRR fragments were individually amplified by PCR and purified by spin column. Equimolar amounts of each fragment were then site-overlap-extensioned together using primers A and D. The resulting 3-kb PCR product coding for the CSF-1R/IRR chimera was purified by spin column. This chimera was subsequently digested with EcoRI and XhoI and ligated to pWZLneo digested with the same enzymes.

To transfer CSF-1R/IR in pcDNA3 and CSF-1R/K1030M IR in pEF-1 to pWZLneo, the plasmids were digested with XbaI and filled in using T4 DNA polymerase, and then each plasmid was digested with EcoRI. The double digested constructs were ligated to pWZLneo double digested with EcoRI and SnaBI.

CSF-1R/IRR complementary DNA (cDNA)
A schematic is shown for the construction of the CSF-1R/IRR cDNA chimeric receptor (Fig. 1Go).



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  		Figure 1. Schematic for the construction of the CSF1R/IRR cDNA chimeric receptor.

 
Retroviral infections and cell culture
3T3-L1 fibroblasts were infected with either the empty pWZLneo retroviral vector or the retroviral vectors encoding CSF-1R/IR, CSF-1R/IRR, or CSF-1R/K1030M IR as previously described (24). In short, Phoenix cells were plated at 4 x 106 cells/60-mm plate in DME-H21 with 10% FBS and antibiotics. On the following day, they were transfected with the appropriate retroviral vector by the calcium phosphate method. The media were changed 8 h after transfection and again 24 h after transfection, at which time the cells were transferred to a 32 C, 5% CO2 incubator for 2 days. The supernatants, containing the retroviral particles, were then clarified by centrifugation and added to the 3T3-L1 fibroblasts, which had been plated at 7.5 x 104 cells/six-well plate the night before. Polybrene was added to the cells at a final concentration of 5 µg/ml. The six-well plates were then spun at 2500 rpm for 90 min at 32 C in a tabletop centrifuge to increase infection efficiency. After an 18-h incubation in a 32 C incubator, the media were changed, and the cells were transferred to a 37 C incubator. Selection with 1 mg/ml neomycin began the following day. The drug-resistant total cell population was used in all experiments.

3T3-L1 fibroblasts were maintained in DMEM-H21 containing 10% calf serum and antibiotics. To differentiate fibroblasts into adipocytes, cells were grown for 2 days postconfluence. The media were then changed to DME-H21 containing 10% FBS, 1 µg/ml insulin, 0.1 µg/ml dexamethasone, and 112 µg/ml isobutylmethylxanthine. After 4 days, cells were maintained in DME-H21, 10% FBS, and 1 µg/ml insulin for 3–4 additional days. Finally, the media were replaced with DME-H21 containing only 10% FBS for at least 2 days, after which the cells were used. At least 90% of the cell population showed the adipocyte phenotype with the accumulation of lipid droplets.

Tyrosine kinase assays
Serum-starved 3T3-L1 adipocytes in six-well plates were treated with 0.3–10 nM CSF-1, 10 nM insulin, or buffer only for 25 min at 37 C; washed once with ice-cold PBS; and lysed in 500 µl lysis buffer [50 mM HEPES (pH 7.6), 150 mM NaCl, 10% (vol/vol) glycerol, 1% (vol/vol) Triton X-100, 1 mg/ml bacitracin, 1 mM phenylmethylsulfonylfluoride, 1 mM benzamidine, 1 mM Na3VO4, 30 mM NaPPi, 10 mM NaF, 1 mM EDTA, 1 mM dithiothreitol (DTT), and 100 nM okadaic acid]. The lysates were centrifuged for 10 min at 14,000 rpm, and the supernatants were immunoprecipitated overnight at 4 C with 30 µl (packed) protein G-Sepharose beads preincubated with anti-CSF-1R antibody (Zymed), control Ig, or anti-IR intracellular domain (2G7). The beads were washed three times with WGBT [50 mM HEPES (pH 7.6), 0.1% Triton X-100, 0.1% BSA, and 150 mM NaCl], then 20 µl of the kinase reaction buffer [50 mM HEPES (pH 7.6), 150 mM NaCl, 0.1% (vol/vol) Triton X-100, plus 5 mM MgCl2, 5 mM MnCl2, 1 mg/ml poly(Glu:Tyr) (4:1), and 0.8 µCi carrier-free [{gamma}-32P]ATP] were added, and the reaction was continued for 30 min at 25 C. The reactions were stopped by spotting 10 µl onto a Whatman 3 MM filter strip (Clifton, NJ) and putting the paper in ice-cold 10% trichloroacetic acid containing 10 mM sodium pyrophosphate for 30 min, boiling in 5% trichloroacetic acid for 5 min, and washing once with 70% ethanol, once with distilled water, and once with acetone. Samples were then counted. The values for the control Ig precipitates (typically 200–450 cpm) were subtracted from the values for the precipitations with the anti-CSF-1R and IR antibody precipitates.

AKT kinase assays
Serum-starved 3T3-L1 adipocytes were treated with hormone for 20 min at 37 C, chilled on ice, washed once with cold PBS, and lysed in 800 µl lysis buffer as described above for the kinase assays. The supernatants were immunoprecipitated for 2 h with a polyclonal anti-Akt antibody bound to protein A-Sepharose beads. The beads were washed three times with 1 ml buffer [25 mM HEPES (pH 7.8), 1% BSA, 10% glycerol, 1 mM DTT, 1% Triton X-100, and 1 M NaCl] and twice with kinase buffer [50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, and 1 mM DTT]. The reactions were started by addition of 30 µl kinase mix [50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 1 mM DTT, 5 µM ATP, 100 µM GSK-3 peptide, and 2 µCi [{gamma}-32P]ATP] and allowed to proceed for 30 min at 30 C. The reactions were stopped by addition of 10 µl gel loading buffer [0.25 M Tris-HCl (pH 6.8) and 12 M urea], and the reaction mixtures were electrophoresed on a 40% polyacrylamide gel containing 6 M urea. The gels were dried and autoradiographed (2–12 h), and the phosphopeptide bands were excised and counted. The GSK-3 peptide was used as substrate instead of the myelin basic protein used previously (24) because it is a better substrate for Akt (25) (our unpublished studies). The immunoprecipitates were subjected to 10% SDS-PAGE, transferred onto nitrocellulose, and immunoblotted with the anti-Akt antibody to verify the amounts of AKT protein in the assay.

Measurements of glucose uptake
Serum-starved 3T3-L1 adipocytes in six-well plates were incubated for 3 h in 0.9 ml Krebs-Ringer bicarbonate (KRB) buffer without BSA. Cells were then treated with 100 µl 1–1000 nM CSF-1, 100 nM insulin, or KRB buffer. After 20 min at 37 C, 100 µl 1 mM 2-deoxyglucose containing 0.5 µCi [3H]2-deoxyglucose were added. After 5 min, cells were washed three times with ice-cold PBS containing 100 nM phloretin, lysed by addition of SDS, and counted.

Measurements of glycogen synthase kinase activation
Serum-starved 3T3-L1 adipocytes in six-well plates were treated with 1–30 nM CSF-1, 10 nM insulin, or KRB buffer for 30 min at 37 C. The cells were then washed once with ice-cold HEPES-buffered saline, and 300 µl solution 1 [50 mM Tris (pH 7.8), 10 mM EDTA (pH 8.0), and 100 mM potassium fluoride] were added to each well. Cells were removed from the well, sonicated for 60 sec, and centrifuged. Fifty microliters of the supernatants were added to 50 µl of a reaction mixture [50 mM Tris (pH 7.8), 10 mM EDTA (pH 8), 100 mM potassium fluoride, 5 µCi [14C]UDP glucose, 15 mg/ml glycogen, and 1 mM UDP-glucose]. To measure total glycogen synthase activity, parallel reactions were performed with each supernatant in the presence of 50 mM glucose-6-phosphate. All reactions were performed in triplicate. Reactions were allowed to proceed at 37 C for 20 min, transferred to 0 C, and then spotted on a Whatman 6F/A filter and immediately immersed in ice-cold 70% ethanol for 40 min. Filters were washed twice in 70% ethanol and counted.

Measurements of the antilipolytic response
Serum-starved 3T3-L1 adipocytes in six-well plates were incubated for 0.5 h in 0.9 ml KRB buffer with 0.1% BSA. Cells were treated with 100 nM isoproterenol or vehicle plus either 3–30 nM CSF-1, 10 nM insulin, or KRB buffer with 0.1% BSA. After 3 h, the supernatant was removed, and the BSA was precipitated with trichloroacetic acid. The glycerol content in 100 µl of the supernatants was determined by adding 100 µl of a periodate solution [0.5% acetic acid (vol/vol), 0.5 M ammonium acetate, and 15 mM sodium periodate] and 200 µl isopropanol with 1% acetylacetone (vol/vol). After 15 min at 37 C, the ODs at 410 nm were determined (26).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of CSF-1R/IR and CSF-1R/IRR in 3T3-L1 adipocytes
To compare the abilities of the cytoplasmic domains of the IRR and IR to mediate various insulin-specific responses, we constructed retroviral constructs encoding chimeric receptors expressing the extracellular domain of the CSF-1 receptor, the transmembrane of the IR, and the cytoplasmic domain of either the IRR or the IR (called CSF-1R/IRR or CSF-1R/IR, respectively; Fig. 2Go). The control constructs used included a chimeric receptor expressing a kinase dead IR cytoplasmic domain (in which lysine 1030 was changed to methionine, called CSF-1R/K1030M IR) and the empty retroviral vector (pWZLneo). 3T3-L1 fibroblasts infected with the retroviruses were drug se- lected and then tested for the presence of the different receptors after allowing the cells to differentiate into adipocytes. To this end, the infected 3T3-L1 adipocytes were treated with buffer, 10 nM CSF-1, or 10 nM insulin. The cells were then lysed, and the lysates were immunoprecipitated with antibodies to the extracellular domain of the CSF-1 receptor, the intracellular domain of the insulin receptor, or control Ig. The precipitates were then tested for tyrosine kinase activity using an in vitro kinase assay. As expected, 10 nM CSF-1 stimulated enzymatic activity in the anti-CSF-1 receptor precipitates from the cells expressing CSF-1R/IR and CSF-1R/IRR, and the amounts of activities from these two cells were approximately the same; that of CSF-1R/IR was slightly higher (Fig. 3AGo). In contrast, no increase in enzymatic activity was observed in the anti-CSF-1R precipitates from these cells after they were treated with 10 nM insulin, indicating that there was not a significant level of cross-phosphorylation and activation of the chimeric receptors by the endogenous IR present in these cells. In addition, there was no increase in enzymatic activity in the anti-CSF-1R precipitates from either the cells expressing the kinase dead chimera (CSF-1R/K1030M IR) or the cells infected with the control retrovirus (pWZL; Fig. 3AGo). The same cells were also treated with CSF-1, insulin, or buffer and lysed, and the lysates were immunoprecipitated with an antibody directed against the cytoplasmic domain of the IR (21). This antibody will recognize both the endogenous IR present in these cells as well as the CSF-1R/IR chimera, but not the CSF-1R/IRR chimera. As expected, insulin stimulation of all cells showed an increase in the amount of enzymatic activity precipitated with this antibody due to the endogenous receptors present in these cells (Fig. 3BGo). In contrast, only the cells expressing the active CSF-1R/IR chimera exhibited an increase in activity after treatment with CSF-1 in these anti-IR antibody precipitates (Fig. 3BGo). Importantly, the amount of enzymatic activity observed with the endogenous insulin receptors was comparable to that observed with the chimeric receptor (Fig. 3BGo), indicating that the level of the expressed chimeric receptor was not substantially greater than that of the endogenous receptor.



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  		Figure 2. Schematic representation of the CSF-1R/IR and CSF-1R/IRR chimeric receptors. Also shown are the percent sequence identities of the cytoplasmic regions of the IRR. JM, Juxtamembrane; CT, carboxy-tail.

 


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  		Figure 3. Tyrosine kinase activities of the CSF-1R/IR and CSF-1R/IRR chimeric receptors. A, 3T3-L1 adipocytes expressing the indicated constructs were treated with buffer, 10 nM CSF-1, or 10 nM insulin for 25 min at 37 C and lysed, and the lysates were immunoprecipitated with either antibodies to the extracellular domain of the CSF-1 receptor or control Ig. The immunoprecipitates were then tested for the ability to phosphorylate an exogenous substrate in vitro, and the values for the control Ig were subtracted from those with the specific antibody. Results shown are the mean ± SEM for three experiments. When not present, the means and/or error bars were too small to be visible. B, The same experiment was performed as in A, except that the lysates were immunoprecipitated with an antibody directed against the cytoplasmic domain of the IR.

 
Activation of Akt/PKB by CSF-1R/IR and CSF-1R/IRR
Recent studies have implicated the Ser/Thr kinase called Akt/PKB in a variety of biological responses, including the insulin-stimulated increase in glucose uptake (27, 28, 29). These two receptors were therefore compared for their ability to mediate the activation of this kinase. 3T3-L1 adipocytes expressing the various chimeric receptors were treated with buffer, various concentrations of CSF-1, or 10 nM insulin. Cells expressing CSF-1R/IR and CSF-1R/IRR exhibited a similar dose-dependent stimulation of Akt/PKB kinase activity (Fig. 4Go). The maximal stimulation observed with CSF-1 in these two cells was only slightly less (~10%) than that observed with insulin. In contrast, the cells expressing the kinase dead chimera or infected with the empty virus (pWZLneo) did not exhibit any CSF-1-stimulated increase in Akt/PKB kinase activity (Fig. 4Go).



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  		Figure 4. Activation of Akt/PKB by CSF-1R/IR and CSF-1R/IRR. 3T3-L1 adipocytes expressing the different constructs were treated with buffer, the indicated concentration of CSF-1, or 10 nM insulin for 20 min at 37 C. The cells were chilled and lysed, and the lysates were immunoprecipitated with an anti-Akt antibody. The precipitates were then assayed for Akt activity. The results were normalized for each experiment by expressing the activity present as a percentage of that observed with 10 nM insulin. Shown are the mean ± SEM for three experiments, except for that with 30 nM CSF-1 (due to limited amounts of CSF-1). In a typical experiment, 1490 and 6940 cpm were incorporated into the GSK-3 peptide via the Akt precipitated from lysates of noninsulin-treated and insulin-treated cells, respectively.

 
Stimulation of glucose uptake by CSF-1R/IR and CSF-1R/IRR
The ability of CSF-1R/IR and CSF-1R/IRR to mediate an increase in glucose uptake was examined by treating 3T3-L1 adipocytes expressing these different receptors with buffer, various concentrations of CSF-1, or 10 nM insulin. CSF-1 stimulated a dose-dependent increase in glucose uptake in the cells expressing these two chimeric receptors, with an EC50 and maximal response that were essentially identical (Fig. 5Go). The maximal responses observed with these two chimeric receptors were approximately 90% of that observed with insulin. In contrast, the cells expressing the kinase dead chimera or infected with the empty virus did not exhibit any CSF-1-stimulated increase in glucose uptake.



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  		Figure 5. Stimulation of glucose uptake by CSF-1R/IR and CSF-1R/IRR. 3T3-L1 adipocytes expressing the different constructs were treated with buffer, the indicated concentration of CSF-1, or 10 nM insulin for 20 min at 37 C, and then [3H]2-deoxyglucose was added. After 5 min, the cells were washed and processed as described, then lysed, and the amount of cell-associated radioactivity was determined. The results were normalized for each experiment by expressing the glucose uptake as a percentage of that observed with 10 nM insulin. Shown are the mean ± SEM for three experiments, except for that with 100 nM CSF-1 (due to limited amounts of CSF-1). In a typical experiment, 2,480 and 18,140 cpm [3H]2-deoxyglucose were taken up via the noninsulin-treated and insulin-treated cells, respectively.

 
Activation of glycogen synthase by CSF-1R/IR and CSF-1R/IRR
The ability of CSF-1R/IR and CSF-1R/IRR to mediate an increase in glycogen synthase activity was examined by treating 3T3-L1 adipocytes expressing these different receptors with buffer, various concentrations of CSF-1, or 10 nM insulin. The cells were then lysed, and glycogen synthase was measured directly in the lysates. Both receptors mediated a CSF-dependent increase in this enzymatic activity with almost identical potencies. In addition, the maximal responses observed with these chimeric receptors were comparable to those observed with that stimulated via the endogenous insulin receptors (Fig. 6Go). Again, the kinase dead chimeric receptor and the cells infected with the empty virus did not exhibit any significant CSF-1-stimulated increase in glycogen synthase activity.



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  		Figure 6. Activation of glycogen synthase by CSF-1R/IR and CSF-1R/IRR. 3T3-L1 adipocytes expressing the different constructs were treated with buffer, the indicated concentration of CSF-1, or 10 nM insulin for 30 min at 37 C and then lysed. Aliquots of the lysates were assayed for glycogen synthase activity in the presence or absence of glucose-6-phosphate. The amount of activity present in each lysate was normalized for the total amount of glycogen synthase present (the activity measured in the presence of glucose-6-phosphate). The results were then normalized for each experiment by expressing the glycogen synthase activity measured after the different stimulations as a percentage of that observed with 10 nM insulin. Shown are the mean ± SEM for three experiments, except for that with 100 nM CSF-1. In a typical experiment, 11 and 20% of the total (glucose-6-phosphate activatable) glycogen synthase activity were present in the lysates of noninsulin-treated and insulin-treated cells, respectively.

 
Both CSF-1R/IR and CSF-1R/IRR mediate an inhibition of the isoproterenol stimulation of lipolysis
The ability of CSF-1R/IR and CSF-1R/IRR to inhibit the isoproterenol-mediated increase in lipolytic activity was examined by treating 3T3-L1 adipocytes expressing these different receptors with isoproterenol together with buffer, various concentrations of CSF-1, or 10 nM insulin. The amount of glycerol released by these cells was then measured. Both receptors were capable of antagonizing the isoproterenol-stimulated increase in lipolysis. The maximal response and sensitivity to CSF-1 were essentially the same for the cells expressing the two chimeric receptors (Fig. 7Go). In addition, the maximal responses observed with these chimeric receptors were comparable to that observed with the receptor stimulated via the endogenous IRs (Fig. 7Go). Again, the kinase dead chimeric receptor did not exhibit any significant CSF-stimulated inhibition of lipolysis.



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  		Figure 7. Both CSF-1R/IR and CSF-1R/IRR mediate an inhibition of the isoproterenol stimulation of lipolysis. 3T3-L1 adipocytes expressing the different constructs were treated with isoproterenol and buffer, the indicated concentration of CSF-1, or 10 nM insulin for 3 h at 37 C. The amount of glycerol present in the medium was assayed as described. Isoproterenol stimulated lipolysis 5-fold in these experiments. The extent of inhibition by the different treatments was expressed as the percentage of the response observed with 10 nM insulin. Shown are the means for three experiments. When not present, the means and/or error bars were too small to be visible. In a typical experiment, OD410 of 0.11 and 0.040 were observed for the noninsulin-treated and insulin-treated cells, respectively.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Recently Chaika et al. (20) described the expression of a chimeric receptor with the extracellular domain of the CSF-1 receptor and the intracellular domain of the IR in 3T3-L1 preadipocytes. This receptor was shown to exhibit a CSF-stimulated increase in receptor autophosphorylation and to mediate a CSF-stimulated increase in IRS-1 and Shc tyrosine phosphorylation and a CSF-stimulated differentiation of these cells into adipocytes. In the present work we have confirmed and extended these findings. First, we used a retroviral expression system to facilitate the attainment of a population of 3T3-L1 cells that are still capable of differentiating into adipocytes. Second, we found that this expressed chimeric receptor can also mediate a CSF-stimulated increase in Akt/PKB Ser/Thr kinase activity, glucose uptake, glycogen synthase activity, and inhibition of the isoproterenol-induced increase in lipolysis. Thus, this chimeric receptor appears capable of mediating a variety of insulin-specific responses in the 3T3-L1 adipocytes, indicating that this approach will allow testing of the abilities of different regions of the IR to elicit different biological responses.

In the present work, we used this approach to examine the signaling capabilities of another member of the IR family, the IRR. Although the ligand for this receptor is not known, the receptor shows overall sequence homology with the IR and IGF-I receptor and can form hybrid receptors with these two other receptors (11, 23, 30, 31). Unlike the IR and the IGF-I receptor, this receptor is expressed in a limited number of tissues (14, 15, 16, 17, 18). It also differs substantially in sequence from that of the IR, with a carboxy-tail that is only 19% identical to that of the IR and is significantly shorter than the comparable region of either the IR or the IGF-I receptor (11). The carboxyl-tail regions of the IR and IGF-I receptor contain both tyrosine and Ser/Thr phosphorylation sites which have been proposed to be important in signaling various biological responses (12, 13). Thus, it was surprising to find in the present studies that the intracellular domain of the IRR appeared essentially indistinguishable from that of the IR in mediating the activation of a variety of biological responses that are considered characteristic of the IR. The responses examined include the stimulation of glucose uptake, the activation of glycogen synthase, as well as the ability to antagonize the isoproterenol-stimulated increase in lipolysis. These responses were chosen because the mechanisms by which insulin elicits these responses differ (32, 33, 34). In each case, the chimeric receptor containing the cytoplasmic domain of the IRR was comparable to that containing the cytoplasmic domain of the IR in terms of both the maximal responses elicited as well as the sensitivity to a particular level of stimulation. In addition, in each case, the intrinsic tyrosine kinase activity of the IR cytoplasmic domain was necessary because the chimeric receptor that contained the inactivating mutation (CSF-1R/K1030M IR) could not stimulate any of these responses. It should also be noted that the ability of these chimeric receptors to be stimulated by CSF-1 to give a response comparable to that mediated by the stimulation of the endogenous receptors by insulin indicates that the internalization of the insulin molecule per se is not required for eliciting the subsequent biological responses.

Recent studies have indicated that insulin stimulates the activity of the Ser/Thr kinase Akt/PKB (35). Moreover, this enzyme may be responsible for inducing some of the subsequent biological responses, such as stimulation of glucose uptake and activation of glycogen synthesis (27, 28, 29, 36). The present studies are also consistent with this hypothesis, as the cytoplasmic domain of IRR was found to be capable of activating this enzyme comparably to that observed with the chimeric receptor containing the cytoplasmic domain of the IR. These results are consistent with the similar abilities of the two receptors to activate the above-mentioned biological responses.

Finally, it should be noted that the reported inability of the IGF-I receptor cytoplasmic domain to stimulate glycogen synthesis comparably to that observed with the IR (19) would suggest that this receptor is lacking some critical residues present in the cytoplasmic domains of both the IR and IRR. By construction of additional chimeric receptors with various portions of the cytoplasmic domain of the IGF-I receptor replaced with comparable regions of either the IR or IRR, it should be possible to delineate the region responsible for this activity.


   Acknowledgments
 
We are grateful to Drs. O. Chaika and R. Lewis for the CSF-1R/IR and CSF-1R/K1030M IR plasmids, to the Genetics Institute for the generous gifts of CSF-1, to Dr. Garry Nolan for the Phoenix retroviral packaging cell line and the pWZLneo retroviral vector, and for advice from Drs. J. Li and Kristina Kovacina.


   Footnotes
 
1 This work was supported by NIH Grants DK-41765 and DK-34926, a Feodor Lynen fellowship of the Alexander von Humboldt Stiftung (to A.B.), and a Howard Hughes undergraduate summer fellowship (to A.A.D.) Back

2 These two authors contributed equally to this work. Back

Received February 13, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Lee J, Pilch PF 1994 The insulin receptor: structure, function, and signaling. Am J Physiol 266:C319–C334
  2. Draznin B 1996 Insulin signaling network–waiting for Copernicus. Endocrinology 137:2647–2648[CrossRef][Medline]
  3. Saltiel AR 1996 Diverse signaling pathways in the cellular actions of insulin. Am J Physiol 270:E375–E385
  4. Cheatham B, Kahn CR 1995 Insulin action and the insulin signaling network. Endocr Rev 16:117–142[Abstract/Free Full Text]
  5. Yenush L, White MF 1997 The IRS-signalling system during insulin and cytokine action. BioEssays 19:491–500[CrossRef][Medline]
  6. Seta KA, Kovacina KS, Roth RA 1994 The insulin receptor family. In: LeRoith D, Raizada M (eds) Current Directions in Insulin-Like Growth Factor Research. Plenum Press, New York, pp 113–132
  7. Blakesley VA, Scrimgeour A, Esposito D, LeRoith D 1996 Signaling via the insulin-like growth factor I receptor: does it differ from insulin receptor signaling? Cytokine Growth Factor Rev 7:153–159[CrossRef][Medline]
  8. Siddle K 1992 The insulin receptor and type I IGF receptor: comparison of structure and function. Prog Growth Factor Res 4:301–320[CrossRef][Medline]
  9. Ish-Shalom D, Christoffersen CT, Vorwerk P, Sacerdoti-Sierra N, Shymko RM, Naor D, DeMeyts P 1997 Mitogenic properties of insulin and insulin analogues mediated by the insulin receptor. Diabetologia 40:S25–S31
  10. Ullrich A, Gray A, Tam AW, Yang-Feng T, Tsubokawa M, Collins C, Hanzel W, Le Bon T, Kathuria S, Chen E, Jacobs S, Francke U, Ramachandran J, Fujita-Yamaguchi Y 1986 Insulin-like growth factor I receptor primary structure: comparison with insulin receptor suggests structural determinants that define functional specificity. EMBO J 5:2503–2512[Medline]
  11. Shier P, Watt VM 1989 Primary structure of a putative receptor for a ligand of the insulin family. J Biol Chem 264:14605–14608[Abstract/Free Full Text]
  12. O’Connor R, Kauffmann-Zeh A, Liu Y, Lehar S, Evan GI, Baserga R, Blattler WA 1997 Identification of domains of the insulin-like growth factor I receptor that are required for protection from apoptosis. Mol Cell Biol 17:427–435[Abstract]
  13. Esposito DL, Blakesley VA, Koval AP, Scrimgeour AG, LeRoith D 1997 Tyrosine residues in the C-terminal domain of the insulin-like growth factor-I receptor mediate mitogenic and tumorigenic signals. Endocrinology 138:2979–2988[Abstract/Free Full Text]
  14. Reinhardt RR, Chin E, Zhang B, Roth RA, Bondy CA 1993 Insulin receptor-related receptor messenger ribonucleic acid is focally expressed in sympathetic and sensory neurons and renal distal tubule cells. Endocrinology 133:3–10[Abstract/Free Full Text]
  15. Shier P, Watt VM 1992 Tissue-specific expression of the rat insulin receptor-related receptor gene. Mol Endocrinol 6:723–729[Abstract/Free Full Text]
  16. Tsujimoto K, Tsuji N, Ozaki K, Ohta M, Itoh N 1995 Insulin receptor-related receptor messenger ribonucleic acid in the stomach is focally expressed in the enterochromaffin-like cells. Endocrinology 136:558–561[Abstract]
  17. Reinhardt RR, Chin E, Zhang B, Roth RA, Bondy CA 1994 Selective coexpression of insulin receptor-related receptor (IRR) and TRK in NGF-sensitive neurons. J Neurosci 14:4674–4683[Abstract]
  18. Mathi SK, Chan J, Watt VM 1995 Insulin receptor-related receptor messenger ribonucleic acid: quantitative distribution and localization to subpopulations of epithelial cells in stomach and kidney. Endocrinology 136:4125–4132[Abstract]
  19. Kalloo-Hosein HE, Whitehead JP, Soos M, Tavare JM, Siddle K, O’Rahilly S 1997 Differential signaling to glycogen synthesis by the intracellular domain of the insulin vs. the insulin-like growth factor-1 receptor. Evidence from studies of trkc-chimeras. J Biol Chem 272:24325–24332[Abstract/Free Full Text]
  20. Chaika OV, Chaika N, Volle DJ, Wilden PA, Pirrucello SJ, Lewis RE 1997 CSF-1 receptor/insulin receptor chimera permits CSF-1-dependent differentiation of 3T3–L1 preadipocytes. Biol Chem 272:11968–11974[Abstract/Free Full Text]
  21. Morgan DO, Roth RA 1986 Mapping surface structures of the human insulin receptor with monoclonal antibodies: localization of the main immunogenic regions to the receptor kinase domain. Biochemistry 25:1364–1371[CrossRef][Medline]
  22. Krook A, Kawano Y, Song XM, Efendic S, Roth RA, Wallberg-Henriksson H, Zierath JR 1997 Improved glucose tolerance restores insulin-stimulated Akt kinase activity and glucose transport in skeletal muscle from diabetic Goto-Kakizaki (GK) rats. Diabetes 46:2110–2114[Abstract]
  23. Zhang B, Roth RA 1992 The insulin receptor-related receptor. Tissue expression, ligand binding specificity, and signaling capabilities. J Biol Chem 267:18320–18328[Abstract/Free Full Text]
  24. Kohn A, Takeuchi F, Roth RA 1996 Akt, a pleckstrin homology domain containing kinase, is activated primarily by phosphorylation. J Biol Chem 271:21920–21926[Abstract/Free Full Text]
  25. Alessi DR, Caudwell FB, Andjelkovic M, Hemmings BA, Cohen P 1996 Molecular basis for the substrate specificity of protein kinase B; comparison with MAPKAP kinase-1 and p70 S6 kinase. FEBS Lett 399:333–338[CrossRef][Medline]
  26. Shechter Y 1984 Differential effects of two phosphodiesterase inhibitors on fat cell metabolism. Endocrinology 115:1787–1791[Abstract/Free Full Text]
  27. Kohn AD, Summers SA, Birnbaum MJ, Roth RA 1996 Expression of a consitutively active Akt Ser/Thr kinase in 3T3–L1 adipocytes stimulates glucose uptake and GLUT4 translocation. J Biol Chem 271:31372–31378[Abstract/Free Full Text]
  28. Tanti J, Grillo S, Gremeaux T, Coffer P, Van Obberghen E, Le Marchand-Brustel Y 1997 Potential role of protein kinase B in glucose transporter 4 translocation in adipocytes. Endocrinology 138:2005–2010[Abstract/Free Full Text]
  29. Cong LN, Chen H, Li Y, Zhou L, McGibbon MA, Taylor SI, Quon MJ 1997 Physiological role of Akt in insulin-stimulated translocation of GLUT4 in transfected rat adipose cells. Mol Endocrinol 11:1881–1890[Abstract/Free Full Text]
  30. Jui HY, Accili D, Taylor SI 1996 Characterization of a hybrid receptor formed by dimerization of the insulin receptor-related receptor (IRR) with the insulin receptor (IR): coexpression of cDNAs encoding human IRR and human IR in NIH-3T3 cells. Biochemistry 35:14326–14330[CrossRef][Medline]
  31. Jui H-Y, Suzuki Y, Accili D, Taylor SI 1994 Expression of a cDNA encoding the human insulin receptor-related receptor. J Biol Chem 269:22446–22452[Abstract/Free Full Text]
  32. Czech MP, Erwin JL, Sleeman MW 1996 Insulin action on glucose transport. In: LeRoith D, Taylor SI, Olefsky JM (eds) Diabetes Mellitus. Lippincott-Raven, Philadelphia, pp 205–212
  33. Skurat AV, Roach PJ 1996 Regulation of glycogensynthesis. In: LeRoith D, Taylor SI, Olefsky JM (eds) Diabetes Mellitus. Lippincott-Raven, Philadelphia, pp 223–236
  34. Londos C 1996 Hormone-sensitive lipase and the control of lipolysis in adipocytes. In: LeRoith D, Taylor SI, Olefsky JM (eds) Diabetes Mellitus. Lippincott-Raven, Philadelphia, pp 223–226
  35. Kohn AD, Kovacina KS, Roth RA 1995 Insulin stimulates the kinase activity of RAC-PK, a pleckstrin homology domain containing Ser/Thr kinase. EMBO J 14:4288–4295[Medline]
  36. Shaw M, Cohen P, Alessi DR 1997 Further evidence that the inhibition of glycogen synthase kinase-3ß by IGF-I is mediated by PDK1/PKB-induced phosphorylation of Ser-9 and not by dephosphorylation of Tyr-216. FEBS Lett 416:307–311[CrossRef][Medline]



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