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Endocrinology Vol. 140, No. 3 1329-1337
Copyright © 1999 by The Endocrine Society

ARTICLES

Cloning, Tissue Expression, and Chromosomal Location of the Mouse Insulin Receptor Substrate 4 Gene1

Valeria R. Fantin, Brian E. Lavan2, Qing Wang3, Nancy A. Jenkins, Debra J. Gilbert, Neal G. Copeland, Susanna R. Keller and Gustav E. Lienhard

Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755; and the Mammalian Genetics Laboratory, ABL-Basic Research Program, National Cancer Institute-Frederick Cancer Research and Development Center (N.A.J., D.J.G., N.G.C.), Frederick, Maryland 21702

Address all correspondence and requests for reprints to: Dr. Gustav E. Lienhard, Department of Biochemistry, Vail Building, Dartmouth Medical School, Hanover, New Hampshire 03755. E-mail: gustav.e.lienhard{at}dartmouth.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The insulin receptor substrates (IRSs) are key proteins in signal transduction from the insulin receptor. Recently, we discovered a fourth member of this family, designated IRS-4, cloned its complementary DNA from the human embryonic kidney 293 cell line, and characterized its signaling properties in this cell line. As part of an investigation of the physiological role of this IRS, we have now cloned the mouse IRS-4 gene and determined its tissue expression and chromosomal location. The coding region of the mouse IRS-4 gene contains no introns, and in this regard is the same as that of the genes for IRS-1 and -2. The predicted amino acid sequence of mouse IRS-4 is highly homologous with that of human IRS-4; the pleckstrin homology domain, the phosphotyrosine-binding domain, and the tyrosine phosphorylation motifs are especially well conserved. The tissue distribution of IRS-4 in the mouse was determined by analysis for the expression of its messenger RNA by RT-PCR and for the protein itself by immunoprecipitation and immunoblotting. The messenger RNA was detected in skeletal muscle, brain, heart, kidney, and liver, but the protein itself was not detected in any tissue. These results indicate that IRS-4 is a very rare protein. The chromosomal locations of the mouse IRS-4 and IRS-3 genes were determined by interspecific backcross analysis and were found to be on chromosomes X and 5, respectively. As the mouse genes for IRS-1 and -2 are on chromosomes 1 and 8, respectively, each IRS gene resides on a different chromosome.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE INSULIN receptor substrates (IRSs) play key roles in signal transduction from the insulin receptor as well as other receptors, including those for insulin-like growth factor I, some interleukins, and GH (reviewed in Refs. 1, 2, 3). The IRSs are phosphorylated on tyrosine by the activated receptors, and the phosphotyrosine forms of the IRSs then bind to and thereby activate a group of SH2 domain-containing signaling proteins. These latter include phosphatidylinositol 3-kinase (PI 3-kinase), Grb2 the adaptor protein associated with Sos, the guanine nucleotide (nt) exchange protein for Ras, and the phosphotyrosine phosphatase SHP-2. The activation of PI 3-kinase and Ras/SHP-2, in turn, leads to the stimulation of kinase cascades involving protein kinase B and mitogen-activated protein kinase, respectively. These stimulations trigger many of the well known cellular effects of insulin and other hormones.

To date, four members of the IRS family, designated IRS-1 through IRS-4, have been described. These are characterized by their similar architecture. Each consists of an amino-terminal pleckstrin homology (PH) domain, followed by a phosphotyrosine binding (PTB) domain and then a large domain containing many motifs for tyrosine phosphorylation and the binding of SH2 domain proteins (4, 5, 6, 7). In the case of IRS-1, it has been found that both the PH and PTB domains are required for efficient phosphorylation by the insulin receptor (8), and presumably this is the case for the other IRSs.

Previously, we cloned the complementary DNA (cDNA) for human IRS-4 from human embryonic kidney (HEK) 293 cells (7) and studied the association of IRS-4 with SH2 domain proteins in these cells (9). As part of the investigation of the physiological role of this IRS, we have now cloned the mouse gene for IRS-4 and determined its tissue expression and chromosomal location. This information is compared with similar information previously obtained for the other IRSs.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cloning and sequencing of the mouse IRS-4 gene
An EcoO1091-BssHII cDNA fragment (417 bp) and a PCR-generated fragment (468 bp) from the human IRS-4 cDNA, which encode amino acids 72–209 and 451–605, respectively (7), were used as probes to screen a mouse genomic library. The probes were labeled with [32P]deoxy-CTP by random primed labeling with the Prime-It RmT kit (Stratagene, La Jolla, CA). A mixture of the two probes was used to screen a 129SJV mouse genomic library in the {lambda} FIXII vector (Stratagene), according to the manufacturer’s instructions and standard methodologies (10). Approximately one million plaques were screened, and one positive was obtained. The phage DNA was isolated from the positive phage as described previously (11).

The IRS-4 genomic DNA was excised from the phage DNA in several pieces by digestion with SacI or SalI. The total size of the genomic insert was approximately 13.3 kb. By a combination of restriction mapping, Southern blotting with probes derived from human IRS-4 (hIRS-4) cDNA, and DNA sequencing, it was established that the coding region for mouse IRS-4 (mIRS-4) (3.6 kb; see Fig. 1Go) was located approximately in the middle of the 13.3-kb fragment, starting approximately 5 kb from the 5'-end. Two SacI fragments, one of 7.5 kb starting at the 5'-end of the genomic piece and an adjoining 3-kb one, encompassed the coding region. In addition, two SalI fragments, one of 8.3 kb starting at the 5'-end of the genomic piece and a second of 5 kb, also encompassed the coding region and accounted for the entire genomic piece. The coding region shows the expected SacI and SalI sites at nt 2550 and 3262, respectively (see Fig. 1Go). The SacI and SalI fragments were subcloned into the pBluescript SKII+ vector (Stratagene). The coding region together with short segments of the adjoining 5'- and 3'-noncoding regions were sequenced. Each part of the final sequence was obtained by sequencing at least two different pBluescript clones; generally, each clone was sequenced in both directions. Sequencing was performed on the PE Applied Biosystems 373 system using the Perkin-Elmer Sequencing Kit FS (Foster City, CA). Data were analyzed with the PE Applied Biosystems software.



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  		Figure 1. The nt and predicted amino acid sequence of the mIRS-4 gene. The in-frame stop codon at nt 100–102 and the start codon at nt 217–219 are underlined. The nt sequence has been submitted to the GenBank with accession no. AF087797.

 
RT-PCR analysis for IRS-4 messenger RNA (mRNA)
Polyadenylated [poly(A)+] RNA from various mouse tissues was purchased from Clontech (Palo Alto, CA). RT was carried out in a 20-µl volume with 0.5 µg RNA, a primer corresponding to nt 3926–3945 of the mIRS-4 gene (see Fig. 1Go), and 200 U SuperScript II reverse transcriptase (Life Technologies, Gaithersburg, MD) according to the Life Technologies manual. In the control reactions, water replaced the reverse transcriptase. Aliquots (2 µl) of the RT and control reactions were amplified by PCR using primers corresponding to nt 3632–3649 and 3788–3807 of the mIRS-4 gene (see Fig. 1Go) and 2.5 U AmpliTaqGold (Perkin Elmer) in a 50-µl volume, according to the manufacturer’s manual. Amplification consisted of 30 cycles of 30 sec at 94 C, 1 min at 58 C, and 1 min at 72 C, with a final 7 min at 72 C. An aliquot (5 µl) of each PCR reaction was reamplified for 30 cycles using the same procedure as the first amplification. The PCR products (10 µl of the reaction mixtures) were separated on a 1.5% agarose gel and visualized with ethidium bromide staining.

Poly(A)+ RNA from mouse embryos on day 7, 11, 15, and 21 of embryonic development, purchased from Clontech, was analyzed for mIRS-4 mRNA as described above with one exception. Because a preliminary experiment showed that the RNA contained sufficient genomic DNA to interfere with the analysis, it was first treated with deoxyribonuclease I (Life Technologies) according to the Life Technologies manual for the preparation of an RNA sample before RT-PCR.

Immunoprecipitation and immunoblotting of IRS-4
The preparation of antibodies against mouse IRS-4 used for these experiments was described previously (9). Briefly, a rabbit antiserum was raised against a glutathione-S-transferase (GST) fusion protein with amino acids 994-1197 of mouse IRS-4. Antibodies in the serum against the GST portion were first removed by adsorption of the serum with immobilized GST, and then antibodies against the mouse IRS-4 portion were affinity purified by chromatography of this adsorbed serum on the immobilized GST-IRS-4 fusion protein.

Normal male mice and male littermates with targeted disruption of the IRS-4 gene (129 x BALB/c mice) at 10 weeks of age were killed. Tissues were removed, washed with cold PBS, frozen in liquid nitrogen, weighed, and stored at -70 C until processed. The tissues were homogenized at 4 C in sufficient homogenization buffer (1–10 vol) to yield about 15 mg protein/ml with a Tekmar (Cincinnati, OH) Tissumizer at setting 80 for 30–60 sec. The homogenization buffer consisted of 40 mM HEPES, 150 mM NaCl, 10 mM sodium pyrophosphate, 10 mM NaF, 2 mM EDTA, and 1 mM sodium vanadate, pH 7.5, with a mixture of protease inhibitors (10 µM leupeptin, 10 µM EP475, 1 µM pepstatin, 10 µg/ml aprotinin, and 2 mM phenylmethanesulfonyl fluoride). An aliquot of each homogenate was solubilized with 4% SDS and 20 mM dithiothreitol, and the protein concentration was determined by a precipitating Lowry assay (12). The homogenates were diluted to 5 mg/ml with the homogenization buffer, made 1.8% in octaethyleneglycol dodecyl ether (Thesit, Boehringer Mannheim, Indianapolis, IN) to solubilize the membranes, and centrifuged at 140,000 x g for 30 min to remove insoluble material. Aliquots of the detergent lysate derived from 2 mg protein (400 µl) were immunoprecipitated with 5 µg affinity-purified antibodies against mouse IRS-4. The immune complexes were collected on 20 µl protein A-Sepharose (Pharmacia, Piscataway, NJ), washed, and released into 50 µl SDS sample buffer by holding them at 100 C for 5 min. The SDS samples were immunoblotted with the antibodies against mouse IRS-4 as described previously (9).

Interspecific mouse backcross mapping
Interspecific backcross progeny were generated by mating (C57BL/6J x M. sprectus)F1 females and C57BL/6J males as previously described (13). A total of 205 N2 mice were used to map the mouse IRS-4 and IRS-3 genes (designated Irs4 and Irs3). DNA isolation, restriction enzyme digestion, agarose gel electrophoresis, Southern blot transfer, and hybridization were performed essentially as previously described (14). The probes consisted of a 500-bp PstI/NheI fragment from the 5'-noncoding region of Irs4 and a 700-bp SacI/NdeI fragment from the 5'-noncoding region of Irs3 (our unpublished results). The Irs4 probe detected fragments of 3.9 and 4.3 kb in PvuII-digested C57BL/6J and M. spretus DNA, respectively. The Irs3 probe detected fragments of 7.0 and 6.0 kb in SphI-digested C57BL/6J and M. spretus DNA, respectively. The presence or absence of the M. spretus-specific fragments were followed in the backcross mice.

A description of the probes and restriction fragment length polymorphisms (RFLP) for the loci linked to Irs4 and Irs3 have been reported previously (15, 16, 17). Recombination distances were calculated using Map Manager, version 2.6.5. (http://mcbio.med.buffalo.edu/mapmgr.html) Gene order was determined by minimizing the number of recombination events required to explain the allele distribution patterns.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cloning the mouse IRS-4 gene
Screening of a mouse genomic library with probes derived from hIRS-4 cDNA yielded a single clone that contained the complete coding region of the mouse gene. Sequencing of this region of the gene showed an open reading frame encoding mouse IRS-4 protein (Fig. 1Go). Thus, as is the case for the IRS-1 and IRS-2 genes (18, 19), the IRS-4 gene contains no introns in the coding region. We have taken the first AUG codon downstream of the in-frame stop codon at nt 100–102 as the site of initiation of translation. This AUG is located within a mouse Kozak consensus sequence for initiation of translation (20). Moreover, the predicted initial amino acid sequence for IRS-4, Met-Ala-Ser, is identical to that for mIRS-1 and IRS-2 (18, 19). With this assignment of the site for initiation of translation, mIRS-4 consists of 1216 amino acids.

Sequence comparison of mIRS-4 and hIRS-4
Previously, we found that hIRS-4, like IRS-1, -2, and -3, has an architecture consisting of an N-terminal PH domain, followed by a PTB domain, followed by a region with many short motifs for tyrosine phosphorylation and SH2 domain binding (7). Comparison of the sequences of mouse and human IRS-4 (Fig. 2Go) showed that the PH and PTB domains are highly conserved. There is 97% identity of amino acids in these domains. The region carboxyl-terminal to the PTB domain is less highly conserved; there is only 70% identity of amino acids in this region, although most of the potential tyrosine phosphorylation/SH2 domain binding motifs in this region (see below and Discussion) are highly conserved. This difference in the percent identity between the different regions of IRS-4 suggests that changes in amino acids in segments interspersed between the tyrosine phosphorylation/SH2 domain binding motifs have less effect on function than changes in amino acids in the PH and PTB domains.



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  		Figure 2. Alignment of the mIRS-4 and hIRS-4. The two sequences were aligned by means of the PILEUP program. Gaps are designated by dashes, and identical amino acids in hIRS-4 are denoted by dots. The PH and PTB domains, previously identified in hIRS-4 (7 ), are overlined, and the tyrosine phosphorylation/SH2 binding motifs (see Table 1Go) are underlined.

 
The potential tyrosine phosphorylation/SH2 domain binding motifs in mouse and human IRS-4 are listed in Table 1Go. The potential sites for tyrosine phosphorylation were selected by the criteria that there be one or more glutamate or aspartate in the five amino acids just amino-terminal to the tyrosine and that the tyrosine not be in the PH or PTB domain. These criteria are derived from the specificity of the insulin receptor for tyrosine phosphorylation of IRS-1; all eight sites of tyrosine phosphorylation that have been identified in IRS-1 conform to these criteria (21). The motifs are given as the tyrosine residue and the following three amino acids, as it is this set of four amino acids that mainly interacts with the SH2 domain (22). The identity of the SH2 domain likely to bind to each motif is based upon the reported specificities of various SH2 domains for such motifs (22, 23). Further analysis of the tyrosine phosphorylation/SH2 domain binding motifs is presented in Discussion.


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  		Table 1. Potential tyrosine phosphorylation/SH2 domain binding motifs in mouse and human IRS-4

 
Tissue distribution of mouse IRS-4
Previously, we were unable to detect the mRNA for human IRS-4 on a Northern blot of mRNA from human tissues (9). This result suggested that the mRNA for mIRS-4 in any tissue would probably be rare, and consequently, we analyzed for expression of the mIRS-4 mRNA using the more sensitive method of RT-PCR with mRNA from various mouse tissues. Preparations of mRNA often contain traces of genomic DNA, and PCR amplification from genomic DNA as the template is usually detected by selection of primers that span an intron. As the coding region of the mIRS-4 gene contains no introns, this experimental design could not be used. Instead, PCR product arising from traces of genomic DNA in the mRNA was detected by running control reactions in which the reverse transcriptase was omitted. As shown in Fig. 3Go, RT and a single PCR amplification yielded a fragment of the expected 176 bp size in skeletal muscle, brain, heart, kidney, and liver that was absent from the control. Testis showed the expected fragment only after a second PCR amplification, and its amount was somewhat greater in the reaction containing reverse transcriptase than in the control. On the other hand, no RT-specific product was detected in pancreas, lung, or spleen. Thus, a detectable amount of IRS-4 mRNA was expressed in skeletal muscle, brain, heart, kidney, liver, and probably testis, but not in pancreas, lung, or spleen.



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  		Figure 3. Tissue distribution of mouse IRS-4 mRNA. Poly(A)+ RNA from several mouse tissues was analyzed for IRS-4 mRNA by RT PCR, as described in Materials and Methods. PCR 1 and 2 denote the first and second amplifications. RT + and - denote the presence or absence of reverse transcriptase in the RT step. A repetition of this experiment gave similar results.

 
Subsequent to the detection of IRS-4 mRNA in these several tissues, we attempted to detect the IRS-4 protein. Detergent extracts of mouse tissues were immunoprecipitated with affinity-purified antibodies against mIRS-4 and then immunoblotted with these same antibodies. As a negative control, extracts of tissues from mice with targeted disruption of the IRS-4 gene were used. The generation and characterization of these IRS-4 knockout mice will be reported later; however, the targeting vector was constructed in such a way that the disrupted gene contains only the coding region for the carboxyl-terminal 152 amino acids of IRS-4, and consequently, no IRS-4 can be expressed. As a positive control, a small amount of HEK 293 cell lysate containing human IRS-4, which we have previously shown is also immunoprecipitated and immunoblotted by the antibodies against mIRS-4 (9), was put into lysate from the liver of knockout mice and immunoprecipitated. The results in Fig. 4Go show that IRS-4 protein was not detectable in any of the tissues. A few tissues (lung, brain, and pancreas) showed a weak band of approximately the expected size, but a band of equal intensity was also present in the samples from the knockout mice. The positive control (lanes 4 and 8) showed the expected human IRS-4 band. Comparison of the intensity of the IRS-4 band in the positive control with that of the IRS-4 band in known amounts of HEK 293 cell lysate (lanes 1–3 and 5–7) indicated that approximately 50% of the IRS-4 in the positive control was recovered upon immunoprecipitation. As there were approximately 5 ng human IRS-4 in the HEK 293 cell lysate used in the positive control, we estimate that the mouse tissues contained less than 3 ng IRS-4/mg protein. We have also attempted to find IRS-4 protein by directly immunoblotting SDS lysates of the mouse tissues with SDS samples of tissues from the knockout mice as the negative control. No IRS-4 was detected in any tissue (data not shown). Since in this experiment 1 ng human IRS-4 in a sample of HEK 293 cell lysate gave a moderate IRS-4 band, whereas 150 µg of the tissue samples gave no IRS-4 band, again we estimate that there was less than 3 ng IRS-4/mg tissue protein. The results indicate that IRS-4 is a nonabundant protein in the tissues. By contrast, we have found that HEK 293 cells contain approximately 300 ng/mg protein (9) and several human breast cancer cell lines (T47, ZR751, and ZR7530) contain approximately 5 ng/mg (Fantin, V., unpublished results).



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  		Figure 4. Immunoprecipitation and immunoblotting of IRS-4 from mouse tissues. Nonionic detergent lysates derived from 2 mg mouse tissues from wild-type (W) and IRS-4 knockout (K) mice were immunoprecipitated and immunoblotted as described in Materials and Methods. Lanes 1 and 5 contained 9 µg HEK 293 cell lysate, which contains about 5 ng human IRS-4 (9 ); lanes 2 and 6, and lanes 3 and 7 contained one half and one fourth this load, respectively. Lanes 4 and 8 contained the immunoprecipitate of the lysate derived from 2 mg liver from knockout mice to which 9 µg HEK 293 cell lysate had been added. WAT, White adipose tissue; BAT, brown adipose tissue; SKM, skeletal muscle (quadriceps); HRT, heart; LUN, lung; PAN, pancreas; LIV, liver; KID, kidney; BRN, brain; SPL, spleen; THY, thymus; TES, testis. The positions of molecular mass standards in kilodaltons are given to the right of the blots. Two repetitions of this experiment gave similar results.

 
Expression of IRS-4 during embryonic development
The expression of IRS-4 mRNA during mouse embryonic development was examined using the RT-PCR method. IRS-4 mRNA was expressed in day 15 and day 17 embryos, but not in day 7 or day 11 embryos (Fig. 5Go). Previously, the expression of IRS-1 and IRS-3 mRNA on days 7, 11, 15, and 17 was analyzed (24). In contrast to IRS-4, IRS-1 mRNA was first found on day 11, and IRS-3 mRNA was first found on day 7. Thus, although the nonabundance of IRS-4 in adult mouse tissues raised the possibility that IRS-4 was expressed mainly in early embryonic development, these results show that this is not the case.



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  		Figure 5. Expression of mIRS-4 mRNA during embryonic development. mRNA from mice on days 7, 11, 15, and 17 were analyzed for IRS-4 mRNA, as described in Materials and Methods. PCR 1 and 2 denote the first and second amplifications. RT + and - denote the presence or absence of reverse transcriptase in the RT step. A repetition of this experiment gave similar results.

 
Chromosomal localization of the mouse IRS-4 and IRS-3 genes
The mouse chromosomal location of Irs4 was determined by interspecific backcross analysis using progeny derived from matings of (C57BL/6J x Mus spretus)F1 x C57BL/6J mice. A similar analysis was performed for Irs3, another mouse IRS gene that we have cloned but have not reported in the literature because while the work was in progress another group described the mouse IRS-3 gene (24). This interspecific backcross mapping panel has been typed for over 2400 loci that are well distributed among all the autosomes as well as the X chromosome (13). C57BL/6J and M. spretus DNAs were digested with several enzymes and analyzed for informative RFLPs using mouse genomic probes. A 4.3-kb PvuII and a 6.0-kb SphI RFLP from M. Spretus were used to follow the segregation of the Irs4 and Irs3 loci, respectively, in backcross mice. The mapping results showed that Irs4 is located in the distal region of the mouse X chromosome linked to Btk and DXPas1 and that Irs3 is located in the distal region of mouse chromosome 5 linked to Gus, Epo, and Pdgfa. In the case of Irs4, 116 mice were analyzed for every marker and are shown in the segregation analysis (Fig. 6Go). Moreover, up to 185 mice were typed for some pairs of markers. Each locus was analyzed in pairwise combinations for recombinant frequencies using the additional data. Similarly, in the case of Irs3, 182 mice were analyzed for every marker and are shown in the segregation analysis (Fig. 7Go); in addition, up to 191 mice were typed for some pairs of markers. The ratios of the total number of mice exhibiting recombinant chromosomes to the total number of mice analyzed for each pair of loci and the most likely gene order are: for Irs4, centromere -Btk –11/185 –Irs4 –9/118 –DXPas1; for Irs3, centromere –Gus –15/185 –Irs3 –0/184 –Epo –7/191 –Pdgfa. The recombination frequencies (expressed in genetic distances in centimorgans (cM) ± the SE) are: for Irs4, -Btk –5.9 ± 1.7 –Irs4 –7.6 ± 2.4 –DXPas1; for Irs3, -Gus –8.1 ± 2.0 –(Irs3, Epo) –3.7 ± 1.4 –Pdgfa. No recombinants were detected between Irs3 and Epo in 184 animals typed in common; this result indicates that the two loci are within 1.6 cM of each other (upper 95% confidence limit).



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  		Figure 6. Irs4 maps in the distal region of the mouse chromosome X. The segregation patterns of Irs4 and flanking genes in 116 backcross animals that were typed for all loci are shown at the top of the figure. For individual pairs of loci more than 116 animals were typed (see text). Each column represents the chromosome identified in the backcross progeny that was inherited from the (C57BL/6J x M. spretus)F1 parent. The black boxes represent the presence of a C57BL/6J allele, and the white boxes represent the presence of a M. spretus allele. The number of offspring inheriting each type of chromosome is listed at the bottom of each column. A partial X chromosome linkage map showing the location of Irs4 in relation to linked genes is shown at the bottom of the figure. Recombination distances in centimorgans are shown to the left of the chromosome, and the positions of loci in human chromosomes, where known, are shown to the right. References for the human map positions of loci can be obtained from the Genome Data Base, a computerized database of human linkage information maintained by The William H. Welch Medical Library of The Johns Hopkins University (http://gdbwww.gdb.org/gdb/).

 


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  		Figure 7. Irs3 maps in the distal region of mouse chromosome 5. See Fig. 6Go for details. In this case the segregation patterns of Irs3 and flanking genes in 182 backcross animals were typed for all loci.

 
Our localization of Irs3 on chromosome 5 agrees with the previous localization of the gene on this chromosome by the method of fluorescence in situ hybridization (24). Moreover, the position on chromosome 5 determined by linkage mapping agrees with determination from fluorescence in situ hybridization that the gene is located at a position that is 89% of the distance from the heterochromatic-euchromatic boundary to the telomer of chromosome 5, in the area corresponding to band 5G2 (information from the Mouse Genome Database (MGD), a computerized database maintained at The Jackson Laboratory (Bar Harbor, ME; http://www.informatics.jax.org/).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have cloned and sequenced the mouse IRS-4 gene. Its lack of introns in the coding region allowed determination of the amino acid sequence of mouse IRS-4. The high degree of conservation between human and mouse IRS-4 in the PH and PTB domains is further evidence for the roles of these in the function of IRS-4. In addition, most of the tyrosine phosphorylation/SH2 domain binding motifs are conserved. Both mouse and human IRS-4 contain six YXXM motifs likely to bind to the SH2 domains of PI 3-kinase; five of these are identical, whereas the sixth is neither identical nor located in the same region of the protein. Mouse and human IRS-4 also contain a conserved, but not identical, YXNX motif specific for binding the SH2 domain of the adaptor protein Grb2 and an identical motif specific for binding the SH2 domains of the tyrosine phosphatase SHP-2 and phospholipase C{gamma}. Previously, we found that tyrosine-phosphorylated hIRS-4 in HEK 293 cells associates with PI 3-kinase and Grb2, but not SHP-2 or phospholipase C{gamma} (9). Possibly the motif specific for SHP-2/phospholipase C{gamma} is not tyrosine phosphorylated by the insulin receptor. Recently, it has been reported that the adaptor proteins CrkL and CrkII associate by their SH2 domain with tyrosine-phosphorylated IRS-4 in HEK 293 cells (25). The Crk SH2 domain has been reported to have high affinity for the YXXP motif (22), but this motif does not occur in IRS-4, so there is no evident site for Crk binding. Lastly, mouse and human IRS-4 contain an identical YRAR motif at the carboxyl-terminal border of the PTB domain and a conserved YDAQ(E) motif near the carboxyl-terminus. These motifs do not conform to the specificity known for any SH2 domain (22, 23), but may be sites to which as yet unidentified SH2 domain proteins bind.

The cloning and sequencing of the IRS-4 gene enabled generation of the primers, antibodies, and probe needed to analyze for the expression of IRS-4 mRNA and protein and for the chromosomal location of the gene in the mouse. RT-PCR showed detectable amounts of mRNA in some, but not all, of the major mouse tissues. On the other hand, the IRS-4 protein was too low in abundance to detect in any tissue. Previously, the tissue expressions of mouse IRS-1, -2, and -3 have been examined by Northern blotting (5, 24) and in the case of IRS-1 and -2 for some tissues by immunoblotting (18). IRS-1 and -2 mRNA were expressed in all tissues examined (skeletal muscle, brain, heart, kidney, liver, lung, testis, and spleen), whereas the mRNA for IRS-3 had a more limited tissue distribution (detectable in heart, kidney, liver, and lung, but not in skeletal muscle, brain, testis, or spleen). Thus, IRS-3 and IRS-4 are the members of the family with somewhat selective tissue expression.

In addition to the issue of sites of tissue expression for the various IRSs, there is the issue of relative abundance of the IRS proteins in the tissues where they are expressed. A rigorous answer to this question will require immunoblotting each tissue for each IRS together with known amounts of the recombinant IRS, so that the signal from the tissue can be converted to nanograms of IRS. This type of analysis has not yet been performed. However, on the assumption that the available antibodies to each of the IRSs can detect it in the nanogram range, which is the case with our antibodies against IRS-4 (see Results), then IRS-1 and IRS-2 are considerably more abundant than IRS-4. In contrast to IRS-4, IRS-1 and IRS-2 protein have been detected by immunoprecipitation and immunoblotting in mouse liver, skeletal muscle, brain, adipocyte, and testis (18). In the case of IRS-3, IRS-3 protein has been detected in rat adipocytes by immunoprecipitation and immunoblotting (26), but other mouse tissues have not yet been examined. It is possible that the nonabundance of IRS-4 protein in some tissues reflects its expression in only a single cell type in that tissue. In the future it should be possible to determine whether this is the case by immunofluorescence analysis of tissues from wild-type and IRS-4 knockout mice. Unfortunately, the inability to detect the IRS-4 protein in mouse tissues means that it will be more difficult to analyze tissues for tyrosine phosphorylation of IRS-4 and the association of IRS-4 with SH2 domain proteins in response to insulin treatment of mice. To date, the IRS-4 protein has only been detected in HEK 293 cells (7) and in some human breast cancer cell lines (Fantin, V., unpublished results).

The mouse IRS-4 and IRS-3 genes were localized on chromosomes X and 5, respectively. We have compared the chromosomal sites of Irs4 and Irs3 with a composite linkage map that reports the map location of many uncloned mouse mutations (provided from MGD). Both Irs4 and Irs3 map in regions of the composite map that lack mouse mutations with a phenotype that might be expected for alterations in these loci. The distal region of the mouse X chromosome where Irs4 is located shares a region of homology with both the long and short arms of the human chromosome (provided from MGD). Consequently the human IRS-4 gene (IRS4) was expected to map to the human X chromosome. In agreement with this expectation, a recent abstract has reported that IRS4 is located on the long arm of chromosome X (27); this study and another study (28) report that genetic variability at the IRS4 locus is unlikely to play a major role in the etiology of type 2 diabetes. The distal region of mouse chromosome 5 where Irs3 is located shares homology with human chromosome 7. The tight linkage in mouse between Irs3 and Epo, which has been mapped to human 7q21.3-q22.1, suggests that the human IRS-3 gene (IRS3) will map to this region of the long arm of human chromosome 7 as well. We have looked at the genetic diseases known to map in this region (the Online Mendelian Inheritance in Man database of the National Center for Biotechnology Information; http://www.ncbi.nlm.nih.gov/). None has characteristics that suggest a mutation in IRS3. Previously it was determined that the mouse genes for IRS-1 and IRS-2 are on chromosomes 1 and 8, respectively (18, 29), and thus each mIRS gene is on a different chromosome.

The existence of four members of the IRS family raises the question of the physiological roles of each in insulin action as well as in the action of insulin-like growth factor I and other agents that elicit the tyrosine phosphorylation of IRSs. Targeted disruptions of the IRS-1 and -2 genes have shown partially different roles for these two IRSs (30, 31, 32). Mice lacking IRS-1 are substantially growth retarded and exhibit insulin resistance, but do not develop diabetes. Mice lacking IRS-2 show only slight growth retardation, also exhibit insulin resistance, and in addition develop type 2 diabetes associated with a failure of the pancreatic ß-cells to proliferate. We have recently generated mice with targeted disruptions of the IRS-3 and IRS-4 genes, and now are in the midst of examining their phenotypes (unpublished). The detailed characterization of mice lacking the individual IRSs as well as mice lacking pairs of IRSs should eventually reveal the roles of each IRS.


   Acknowledgments
 
We thank Deborah B. Householder for excellent technical assistance with the interspecific backcross analysis, and Nicholas J. Morris for excellent assistance with computing.


   Footnotes
 
1 This work was supported by NIH Grant DK-42816 (to G.E.L.) and by the National Cancer Institute under contract with ABL (to N.G.C.). Back

2 Present address: Metabolex, 3876 Bay Center Place, Hayward, California 94545. Back

3 Present address: Department of Anesthesiology, Sinai Hospital of Detroit, 6767 Outer Drive, Detroit, Michigan 48235. Back

Received September 8, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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