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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
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Introduction |
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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.
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Materials and Methods |
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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. 1
) 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. 1).
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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), 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. 1) 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.
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Results |
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). 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. 2) 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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. 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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, 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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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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). 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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). 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. 7); 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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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. 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 (9). Possibly
the motif specific for SHP-2/phospholipase C 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.
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Acknowledgments |
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Footnotes |
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2 Present address: Metabolex, 3876 Bay Center Place, Hayward,
California 94545.
3 Present address: Department of Anesthesiology, Sinai Hospital of
Detroit, 6767 Outer Drive, Detroit, Michigan 48235.
Received September 8, 1998.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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