The IRS-2 Gene on Murine Chromosome 8 Encodes a Unique Signaling Adapter for Insulin and Cytokine Action
Xiao Jian Sun1,
Sebastian Pons,
Ling-Mei Wang,
Yitao Zhang,
Lynne Yenush,
Deborah Burks,
Martin G. Myers, Jr.,
Erin Glasheen,
Neal G. Copeland,
Nancy A. Jenkins,
Jacalyn H. Pierce and
Morris F. White
Research Division, Joslin Diabetes Center (X.J.S., S.P., Y.Z.,
L.Y., D.B., M.G.M., E.G., M.F.W.) and Department of Medicine
Harvard Medical School Boston, Massachusetts 02215
Laboratory of Cell and Molecular Biology (L-M.Y., J.H.P.)
National Institutes of Health Bethesda, Maryland 20892
Mammalian Genetics Laboratory (N.G.C., N.A.J.) ABL-Basic
Research Program National Cancer Institute-Frederick Cancer
Research and Development Center Frederick, Maryland 21702
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ABSTRACT
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Signal transduction by insulin and IGF-1, several
interleukins (IL-2, IL-4, IL-9, IL-13), interferons, GH, and other
cytokines involves IRS proteins, which link the receptors for these
factors to signaling molecules with Src homology-2 domains
(SH2-proteins). We recently reported the amino acid sequence of murine
IRS-2; in order to examine a potential genetic role for this molecule
in disease, we isolated the murine IRS-2 gene and compared the
expression pattern of IRS-2 against IRS-1. Like IRS-1, IRS-2 is encoded
by a single exon. Whereas IRS-1 is located on murine chomosome 1, IRS-2
is located on murine chromosome 8 near the insulin receptor. IRS-2 is
expressed together with IRS-1 in many cells and tissues; however, IRS-2
predominates in murine hematopoietic cells where it may be essential
for cytokine signaling; IRS-1 predominates in adipocytes and
differentiated 3T3-L1 cells where it contributes to the normal insulin
response. In 32D cells, IRS-1 and IRS-2 undergo differential tyrosine
phosphorylation during insulin or IL-4 stimulation, as assessed
indirectly by interaction with various recombinant SH2 domains. Thus,
signaling specificity through the IRS proteins may be accomplished by
specific expression patterns and distinct phosphorylation patterns
during interaction with various activated receptors.
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INTRODUCTION
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The identification of downstream elements controlling cellular
growth and metabolism is a central issue in contemporary biology.
Common themes are emerging to explain how these processes are linked to
growth factor, hormone, or cytokine receptors with intrinsic or
associated tyrosine kinase activity (1, 2). Ligand-induced receptor
dimerization stimulates tyrosine autophosphorylation of growth factor
receptors or various components in cytokine receptor complexes. In many
cases, these phosphorylation sites selectively bind to the Src
homology-2 (SH2) domain in the various enzymes or adapter molecules
that mediate biological responses (2, 3, 4, 5).
In a few cases, notably insulin and insulin-like growth factor 1
(IGF-1), receptor autophosphorylation correlates closely with increased
kinase activity but poorly with the recruitment of most SH2-proteins to
the receptor (6, 7). In contrast, tyrosine phosphorylation of the IRS
proteins (IRS-1 and IRS-2) provides an interface between these
receptors and various SH2-proteins (8, 9). IRS proteins are also
phosphorylated by Jak kinases, which are activated by the receptors for
various cytokines, including interleukins (IL-2, IL-4, 3IL-9,
IL-13), interferons (IFN
, IFNß and IFN
), and GH. During insulin
stimulation the phosphorylation of multiple tyrosine residues in the
COOH terminus of IRS-1 enables the binding of several SH2 proteins,
including the PI-3 kinase-regulatory subunits
(p85
/p85ß/p55PIK), GRB-2, nck, c-fyn, and
SHPTP2 (10, 11, 12, 13, 14). As a consequence of these and other interactions,
IRS-1 mediates multiple downstream signals, including the direct
activation of PI-3 kinase and SHPTP2, the indirect stimulation of
mitogen-activated protein kinase and p70s6k, and other
events that regulate gene expression and stimulate protein synthesis,
mitogenesis, and glucose transport (6, 15, 16, 17, 18, 19, 20).
IRS-2 was difficult to identify using several standard approaches,
including expression screening, RT-PCR, and low stringency cDNA or
genomic screening. However, purification of 4PS, an insulin/IL-4
receptor substrate in FDC-P2 cells, allowed the cloning of a cDNA that
encodes a protein with several structural and functional features in
common with IRS-1 (21). IRS-2 appears to be especially important in
mice lacking IRS-1, as IRS1(-/-) mice survive, reproduce,
and display only mild insulin resistance (22, 23). Hepatocytes from
these animals reveal increased tyrosine phosphorylation of IRS-2 and
retain a significant responsiveness to insulin and IGF-1 (22, 23).
However, muscle from the IRS1(-/-) mouse is significantly
insulin resistant and does not display a compensatory increase in
IRS-2 phosphorylation (24). Thus, the distinct expression of IRS-1
and IRS-2 may further contribute to their unique signaling potential
and importance for survival.
In this paper we characterize and analyze the murine IRS-2 gene and
investigate the potential for alternate signaling by IRS-1 and IRS-2 in
different cell types and in response to different upstream signals. The
IRS-2 gene is located on murine chromosome 8 in a location close to the
insulin receptor; the coding region is contained in a single exon.
Although IRS-2 possesses similar structural features to IRS-1, the
tyrosine phosphorylation of IRS-1 and IRS-2 by insulin and IL-4 is
qualitatively different.
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RESULTS
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The structure and Location of the Murine IRS-2 Gene
We recently described an optimized 45-mer nucleotide probe based
on a partial amino acid sequence of 4PS that was used to isolate
overlapping cDNA molecules encoding a consensus cDNA for IRS-2 (21).
This oligonucleotide was also used to isolate a 17-kb genomic fragment
(mG28) from a murine genomic library. Several restriction fragments of
mG28 were isolated, subcloned, and sequenced (Fig. 1A
). Restriction mapping and PCR
analysis confirmed that the fragments obtained from the genomic DNA
corresponded exactly to the partial cDNA sequences obtained previously
from various murine libraries (Fig. 1A
). Translation of the open
reading frame obtained from the genomic sequence revealed 1324 amino
acids, including three additional residues at the COOH terminus that
were not previously described (Fig. 1B
) (21). The 5'-untranslated
region of the IRS-2 gene contains two GC-rich regions that proved
difficult to sequence accurately and remain undefined (Fig. 1B
).


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Figure 1. The Molecular Cloning of IRS-2 Genomic DNA
Panel A; IRS-2 genomic DNA was obtained by screening the mouse genomic
library with IRS-2 cDNA. The solid black bars indicate the
restriction fragments hybridized with IRS-2 cDNA and PCR products.
Shaded bars indicate the regions that have been sequenced.
Start and stop codons and the location of 4PS peptides are indicated.
As usual, positive numbers begin at the start codon and negative
numbers indicate the sequence before the start codon into the
5'-untranslated region. Panel B; Genomic DNA and deduced protein
sequence of mouse IRS-2. The putative 145-kDa open reading frame of
IRS-2 is shown beginning with a Kozac start site and ending at an
in-frame stop codon (*). The IRS-homology domains (IH1PH
and IH2PTB) are shaded; potential tyrosine
phosphorylation sites are highlighted as white characters on
black backgrounds.
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Southern analysis of 129-mouse genomic DNA with a 5'-end IRS-2 probe
(-2525 to 423) revealed a single AflII fragment and two
SacI fragments (2 kb and 6 kb); SmaI digestion
revealed two fragments of 0.72 kb and 3.7 kb, which hybridized with a
3'-end probe (2777 to 4468) (Fig. 2A
). This pattern is
consistent with the conclusion that IRS-2 is encoded by a single gene
in the 129 mouse as diagramed in Fig. 1A
.

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Figure 2. Southern Blot and Chromosome Localization
Panel A, Mouse genomic DNA was obtained from liver and digested with
the indicated restriction enzymes. The restriction fragments were
blotted with IRS-2 cDNA as indicated in the figure. Panel B,
Chromosomal localization of the mouse Irs2 gene.
Irs2 was placed on mouse chromosome 8 by interspecific
backcross analysis. The segregation patterns of Irs2 and
flanking genes in 90 backcross animals that were typed for all loci are
shown at the top of the figure. For individual pairs of
loci, more than 90 animals were typed (see text). Each column
represents the chromosome identified in the backcross progeny that was
inherited from the hybrid parent. Shaded boxes represent the
C57BL/6J allele and white boxes represent the M.
spretus allele. The number of offspring inheriting each type of
chromosome is listed at the bottom of each column. A partial
linkage map of chromosome 8 is shown. Recombination distances between
loci are shown in centimorgans on the left and the human
chromosomal positions, where known, are shown on the right.
References for the human map positions used in this study can be
obtained from GDB, a computerized database of human linkage information
maintained by the William H. Welch Medical Library of the Johns Hopkins
University (Baltimore, MD).
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We mapped the chromosomal location of the murine Irs2 gene
by following a BamHI restriction fragment length
polymorphism (RFLP) during interspecific backcross analysis on
(C57BL/6J x M. spretus)F1 x C57BL/6J
progeny. The mapping results indicated that Irs2 is located
in the proximal region of murine chromosome 8, linked to the
Insr, Col4al, and Plat loci. Although
90 mice were analyzed for every marker (shown in the segregation
analysis, Fig. 2B
), up to 181 mice were characterized for some pairs of
markers; each locus was analyzed in pairwise combinations for
recombination frequencies using the additional data. The ratios of the
total number of mice exhibiting recombinant chromosomes to the
total number of mice analyzed for each pair of loci suggests the
following gene order: centromere
Insr
(1/95)
Irs2 (0/142)
Col4al
(8/181)
Plat (Fig. 2B
). The lack of recombinations
detected between Irs2 and Col4al in this number
of animal suggests that the two loci are within 2.1 centiMorgans (cM)
of each other at the 95% confidence limit. The recombination
frequencies reported as the genetic distance in centiMorgans ±
SE is 1.1 ± 1.1 between the Insr loci and
Irs2/Col4al, and 4.4 ± 1.5 between
Irs2/Col4al and Plat (Fig. 2B
).
Downstream Signaling Specificity of IRS-1 and IRS-2
The amino acid sequence identity in the COOH terminus of IRS-2 and
IRS-1 is only 35%, which arises largely from similar tyrosine
phosphorylation motifs surrounded by variable stretches of amino acid
sequence (21). To explore possible signaling diversity in IRS-1 and
IRS-2 at the level of tyrosine phosphorylation, we prepared a panel of
GST-fusion proteins containing SH2-domains from various signaling
molecules and tested their ability to bind to IRS-2 or IRS-1 expressed
in 32DIR/IL4R cells. After insulin or IL-4 stimulation,
cell lysates were incubated with GST-SH2 fusion proteins and the
associated IRS-2 and IRS-1 were measured by immunoblotting with
PY
(25).
The NH2-terminal SH2 domain of p85 bound strongly to both
IRS-2 and IRS-1 during stimulation with insulin (Fig. 3
); insulin and IL-4 stimulated similar amounts of IRS-2
binding to p85, while insulin stimulated more binding to IRS-1 than
IL-4 did. Similar results were observed with the SH2 domain of
c-fyn. However, the SH2 domains from Crk, phospholipase C,
and GRB-2 revealed functional differences. Each SH2 domain bound IRS-1
more strongly than IRS-2 during insulin and IL-4 stimulation. Moreover,
IRS-1 bound more tightly during insulin stimulation than during IL-4
stimulation, whereas IRS-2 bound equally during insulin and IL-4 (Fig. 3
). The SH2 domains of Abl and SHP-2 associated only with IRS-1, and
then only during insulin stimulation (Fig. 3
). These results suggest
that IRS-2 and IRS-1 may be phosphorylated differently by various
receptors and engage a unique cohort of SH2 proteins.

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Figure 3. Differential Binding of SH2 Domain Proteins to IRS-1
or IRS-2
32DIR,IL4R/IRS-1 and 32DIR,IL4R/IRS-2 cells
matched for expression of IRS-1, IRS-2, IR, and IL4R were incubated
without or with 100 nM insulin for 1 min or 10
nM IL-4 for 10 min; these conditions were shown previously
to give maximal phosphorylation. Cell extracts were precipitated with
the indicated GST-fusion proteins as described in Materials and
Methods, separated by SDS-PAGE, and immunoblotted with PY.
Phosphorylated IRS-1 and IRS-2 are indicated. These data are
representative of two similar experiments.
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Distribution of IRS-1 and IRS-2 in Cells and Tissues
IRS-1 and IRS-2 may regulate unique signaling pathways due, in
part, to their distinct cellular distribution in addition to the unique
interactions with downstream signaling elements. To examine the effect
of differentiation on IRS-1 and IRS-2 expression, three cell culture
systems were investigated, including 3T3-L1 cells, P19 embryonic
carcinoma cells, and Bal-17 lymphocytes. The 3T3-L1 adipocyte is
frequently used to study the mechanism of insulin-stimulated glucose
uptake, as several metabolic responses acquire insulin sensitivity
during differentiation into adipocytes (26). Before differentiation of
3T3-L1 fibroblasts, IRS-1 and IRS-2 were barely detected by
immunoblotting with
PY during insulin stimulation (Fig. 4A
). However, after differentiation, the amount of IRS-1
increased dramatically, and insulin robustly stimulated its tyrosine
phosphorylation; however, IRS-2 was barely detected and its
insulin-stimulated tyrosine phosphorylation was quite low (Fig. 4A
).
Consistent with this observation, IRS-1 was the predominant
phosphorylated IRS protein in isolated rat adipocytes during insulin
stimulation; however, a small amount of p85 was detected in
IRS2
immunoprecipitates after insulin stimulation, suggesting that a small
amount of IRS-2 is expressed in this background (Fig. 4B
). Whether this
low expression of IRS-2 is sufficient to rescue insulin signaling in
murine adipocytes from the IRS-1(-/-) mouse is unknown,
but future experiments should reveal whether other pathways such as the
tyrosine phosphorylation of p60 or Gab1 are involved (27, 28).
IGF-1 plays an important role in neuronal differentiation and survival
(29). Retinoic acid-induced differentiation of P19 embryonic carcinoma
cells provides a cell culture model of neuronal differentiation (30).
Before induction with retinoic acid, IRS-1 and IRS-2 were expressed
approximately equally in P19 cells, and both proteins were tyrosine
phosphorylated and associated with p85 during IGF-1 stimulation.
However, 2 days after retinoic acid removal, while the cells continue
to differentiate, the IRS-2 expression fell whereas IRS-1 expression
was unchanged (Fig. 5
). This change in relative
expression continued until IRS-1 predominated in the fully
differentiated P19 neurons (6 days after retinoic acid removal).
Consistent with these results, IGF-1 strongly mediated IRS-1
phosphorylation in the P19 neurons and IRS-1 associated with p85 (Fig. 5
). It will be interesting to determine whether the reduced expression
of IRS-2 is required for retinoic acid-induced differentiation in P19
cells.
Finally, we investigated the expression and function of IRS-1 and IRS-2
in differentiated, but proliferating, Bal-17 cells. Bal-17 cells are
mature B lymphocytes that are ordinarily activated by cross-linking of
the surface IgM with specific IgM antibodies (31). Insulin strongly
stimulated IRS-2 tyrosine phosphorylation in these cells, whereas IRS-1
was barely detected (Fig. 6
). Interestingly,
cross-linking surface IgM with
IgM induced weak (relative to
insulin) tyrosine phosphorylation of IRS-2 (Fig. 6
, lanes g-i). The
effect of
IgM was specific as nonimmune serum had no effect,
suggesting that IRS-2 may function downstream of the B cell antigen
receptor (Fig. 6
, lanes a-c). We also observed the tyrosine
phosphorylation of IRS-2 in isolated B cells during anti-IgM
stimulation (data not shown).

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Figure 6. Expression and Phosphorylation of IRS-1 and IRS-2 in
Bal-17 Cells
Bal-17 cells were stimulated with 100 nM insulin or 15
µg/ml anti-IgM ( IgM) for 2 min. Cell extracts were
immunoprecipitated with nonimmune serum (control), or specific
antibodies against IRS-1 and IRS-2; tyrosine phosphorylation was
detected by immunoblotting with PY.
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In summary, IRS-2 was expressed in nearly all cells that we analyzed by
RT-PCR or immunoblotting, including lymphoid and myeloid progenitor
cells, B cells, carcinoma cells, fibroblasts, liver, skeletal muscle,
and brain (Table 1
). However, IRS-2 was conspicuously
absent from a few cell lines of lymphoid origin, including Daudi and
ABMC1 (B cell lines), and CT6 and EL4 (T cell lines). IRS-2 was barely
detected in rat testis and surprisingly weak in 3T3-L1 cells and rat
adipocytes (Table 1
). By comparison, IRS-1 was also broadly expressed;
however, it was absent from several lymphoid cell lines and all cells
of the myeloid lineage that were tested (Table 1
). Differential
expression was also observed in several carcinoma cell lines: IRS-2 was
highly expressed in three colon carcinoma cells (clone-A, CX-1, RKO),
whereas only clone A coexpressed IRS-1; both IRS-1 and IRS-2 were
expressed relatively highly in several mammary carcinoma cell lines
where they may contribute to their prolonged survival (Table 1
).
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Table 1. Relative Detection of IRS-1 and IRS-2 in
Various Cell Lines and Tissues from Mice (m), Rats (r), Humans (h), or
Hamsters (ham)
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DISCUSSION
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The identification of IRS-2 and its alignment against
IRS-1 established the modular structure and function of the IRS
proteins (21). Additional proteins in this family are expected to
contain at least one interaction domain, either a pleckstrin homology
(PH) domain or a phosphotyrosine binding (PTB) domain, and a COOH
terminus with multiple tyrosine phosphorylation sites. Thus, mammalian
Gab-1 and Drosophila DOS, each possessing a single PH domain
and a tail with tyrosine phosphorylation sites, fit this general
paradigm and may be considered to be members of the IRS family (28).
Teleologicaly, IRS proteins provide an evolutionary signaling advantage
in several ways: 1) They provide a means for signal amplification by
eliminating the stoichiometric constraints encountered by receptors
that directly recruit SH2 proteins to their autophosphorylation sites.
2) IRS proteins may dissociate the intracellular signaling complex from
the endocytic pathways of the activated receptor. 3) The ability of a
single receptor to engage multiple IRS proteins (each with potentially
different signaling characteristics) expands the repertoire of
signaling pathways that can be regulated. 4) The shared use of IRS
proteins by multiple receptors provides a mechanism to integrate
several receptor systems through one signaling complex to generate a
coordinated cellular response.
IRS-1 and IRS-2 appear to interact with a similar cohort of cellular
membrane receptors, including insulin/IGF-1, several ILs, IFNs, and GH
(9). During stimulation of these receptors with their cognate ligands,
the IRS proteins undergo tyrosine phosphorylation and associate with
signaling proteins that contain SH2-proteins. Although the overall
amino acid sequence identity between IRS-2 and IRS-1 is 43%, the
alignment shows regions of striking homology within the
NH2-terminal third of the molecule (21). Translation of the
coding region of IRS-2 reveals at least three functional regions. Two
highly conserved regions occur in the NH2-terminal portion
of IRS-1 and IRS-2: the first IRS-Homology region
(IH1PH) contains a PH domain; the second,
IH2PTB, contains a PTB domain (32). Recent experiments
indicate that both regions couple IRS-1 to the insulin receptor in 32D
cells; however, the IH1PH region provides the most
efficient coupling (33). Another region (between residues 313 and 462
of IRS-1) was aligned recently to the PTB domain in Shc and designated
the "SAIN" domain (34, 35, 36); however, this region is poorly
conserved between IRS-1 and IRS-2 and does not function as a PTB domain
(33).
By contrast, the COOH-terminal regions of IRS-1 and IRS-2 are poorly
conserved, displaying only 35% identity (21). The middle of IRS-2
contains a unique region that interacts specifically with the
phosphorylated regulatory loop of the insulin receptor (35). This
region, which is absent from IRS-1, may alter the phosphorylation
pattern of the COOH terminus by restricting the flexibility of IRS-2 in
the catalytic domain. On the other hand, several tyrosine
phosphorylation sites in IRS-2 align with similarly spaced sites in
IRS-1 (21). In several cases, the amino acid sequence around the
tyrosine residues are nearly identical in IRS-2 and IRS-1, including
the NH2-terminal acidic residues and the COOH-terminal
hydrophobic residues. In at least half of these cases, however, either
the relative position of the acidic or hydrophobic residues are
different; either change is likely to alter the interaction of the
phosphorylation site with upstream kinases or downstream SH2-proteins,
effectively changing the signal. The comparative association of IRS-1
and IRS-2 with various SH2 domains observed in our experiments is
consistent with these predictions.
The mIRS-2 gene lies near the type 1 procollagen 4a locus
(Col4al), proximal to the centromere on mouse chromosome 8
(37, 38). We have compared our interspecific map of chromosome 8 with a
composite mouse linkage map that reports the map location of many
uncloned mouse mutations (provided from the Mouse Genome Database,
maintained by the Jackson Laboratory, Bar Harbor, ME). Irs2
maps in a region that lacks mouse mutations that might be expected for
an alteration in this locus (data not shown). By contrast,
Irs1 is located on mouse chromosome 1, and the human gene is
on human chromosome 2q36-37 (39). A few point mutations have been found
in the human IRS-1 gene, but their effect on function is unclear (40, 41). It will be important to determine whether mutations in the human
IRS-2 gene contribute to insulin resistance of non-insulin-dependent
diabetes mellitus or to immune system disorders. Toward this end, we
have isolated a partial clone with a high degree of identity to murine
IRS-2, which may be the human counterpart (M. F. White and D. Bernal,
unpublished data).
The proximal region of mouse chromosome 8 shares regions of homology
with human chromosomes 8p, 13q, and 19p. In particular,
Col4al, the locus most closely linked with Irs2,
has been placed on human 13q34, suggesting that Irs2 may
reside there as well. However, it is interesting that Irs2
lies close to the murine insulin receptor. The Drosophila
insulin receptor contains a COOH-terminal extension of 400 residues
with similarity to the first half of the COOH-terminus of IRS-2 (42).
It is possible that the evolutionarily early insulin receptor contained
such a tail, and that during the evolution of higher organisms, this
tail (which remains part of the Drosophila receptor)
separated from the receptor and was reassembled into the IRS-2 gene.
Based on the proximity and functional relation between the insulin
receptor and IRS-2, hIrs2 may be adjacent to the human
insulin receptor on chromosome 19.
Broad distribution of IRS proteins and their interaction with various
receptor systems suggests that they are essential for maintaining cell
survival and growth and normal metabolic regulation (9, 11). However,
our finding that phosphorylated IRS-1, but not IRS-2, increases during
differentiation of 3T3-L1 fibroblasts into adipocytes suggest that
IRS-1 and IRS-2 may not mediate identical signals. Because IRS-1
predominates in adipocytes, it may best mediate metabolic effects of
insulin in this cell context, including the stimulation of glucose
transport and inhibition of lipolysis. The predominance of IRS-2 in
hematopoietic cells suggests that it may be preferred by cytokine
receptors and be best adapted to mediate mitogenesis. This hypothesis
is consistent with our previous finding that IRS-2 mediates a very
sensitive mitogenic response to IL-4 (21). Thus, although IRS-1 and
IRS-2 may be serving redundant roles in some circumstances, they
perform physiologic functions in several instances based upon tissue
distribution. Not only are IRS-1 and IRS-2 differentially expressed,
but our analysis using various SH2 proteins to probe phosphorylation
site characteristics suggests that IRS-1 and IRS-2 are phosphorylated
differently and may recruit alternate downstream SH2-proteins.
Furthermore, the stimulating factor (IL-4 or insulin) alters the
association of SH2 proteins with IRS-1 and IRS-2. Similar differences
may also occur during interaction with other IL, IFN, or GH
receptors.
Thus, signaling by IRS-1 and IRS-2 varies by 1) tissue type, 2) growth
factor, and 3) elements directed by the differing structures of the two
IRS proteins. The ability to switch between IRS-1 or IRS-2 in cell
context-dependent manner provides a unique mechanism for signal
diversity that would be absent from classic receptors that engage SH2
proteins directly at their autophosphorylation sites. While it is
interesting to note that the IRS-1(-/-) mouse displays
disordered growth and metabolism, no immune system dysfunction has been
detected, which is consistent with the general absence of IRS-1 from
hematopoietic cells. However, based on the expression of IRS-1 in
classical insulin target tissues, it is not surprising that IRS-2
cannot completely compensate for the absence of IRS-1 for carbohydrate
metabolism (22, 23). The relative importance of IRS-2 and IRS-1 for
growth and development and metabolic regulation awaits the generation
of IRS-2(-/-) mouse and direct comparison and mating with
the IRS-1(-/-) mouse. Moreover, the recent discovery that
IRS-1, but not IRS-2, may mediate the inhibitory effect of tumor
necrosis factor-
(TNF
) on the insulin receptor suggests that the
IRS proteins define and regulate the crossroads at which many diverse
signaling systems converge and diverge (43).
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MATERIALS AND METHODS
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Cell Culture
All the cells were incubated at 37 °C in a humidified
atmosphere composed of 5% CO2 and 95% air, except 3T3-L1
cells, which were maintained in an incubator with 10% CO2.
IL-3-dependent FDC-P1, FDC-P2, and 32D cells were maintained in
RPMI-1640 medium (GIBCO Life Technologies, Inc., Gaithersburg, MD)
containing 10% FBS (Sigma, St. Louis, MO) and 5% WEHI-3-conditioned
RPMI-1640 medium as a source of IL-3 (44). Cell lines expressing the
human insulin receptor, human IL-4 receptor (IL-4R
), and IRS-1 have
been described (6, 20). Bal-17 B lymphoma cells were maintained in
RPMI-1640 medium supplemented with 10% FBS, L-glutamine (2
mM), and 2-mercaptoethanol (50 µM). The
3T3-L1 cells were maintained in DMEM high-glucose supplemented with
10% bovine calf serum; 2 days post-confluence, differentiation was
induced by incubating the cells for 3 days in DMEM supplemented with
10% FBS, 5 µg/ml insulin, 1 µM dexamethasone.
p19 cultures were performed as previously described (30). Briefly,
undifferentiated p19 cells were grown on tissue culture quality plastic
in DMEM-F12 containing 10% FCS. Differentiation of p19 cells was
induced by plating in the presence of 1 µM retinoic acid
for 4 days on bacterial quality dishes (to which cells do not attach).
At the end of the fourth day the aggregates were decanted, plated on
tissue culture quality plastic, and allowed to differentiate for 4
days.
32D cells or cells expressing the human receptors for insulin
and IL-4 (32DIR/IL4R) were transfected with
pCMVhisIRS-2 or pCMVhisIRS-1 by
electroporation, as previously described (20, 45). Cells were selected
by survival in 5 mM histidinol (Sigma), and expression of
IRS-2 and IRS-1 was monitored by immunoblotting.
Epididymal adipocytes were isolated from Spraque-Dawley rats (280300
g) using a modification of the original methods described by Rodbell
(46). Fat was dissected and collected in modified Krebs Ringer
bicarbonate (buffer A), supplemented with 10 mM HEPES
(Sigma) 2.5% BSA (Sigma), and 200 nM adenosine (Sigma).
The tissue was briefly minced, and then digested for 40 min in a 37
°C shaker bath set at 10 rpm/6 sec by adding 2 mg collagenase
(Worthington Biochemical, Freehold, NJ) per gram of tissues (47). The
digested tissue was passed through a 50-µm nylon screen and rinsed
several times with buffer A to remove the collagenase, then rinsed
several times with BSA-free buffer A. Equal volumes of adipocytes are
treated without or with 80 nM insulin for 5 min with gentle
rotation in a 37 °C water bath.
Southern Analysis of Mouse Genomic DNA
Genomic DNA (10 µg) from mouse liver (48) was digested
overnight with various restriction enzymes, resolved by
electrophoresis, and transferred to Hybond N membranes (Amersham,
Arlington Heights, IL) for Southern analysis. Two specific DNA probes,
which contain the sequences corresponding to approximately -2525 to
423 bp, and 2777 to 4468 bp of IRS-2, were obtained by digesting mG28
with KpnI. The KpnI fragments were isolated and
labeled with [32P]phosphate as previously described (48).
Hybridization was conducted overnight at 40 °C in 5 x saline
sodium citrate (SSC), 40% formamide, 5x Denhardts, 0.1% SDS, and
100 ng/ml of salmon sperm DNA. Final washing of the blots was at 65
°C in 0.5 x SSC containing 0.1% SDS.
Interspecific Mouse Backcross Mapping
Interspecific backcross progeny were generated by mating
(C57BL/6J x M. spretus)F1 females and
C57BL/6J males as described (49). A total of 205 N2 mice
were used to map the Irs2 locus. This mapping panel has been
typed for more than 2000 loci that are distributed among all of the
autosomes and the X chromosome (49). DNA isolation, restriction enzyme
digestion, agarose gel electrophoresis, Southern blot transfer, and
hybridization were performed as described (50). An approximately 300-bp
BamHI/EcoRI mouse cDNA fragment was labeled with
[32P]dCTP using a nick translation kit (Boeringer
Mannheim, Indianapolis, IN) and used as probe; the final wash
stringency was 0.5x saline sodium citrate phosphate, 0.1% SDS, 65
°C. The probe detected 13 kb and 20 kb BamHI fragments
from C57B1/6J and M. spretus DNA, respectively. The 20-kb
M. spretus-specific fragment was followed in backcross
mice.
A description of the probes and RFLPs for the linked insulin receptor
(Insr) and tissue plaminogen activator (Plat)
loci has previously been reported (37). The probe for type IV
1
procollagen (Col4al) has not previously been reported; it
was a 1.9-kb EcoRI fragment which detected 9.7 (C57BL/6J)
and 8.6 (M. spretus) kb XbaI fragments.
Recombination distances were calculated as described (51) using the
SPRETUS MADNESS program. Gene order was determined by minimizing the
number of recombination events required to explain the allele
distribution patterns.
Immunoprecipitation and Immunoblot Analysis
Quiesent cells were incubated for 1 min in the absence or
presence of 100 nM insulin or 10 nM IL-4, and
lysed in homogenization buffer. The lysates were incubated with
polyclonal antibodies, and the immune complexes were collected on
protein A and washed three times with homogenization buffer, denatured,
separated by 7.5% SDS-PAGE, and transferred to nitrocellulose
membranes (Schleicher & Schuell, Keene, NH) for immunoblotting (52).
The antibodies were prepared in rabbits (HRP Corp., Denver, PA) as
previously described (10). The
IRS-2 was obtained using a GST-fusion
protein containing residues 619-746 of mouse IRS-2 (25) as antigen;
IRS-1 was obtained using recombinant rat IRS-1 purified from Sf9
cells infected with a recombinant baculovirus as antigen (25);
IRS-1CT (residues 12211234 of rat IRS-1) were made
with synthetic peptides coupled to Keyhole limpet hemocyanin (53).
PY antibodies were rabbit polyclonal (54) or mouse monoclonal 4G10
(UBI, Lake Placid, NY).
p85 antibodies were purchased from UBI.
Differential Binding of SH2 Domains with the IRS-Proteins
GST-fusion proteins containing nSH2p85
,
SH2fyn, SH2Grb2, nSH2PLC
,
nSH2SHPTP2, SH2abl, and SH2Crk were
prepared as previously described (25). Cell lysates were prepared from
unstimulated, insulin-stimulated, or IL-4-stimulated cells
(32DIR,IL4R, 32DIR,IL4R/IRS-1 and
32DIR,IL4R/IRS-2 cells) in homogenization buffer. The
extracts were clarified by centrifugation at 100,000 x
g for 1 h at 4 °C. The supernatants were incubated
with the 1 µg GST fusion proteins containing SH2 domains as indicated
at 4 °C for 1 h and precipitated with glutathione Sepharose at 4
°C for 1 h, washed twice with 50 mM Tris-HCl (pH
7.4) containing 100 mM NaCl, 250 µg/ml BSA, 0.2
mM vanadate, and 0.4 mM
phenylmethylsulfonylfluoride, and boiled for 5 min in 100 µl of
Laemmli sample buffer containing 0.1 M dithiothreitol.
Samples were separated on 7.5% SDS-PAGE and analyzed by immunoblotting
(25, 52).
 |
ACKNOWLEDGMENTS
|
---|
Many thanks to Mary Elizabeth Patti and Ron Kahn for helpful
discussions and sharing unpublished data. R. Robinson, V. Bailey,
J. Neveu, and D. Lizotte provided valuable expertise in peptide
isolation and microsequencing. Bruce Mayer provided recombinant abl and
crk SH2 domains; Ed Skolnik provided nck; ßCT-3 cells were a gift
from Chris Rhodes. Thanks to Dr Tom Charles for providing Bal-17 cells.
We thank Mary Barnstead for excellent technical assistance.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Morris F. White, Ph.D., Research Division, Joslin Diabetes Center, 1 Joslin Place, Boston, Massachusetts 02215.
This work was supported by NIH Grants DK-38712 and DK-43808 (M.F.W.)
and by the National Cancer Institute, Department of Health and Human
Services under contract with ABL (N.A.J.). X.J.S. is a recipient of the
Juvenile Diabetes Foundation, and M.G.M., Jr. was partially supported
by the Medical Scientist Training Program at Harvard Medical School.
1 Present address: University of Vermont, College of Medicine, Given
C350, Burlington, Vermont 05405. 
Received for publication August 1, 1996.
Revision received October 23, 1996.
Accepted for publication November 6, 1996.
 |
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