(Received for publication, March 8, 1996; and in revised form, March 28, 1996)
From the
Insulin receptor substrate 2 (IRS-2) has recently been shown to be a substrate of the insulin receptor (IR). In this study we utilize the yeast two-hybrid system and assays of in vitro interaction to demonstrate that IRS-2 interacts directly with the IR and the insulin-like growth factor I receptor. We show that, like IRS-1, the region of IRS-2 that contains the putative phosphotyrosine binding and SAIN elements(188-591) is sufficient for receptor interaction and that this interaction is dependent upon the NPX(p)Y (where (p)Y is phosphotyrosine) motifs within the juxtamembrane domains of the receptors. In addition to this amino-terminal NPX(p)Y-binding domain, an additional domain of strong interaction was identified in the central region of IRS-2 and was localized between amino acids 591 and 733. This interaction was found to be dependent upon receptor phosphorylation but was NPX(p)Y-independent. This region does not appear to have either an SH2 or a phosphotyrosine binding domain. Both of the interactions could also be demonstrated in vitro using IRS-2 glutathione S-transferase fusion proteins. We conclude that IRS-2, unlike IRS-1, can interact with tyrosine-phosphorylated receptors such as the IR and insulin-like growth factor I receptor via multiple independent binding motifs. Our findings suggest the existence of a previously unidentified phosphotyrosine-dependent binding domain within the central region of IRS-2.
The insulin receptor (IR) ()and the related
insulin-like growth factor I receptor (IGFIR) are believed to transduce
signals at least in part by phosphorylation of cellular proteins such
as IRS-1 and SHC, which, upon phosphorylation, serve as docking sites
for downstream signaling proteins(3, 4, 5) .
Phosphorylation of IRS-1 upon multiple tyrosines leads to interaction
with SH2 domain-containing proteins including Grb2, Syp, and the p85
subunit of phosphatidylinositol 3-kinase (PI 3-kinase) that lead to
activation of various signaling
cascades(6, 7, 8) . Tyrosyl phosphorylation
of SHC leads to its interaction with Grb2 and mediates activation of
the guanine nucleotide exchange factor SOS and subsequent activation of
the Ras signaling
pathway(9, 10, 11, 12) . Both IRS-1
and SHC have been implicated in insulin-stimulated mitogenic signal
transduction(13, 14, 15) , and the relative
role that each protein plays in mitogenic signaling by the IR and IGFIR
is currently an area of active investigation(12, 16) .
IRS-1 has also been proposed to mediate some of the metabolic effects
of the IR including regulation of glucose transport(17) . The
leading candidate for the regulation of glucose transport is PI
3-kinase, which interacts with and is activated by IRS-1 and to a
lesser extent by the IR(18, 19) .
Recently, mice
have been generated in which the IRS-1 gene has been disrupted by
homologous recombination. Surprisingly these mice were found to have
only a slight insulin-resistant phenotype(20, 21) .
Muscle and adipose tissue from these mice were found to express an
alternative 190-kDa substrate which, like IRS-1, could interact
with and activate PI 3-kinase in an insulin-dependent manner. Recently,
a protein that possesses similar characteristics to IRS-1 was
cloned(22) . This molecule was first identified as a protein
that was tyrosine-phosphorylated in response to interleukin 4
stimulation and was initially termed 4PS(23) . The 4PS protein
was shown to be tyrosine-phosphorylated in myeloid progenitor cells in
response to interleukin 4 and insulin and to interact with and activate
PI 3-kinase (24, 25) . Using the p85 subunit of PI
3-kinase as an affinity matrix, the 4PS protein was purified from
FDC-P2 cells and its cDNA was isolated(22) . Sequence analysis
revealed that 4PS possessed extensive homology with IRS-1, and it was
therefore redesignated as IRS-2. Reconstitution experiments have
confirmed that IRS-2 is a substrate of the IR and that it associates
with PI 3-kinase and Grb2(26) .
We have recently characterized the interactions of the IR and IGFIR with IRS-1 and SHC using the yeast two-hybrid system and other assays of protein-protein interaction(27, 28, 29, 30) . The interactions of IRS-1 and SHC with the IR were found to be phosphotyrosine-dependent and also to require an intact NPX(p)Y motif within the juxtamembrane domains of the IR and IGFIR. We have localized the region within IRS-1 that is involved in interaction with the IR and IGFIR to the amino terminus of IRS-1 between amino acids 160 and 516(30) . Further deletions within this region suggested the presence of an essential region at the amino-terminal end of this domain, since deletion of amino acids 160-174 totally eliminated interaction(27) . This essential region was subsequently shown to be the most highly conserved region within the IRS-1 and IRS-2 proteins and has been suggested to contain a phosphotyrosine binding (PTB) motif(22) . Although it is not yet clear whether this region contains a true PTB domain, these findings nevertheless support our previous identification of this region as an essential element of interaction(27, 30) . We initially termed the region just downstream of the potential PTB domain the SAIN domain (for SHC and IRS-1 NPXY binding) on the basis of a limited homology to the SHC PTB domain(28) . This region appears to be important for high level interaction yet is not essential for the interaction(27, 28, 30) . Since our data clearly show that the SAIN elements in IRS-1 (and IRS-2, see below) are important for interaction but do not appear to contain the core binding element, the mechanism by which this domain enhances the interaction with the IR and IGFIR is currently unclear.
In this study, we characterize the interaction of IRS-2 with the IR and IGFIR. We demonstrate that IRS-2 interacts with the IR via two discrete domains, the first interacting in an NPX(p)Y-dependent manner very similar to IRS-1 via an amino-terminal domain within IRS-2. We also identify a second receptor interaction domain located between 591 and 733 of IRS-2, which interacts in a phosphotyrosine-dependent but NPX(p)Y-independent manner, unlike IRS-1.
Figure 3:
Both receptor binding domains within IRS-2
interact in a phosphotyrosine-dependent manner but only RBD1 binds in
an NPX(p)Y-dependent manner. A, we assayed four IRS-2
hybrids for interaction with a number of IR and IGFIR bait hybrids that
contained specific mutations or deletions. The activities refer to
Miller units. WT refers to wild type. B, interaction
of the IRS-2 hybrid (amino acids 3-591) was assayed with 17 IR
hybrids, which contained single or double amino acid substitutions.
-Galactosidase activity was measured and is reported in Miller
units ± S.E. The data were analyzed by randomized one-way
analysis of variance followed by comparison of each mutant receptor to
the wild-type receptor using the Newman-Keuls test for significance
(**, p < 0.01).
Figure 1:
IRS-2 interacts specifically with the
IR and IGFIR via two independent domains. A, schematic
representation of the hybrid proteins. The entire cytoplasmic domain of
the human IR or IGFIR was fused to the LexA DNA-binding domain (DBD). B, various regions of IRS-2 were fused to the
activation domain and assayed for interaction with wild-type IR or
IGFIR bait hybrids. Transformants were assayed for -galactosidase
activity by either the colony color or the solution assay as described
under ``Experimental Procedures.'' The colony color assay
showed either white(-), light blue (++), or dark blue
(+++) colonies.
-Galactosidase (
-GAL)
activity is reported in Miller units using a minimum of three
independent colonies.
We first focused on the amino-terminal receptor interaction domain that we term RBD1 for receptor-binding domain 1. As shown in Fig. 1B, expression of 10 IRS-2 hybrid proteins within this region provided evidence that the minimal domain possessing full activity was located between amino acids 188 and 591. Deletion of the carboxyl portion of this fragment to amino acid 429 or 326 reduced activity by at least 70%, and further deletion to amino acid 263 eliminated all activity. Amino-terminal deletion of amino acids 188-214 within the 188-591 fragment totally eliminated activity. We conclude that the amino-terminal subdomain within RBD1 forms an essential element for interaction with the IR or IGFIR while the carboxyl portion of this domain is important but not essential for the interaction. These data are entirely consistent with our previous delineation of the IR-binding domain of IRS-1(27, 30) . Not surprisingly, the amino-terminal subdomain of the RBD1 is the most highly conserved region between IRS-1 and IRS-2(22) . Furthermore, this region has been suggested (22) to contain a putative PTB domain (28, 42, 43) and has been shown to interact with the IR in vitro(44) . Our data are in agreement that this region contains an essential element of the RBD1. Whether or not the IRS molecules contain regions that are structurally related to the classical PTB domain remains to be proven.
To better characterize the binding activity observed within the carboxyl end (1) of IRS-2, we expressed nine regions of this protein and determined their activity in the two-hybrid assay. As shown in Fig. 2, the four carboxyl-terminal deletions showed that removal of amino acids 734-1321 had no significant effect upon activity. Further deletion to amino acid 697 eliminated all activity. Amino-terminal deletions showed that removal of amino acids 591-697 or 591-732 markedly reduced activity. These data suggest that IRS-2 contains a receptor binding domain located between amino acids 591 and 733, which mediates a strong interaction with both the IR and IGFIR.
Figure 2: IRS-2 contains a second domain between amino acids 591 and 733 that interacts strongly with the IR and IGFIR. Ten IRS-2 hybrid proteins were produced as activation domain fusions and tested for interaction with either the IR or IGFIR as described under ``Experimental Procedures'' and in the legend of Fig. 1.
To characterize better the
interaction of the RBD1 domain of IRS-2 with the NPXY region
of the IR, we expressed the IRS-2(3-591) hybrid with 17 IR
hybrids that contained single or double amino acid substitutions within
and surrounding the NPXY motif(29) . As shown in Fig. 3B, mutation of Tyr-960 to Glu eliminated the
interaction with this region of IRS-2. We conclude that replacement of
this tyrosine with a negatively charged amino acid is not sufficient to
reconstitute this interaction. Alteration of the Asn or the Pro within
the NPXY motif significantly reduced the interaction of IRS-1
with the IR, as did a double Asn and Pro replacement. Replacement of
the Glu at position -1 from the Tyr had no effect upon IRS-2
interaction. These data are consistent with the idea that the RBD1
domain within IRS-2 interacts with the IR in an
NPX(p)Y-dependent manner. None of the other receptor mutations
had significant effects upon IRS-2 interaction, although the Leu and
Tyr mutations at positions -7 and -8 showed consistently
lower levels of interaction. These data are consistent with our
previous findings (29) regarding the interaction of IRS-1 and
the IR, with a few minor exceptions. First, alteration of either the
Asn or Pro residues within the NPXY motif totally eliminated
IRS-1 interaction, whereas IRS-2 shows significant, though greatly
reduced, interaction. Second, alteration of the Leu and Tyr residues at
amino acids -7 and -8 had a much more pronounced effect
upon IRS-1 interaction (70% reduction in activity) than we
observed with IRS-2 (
40% reduction in activity). These differences
suggest that although both IRS-1 and IRS-2 require an intact
NPX(p)Y motif for interaction with RBD1, subtle differences
between these proteins may exist.
Figure 4: In vitro interaction of both IRS-2 receptor binding domains with the insulin receptor. A, we produced seven IRS-2 GST hybrid proteins (a-g) and assayed their abilities to interact in vitro with the IR expressed in CHO cells. Cellular lysates were prepared from CHO cells, which overexpress the IR before and after insulin (INS) stimulation, and incubated with either a control GST or a GST-IRS-2 fusion protein as described under ``Experimental Procedures.'' B, similar experiments were performed with four (h-k) IRS-1 GST fusion proteins. The data obtained with the same regions of IRS-1 and IRS-2 are shown to the right of each construct. nd, not determined.
For comparison, we also produced four GST fusion proteins containing similar regions of the IRS-1 protein and assayed their ability to interact with the IR in vitro. As shown in Fig. 4B, the two GST fusions that contained the essential domain of interaction within RBD1 (amino acids 21-400 and 108-516) were efficiently coprecipitated with the IR, whereas the region of IRS-1 corresponding to RBD2 of IRS-2 showed no interaction. These data are consistent with our two-hybrid data and suggest that IRS-1 does not contain this second domain of IR interaction. This is not surprising since these regions show only minimal sequence similarity(22) .
Figure 5: Models of IRS-2 interaction with the IR and IGFIR. Our data show that the RBD1 region of IRS-2 interacts with the NPX(p)Y motif of the IR. The mechanism of RBD2 interaction is less clear and may either be mediated by a direct phosphotyrosine interaction (Model 1) or by an interaction with a region of the IR that is exposed upon autophosphorylation but is independent of phosphotyrosine residues. See the last two paragraphs of text for a discussion of these models.
The existence of two distinct phosphotyrosine binding elements within IRS-2 is intriguing in a number of other ways. It seems likely, for example, that such a binding element should allow IRS-2 to interact with additional signaling proteins and receptors that do not contain NPX(p)Y elements thus expanding the repertoire of potential signaling partners utilized by the IRS family of proteins. Whether a receptor has one or two sites of interaction with IRS-2 may also allow for differential signal transduction, which might theoretically be mediated by differences in the stability of the interaction or differential phosphorylation of IRS-2 tyrosine residues. It is also possible that each binding element within IRS-2 may interact in vivo with distinct tyrosyl-phosphorylated substrates. It will be interesting to determine whether the differences which we have observed between IRS-1 and IRS-2 allow these highly related proteins to play distinct functional roles in vivo.