(Received for publication, December 28, 1995)
From the
We compared the interaction between the insulin receptor (IR)
and the IR substrate (IRS) proteins (IRS-1 and IRS-2) using the yeast
two-hybrid system. Both IRS proteins interact specifically with the
cytoplasmic portion of the IR and the related insulin-like growth
factor-I receptor, and these interactions require receptor tyrosine
kinase activity. Alignment of IRS-1 and IRS-2 revealed two conserved
domains at the NH terminus, called IH1
and
IH2
, which resemble a pleckstrin homology (PH) domain and
a phosphotyrosine binding (PTB) domain, respectively. The IH2
binds to the phosphorylated NPXY motif (Tyr-960) in the
activated insulin receptor, providing a specific mechanism for the
interaction between the receptor and IRS-1. Although the IH2
of IRS-2 also interacts with the NPEY motif of the insulin
receptor, it is not essential for the interaction between the insulin
receptor and IRS-2 in the yeast two-hybrid system. IRS-2 contains
another interaction domain between residues 591 and 786, which is
absent in IRS-1. This IRS-2-specific domain is independent of the
IH2
and does not require the NPEY motif; however, it
requires a functional insulin receptor kinase and the presence of three
tyrosine phosphorylation sites in the regulatory loop (Tyr-1146,
Tyr-1150, and Tyr-1151). Importantly, this novel domain mediates the
association between IRS-2 and insulin receptor lacking the
NPXY motif and may provide a mechanism by which the
stoichiometry of regulatory loop autophosphorylation enhances IRS-2
phosphorylation.
IRS ()proteins (IRS-1 and IRS-2) are substrates for
the insulin and IGF-I (insulin-like growth factor-I) receptors and
tyrosine kinases associated with the receptors for various cytokines,
including growth hormone, several interleukins (IL-2, IL-4, IL-9,
IL-13, and IL-15), and interferons (interferon
/
, and
interferon
)(1, 2, 3, 4, 5, 6, 7, 8, 9) .
IRS proteins provide interfaces between these activated receptors and
various signaling proteins, especially those with Src homology-2
domains (SH2 proteins). During insulin stimulation, IRS-1 associates
with several signaling proteins, including the regulatory subunits of
the phosphatidylinositol 3-kinase (p85
/
and
p55
), SH-PTP2, Nck, Grb-2, and Fyn (10, 11, 12, 13) . The use of IRS
proteins rather than receptor autophosphorylation sites to engage
signaling molecules provides a mechanism to dissociate the signaling
complex from the activated receptor and amplify the signal. However,
the use of IRS proteins requires a specific mechanism to select the
appropriate activated receptor.
Previous reports suggested that
Tyr-960 in the NPEY motif of the insulin receptor juxtamembrane region
is essential for insulin signal transmission and substrate
phosphorylation(14, 15) . A similar NPXY
motif in the IL-4 receptor is also required for IRS-1
phosphorylation(16) . The recent identification of the
phosphotyrosine binding (PTB) domain in the IRS proteins
(IH2), which binds to phosphorylated NPXY
motifs, provides a provisional explanation for interaction between IRS
proteins and the receptor for insulin, IGF-I, and
IL-4(17, 18, 19, 20, 21) .
In addition, the pleckstrin homology domain at the NH
terminus of IRS-1 (IH1
) also contributes to its
interaction with the insulin receptor; however, the mechanism of this
interaction is unknown(22) .
Since many receptors that
mediate IRS protein phosphorylation do not contain NPXY
motifs, the IH1 domain or some other mechanism must be
involved. To search more precisely for other regions of interaction
between IRS proteins and the insulin receptor, we have employed the
yeast two-hybrid system. Our results reveal an novel region in IRS-2
that interacts with the kinase domain of the insulin receptor. Unlike
the IH2
domain, this region interacts with tyrosine
residues in the regulatory loop of the kinase domain. Since an
equivalent region was not detected in IRS-1, our results reveal an
important difference between IRS-1 and IRS-2, which may play a role in
signaling specificity between those two IRS proteins.
Synthetic defined dropout yeast media lacking the appropriate amino acids were obtained from BIO 101 (La Jolla, CA). Oligonucleotides were purchased from Genset (Paris, France). All chemical reagents were from Sigma France, and enzymes were from New England Biolabs (Beverly, MA).
The mouse IRS-2 cDNA amino acids 2-1324 were subcloned into the plasmid pACTII, encoding the GAL4 activation domain (GAD). The GAD-IRS-2 construct was generated by the use of convenient restriction sites maintaining the appropriate reading frame.
Several subdomains of IRS-2 were amplified by the
polymerase chain reaction using the Pwo DNA polymerase (Boehringer
Mannheim, France) with different sets of the following primers (5` to
3`): cgcggatccgctaccgcgaggtgtggcag(1) ,
ccggaattctgctgggggggtggccgc(2) ,
cgcggatccgcctagatgaatacactctc(5) ,
ccggaattcgaggccttcactgcctcc(6) ,
ccggaattctcaaagggcctcacctttcacgac (2`),
cgcggatccgcgccaagtgcacttcgtgccgg(7) , and
ccggaattcagaggcagaggaaggctgagg(8) . EcoRI or BamHI sites are underlined. The polymerase chain reaction
products digested with EcoRI and BamHI were cloned
into pACTII (vector containing Leu selection marker),
digested with EcoRI and BamHI. The inserted fragment
was in frame with the activation domain of Gal4. The plasmids obtained
by cloning the DNA fragment were amplified with the primer sets 1/2,
5/6, 5/2`, and 7/8; they encode GAD hybrid proteins with subdomains
containing amino acids 191-350, amino acids 591-786, amino
acids 591-1325, and amino acids 351-590, respectively.
Rat IRS-1 and different subdomains were cloned into the pACTII in the same fashion as IRS-2. The GAD-IRS-1 construct was described previously (19) , and the fragment of IRS-1 was obtained by polymerase chain reaction using the following primers (5` to 3`): cgcggatccgcgacttgagctatgacacgggc (1) and ccggaattctggtttcccacccaccatact(2) . EcoRI or BamHI sites are underlined. The plasmids encode GAD hybrid protein with the subdomain containing amino acids 144-316 with primer sets 1/2.
To
establish whether IRS-2 binds to IR and IGF-IR
, we tested the
interaction of LDBD-IR
and LDBD-IGF-IR
with GAD-IRS-2. When
IRS-2 was expressed with IR
or IGF-IR
, cotransformants were
blue in the X-Gal assay (Fig. 1A). In contrast, when
IRS-2 was coexpressed with lamin, the X-Gal assay was negative as
expected. Moreover, no interaction occurred between GAD-IRS-2 and the
kinase inactive receptor in which lysine 1018 was replaced with alanine
(LDBD-IR
K1018A) (Fig. 1B). Thus, we conclude
that, like for IRS-1(17) , the interaction between IR and IRS-2
is dependent on tyrosine phosphorylation of the insulin receptor. The
insulin receptor undergoes tyrosine autophosphorylation in three
regions including the regulatory loop (Tyr-1146, Tyr-1150, Tyr-1151),
the COOH terminus (Tyr-1316, Tyr-1322), and the juxtamembrane region
(Tyr-960)(28) .
Figure 1:
Characterization of the interaction
between IR and IGF-IR with IRS-2. The yeast reporter strain L40 was
cotransformed with the plasmid encoding GAD-IRS-2 in combination with
plasmids encoding various LDBD receptor constructs. After isolation on
selective plates, transformants were assayed for -galactosidase
activity by performing a color filter assay as described under
``Experimental Procedures.'' A, coexpression of
GAD-IRS-2 with LDBD-IR
(amino acids 944-1343) or LDBD-IGF-IR
(amino acids 933-1337). As a negative control, the IRS-2 hybrid was
coexpressed with the LDBD-lamin hybrid. B, coexpression of
GAD-IRS-2 with LDBD-IR
-mutated forms. K1018A contains the mutation
of lysine 1018 to alanine. This mutation abolishes the ATP-binding site
and the tyrosine kinase activity of IR
. Y960F has the tyrosine 960
mutated to phenylalanine, and
1316-1322 is a IR mutated form
with the amino acids 1316-1322 deleted. A blue color of
the colonies (shown in black) indicates an interaction with
the two hybrid proteins. Similar results were obtained by analyzing
growth on SC plates lacking histidine.
We tested the requirement for tyrosines 1316
and 1322 of the IR in this association by coexpressing GAD-IRS-2 with
LDBD-IR
1316-1322. This mutant IR lacked two
autophosphorylation sites (tyrosines 1316 and 1322) because of the
deletion of residues 1316-1322. This mutant receptor interacted
normally with IRS-2, demonstrating that the tyrosine residues 1316 and
1322 do not mediate the interaction (Fig. 1B).
Since
tyrosine 960 of the juxtamembrane domain of IR has been shown to
correspond to a key binding site of IRS-1, we investigated whether this
residue plays a role in the association with IRS-2. To do this we
coexpressed GAD-IRS-2 with LDBD-IR Y960F, which contains
phenylalanine in place of tyrosine 960. Surprisingly, this mutation did
not prevent the interaction between the LDBD-IR
and the
full-length GAD-IRS-2 (Fig. 1B). These results were
unexpected because the IH2
domain in IRS-2 binds
specifically to phosphorylated NPXY motifs with similar
characteristics to those of IRS-1. (
)Furthermore, a GAD
hybrid protein containing the IH2
domain from IRS-2 and
IRS-1 interacted specifically with the wild type IR but not with the IR
Y960F (Fig. 2). Thus, the interaction of IRS-2 and IRS-1 with
the IR involves their PTB domain, and both IRS proteins seem to
interact in a similar fashion. However, our finding that IRS-2 still
associates with IR Y960F suggests that another domain of IRS-2
interacts with a cytoplasmic region of the IR, which is different from
the juxtamembrane NPXY motif.
Figure 2:
Interaction between the isolated PTB
domain of IRS-2 and IRS-1 with the IR tyrosine 960. The yeast reporter
strain L40 was cotransformed with the plasmids encoding the LDBD fused
to either IR (amino acids 944-1343) or IR
mutated on tyrosine
960 (IR
Y960F) in combination with plasmids encoding GAD fused to
the isolated PTB domain of IRS-2 (GAD-PTB) (A) or isolated PTB
domain of IRS-1 (GAD-PTB) (B). As a negative control, the
IRS-1 and IRS-2 GAD-PTB constructs were coexpressed with the LDBD-lamin
hybrid. Transformants were isolated on selective plates. Activation of
the reporter gene lexA-lacZ was monitored by measuring the
-galactosidase activity in a color filter assay with X-Gal as
substrate. A blue color (shown in black) indicates an
interaction with the two protein hybrids. Similar results were obtained
by analyzing growth on SC plates lacking
histidine.
Figure 3:
Characterization of the interaction of IR
with the second interaction domain of IRS-2. A, schematic
representation of the mouse IRS-2 protein. PH, pleckstrin
homology domain. Amino acids boundaries for each regions are indicated. B, fragments of IRS-2 corresponding to the indicated residues
were obtained and expressed as fusion proteins with GAD. These
GAD-IRS-2 constructs were cotransformed in the reporter strain L40 with
plasmids encoding the indicated LDBD construct. The -galactosidase
activities in cotransformants were measured by the filter color assay
using the substrate X-Gal. As a negative control, the IRS-2 hybrid
constructs were coexpressed with the LDBD-lamin hybrid. C, the
second interaction domain of IRS-2 (amino acids 591-786), IRS-2,
and IRS-1 fused to the GAD were coexpressed with native LDBD-IR or the
indicated mutated forms of LDBD-IR. Y1146F, Y1150F, and Y1151F have the
Tyr-1146, Tyr-1150, and Tyr-1151 mutated to phenylalanine,
respectively. The
-galactosidase activities of cotransformants
were measured as indicated above. A blue color (shown in black) indicates an interaction between the two hybrid
proteins. Similar results were obtained by analyzing growth on SC
plates lacking histidine.
Figure 4:
Contribution of the different IRS-2
domains in the interaction between IR and IRS-2. A, the yeast
reporter strain L40 was transformed with the plasmid encoding the
LDBD-IR in combination with the plasmid encoding the GAD fused to
IRS-2 native form (amino acids 2-1324), PTB domain of IRS-2
(amino acids 190-350), or IRS-2 (amino acids 591-786). The
-galactosidase activities in cell lysates were measured by a
liquid test using the substrate o-nitrophenyl-
-D-galactopyranoside and were
calculated according to Miller(30) . Values represent the
average (±S.E.) of six independent transformants. B,
the yeast reporter strain L40 was transformed with the plasmid encoding
the GAD fused to IRS-2 native or the GAD fused to p85 subunit with the
indicated IR mutated forms, LDBD-IR
Y960F/Y1150F and LDBD-IR
Y960F/Y1150F/Y1151F, which are the tyrosines 960 and 1150 mutated to
phenylalanine and tyrosines 960, 1150, and 1151 mutated to
phenylalanine, respectively. The
-galactosidase activities of
cotransformants were measured as indicated above. A blue color
(shown in black) indicates an interaction between the two
hybrid proteins. Similar results were obtained by analyzing growth on
SC plates lacking histidine.
Finally, to
investigate whether for IRS-2, the PTB domain and the newly identified
domain 591-786 are the only two regions through which binding to
the insulin receptor takes place, we constructed an IR mutated on both
Tyr-1150 and/or Tyr-1151, and on Tyr-960. Co-expression of these
mutants with GAD-IRS-2 indicates that the interaction between IR and
IRS-2 is entirely lost (Fig. 4B). To verify the
expression of IR mutants in yeast, we coexpressed the LDBD-IR
Y960F/Y1150F and LDBD-IR
Y960F/Y1150F/Y1151F with the GAD-p85
subunit of phosphatidylinositol 3-kinase. The transformation with p85
can be considered as a positive control because the SH2 domains of p85
interact with the COOH-terminal tyrosines 1316 and 1322 of the
IR(29) . Taking our results together, we conclude that IRS-2
interacts with the IR with two domains, i.e. the PTB domain
and the domain comprising amino acids 591-786.
In summary,
using the yeast two-hybrid system we have shown that IRS-2 binds to
tyrosine-phosphorylated IR and IGF-IR. The analysis of IRS-2 binding to
IR shows that IRS-2 associates via its PTB domain to the
IR-subunit at the level of the NPXY
motif
in the juxtamembrane domain. However, the key observation of our study
is that IRS-2 interacts with the IR via a second domain comprising
amino acids 591-786, which is different from the PTB domain. This
newly identified domain associates with the catalytic domain of IR
containing Tyr-1146, Tyr-1150, and Tyr-1151 residues. The IR
interaction of IRS-2 through its PTB domain appears to be less tight
compared with the interaction through the other IRS-2 domain (Fig. 4A). In addition, the interaction through the PTB
domain does not abolish the binding of native IRS-2 to the IR (Fig. 1).
Taking these observations together we would like to suggest that the two domains might have different functions. Indeed, the IRS-2 domain comprising amino acids 591-786 would be the primary anchor of IRS-2 to the insulin receptor, while the PTB domain would have a stabilizing action on the interaction with the insulin receptor.