(Received for publication, August 10, 1995; and in revised form, December 4, 1995)
From the
A structural analysis has been carried out to determine which
part of the intracellular domain of the insulin receptor (IR)
subunit is involved in direct interaction with the receptor substrates
IRS-1 and Shc. Toward this end, the juxtamembrane (JM) domain (amino
acids 943-984) and the carboxyl-terminal (CT) region (amino acids
1245-1331) of IR were expressed in bacteria as
(His)
-fusion peptides, and their interaction with IRS-1 and
Shc was studied. We could demonstrate that the CT region of IR was
sufficient to bind Shc, although significant, but much lower binding of
Shc to the JM region could be detected as well. Furthermore, in
vitro Tyr phosphorylation of the CT region potentiated its
interactions with Shc 2-fold. In contrast, the JM region, but not the
CT domain of the IR, was sufficient to mediate interactions between the
IR and IRS-1. These interactions did not involve the pleckstrin
homology (PH) region of IRS-1, since an IRS-1 mutant, in which four
``blocks'' of the PH domain
(Pro
-Pro
) were deleted, interacted with the JM
region of IR with the same efficiency as native IRS-1. These results
suggest that the IR interacts with its downstream effectors through
distinct receptor regions, and that autophosphorylation of Tyr residues
located at the CT domain of the IR can modulate these interactions.
The insulin receptor (IR) ()is an heterotetrameric
transmembrane glycoprotein composed of two extracellular
subunits
and two transmembrane
subunits linked by disulfide bonds. The
subunits contain the insulin-binding domain, while the
transmembrane
subunits function as a tyrosine-specific protein
kinase that undergoes autophosphorylation following insulin binding
(for reviews, see (1) and (2) ). Autophosphorylation
activates the insulin receptor kinase (3) and enables it to
phosphorylate endogenous proteins, including insulin receptor
substrate-1 (IRS-1) (4) , IRS-2(5) , and
Shc(6) .
Several structural regions have been defined within
the intracellular part of the subunit. These are the
juxtamembrane (JM) region, the kinase region, and the carboxyl-terminal
(CT) region(7, 8) . The JM region
(Arg
-Leu
) contains at least one
autophosphorylation site (Tyr
), which resides in an
LX
NPXYXSXSD motif
(numbering of IR amino acids is according to Ullrich et
al.(7) ). Replacement of Tyr
with Phe or Ala
impairs receptor signal transmission and abolishes both the metabolic
and growth-promoting effects of insulin, even though
autophosphorylation in other regions is normal and the kinase is fully
active in vitro(9, 10) . This appears to be
due to an inability of the mutant receptors to mediate the
phosphorylation of endogenous receptor substrates, including IRS-1.
Indeed, overexpression of IRS-1 can rescue certain biological effects
in cells overexpressing the mutated receptor(1) . Similarly,
the IGF-1 and the IL-4 receptors, which contain
LX
NPXYXS motifs, also
phosphorylate IRS-1(11, 12) . These findings strongly
suggest that the JM region of IR is involved in the interactions with
IRS-1.
The CT region (Leu-Ser
)
possesses only limited (44%) homology with the related IGF-1 receptor
or with other protein tyrosine kinases(13) . It contains two
autophosphorylation sites at Tyr
and Tyr
whose role in receptor signaling is still
unresolved(14) . Mutations of these residues augments
insulin-dependent activation of mitogen-activated protein kinase and
phosphatidylinositol 3-kinase, suggesting that these tyrosine residues
negatively regulate the growth-promoting effects of
insulin(15) . Similarly, cells expressing a receptor mutant in
which the carboxyl-terminal 43 amino acids (including these Tyr
residues) were deleted (IR
) exhibit impaired
metabolic effects (16, 17) and impaired induction of
c-fos(18) but have augmented mitogenic
signaling(16, 17) .
The best studied IR substrates
are IRS-1 and Shc. IRS-1 undergoes phosphorylation on multiple Tyr
residues(1) , which serve as docking sites for SH2-containing
proteins like the p85 regulatory subunit of phosphatidylinositol
3-kinase, GRB2, Nck, and the protein tyrosine phosphatase SH-PTP2.
IRS-1 contains a pleckstrin-homology (PH) domain at its extreme
amino-terminal region(19, 20) , whose deletion
markedly impairs Tyr phosphorylation of IRS-1 in vivo and the
capability of IRS-1 to associate with, and activate,
phosphatidylinositol 3-kinase(21, 22) . A
phosphotyrosine binding (PTB) domain resides at the carboxyl-terminal
end of the PH region of IRS-1. This domain, which is present in several
signaling molecules, interacts with NPXY motifs of
Tyr-phosphorylated growth factor
receptors(23, 24, 25, 26) .
Another substrate of the insulin receptor kinase is Shc(6) , which has three different isoforms of 46, 52, and 66 kDa. Tyr-phosphorylated Shc forms specific complexes with the SH2 domain of Grb2 to further propagate the insulin signal by activation of mSOS/Ras/mitogen-activated protein kinase signaling pathway(27, 28, 29) . Yonezawa et al.(30) suggested that the CT region of the IR might mediate the interactions between IR and Shc. In contrast, the presence of a PTB domain at the amino-terminal region of Shc (23, 24, 25, 26, 31) suggests that Shc has the potential to interact either with the JM or the CT region of the IR.
To better characterize the interactions between the IR and its downstream effectors, we examined the ability of individually expressed receptor domains to interact with IRS-1 and Shc. Our findings suggest that the IR can directly interact with its downstream effectors through distinct receptor regions and that autophosphorylation of Tyr residues located in the CT domain can modulate these interactions.
Figure 1:
Purification of epitope-tagged
JM. Top panel, the pTrcHis-JM vector was
expressed in E. coli Top-10 cells. After IPTG induction, the
bacteria were lysed, and supernatants were prepared as described under
``Experimental Procedures.'' 10 ml of supernatant was applied
to a ProBond Ni
column and eluted with 0-0.6 M imidazole gradient. From the total, effluent, and each
eluted fraction, 50 µl were run on 12% SDS-polyacrylamide gel,
transferred to nitrocellulose and immunoblotted with T7
Tag
antibodies. Bottom panel, fractions 5-8, eluted from the
column, were pooled and dialyzed, and samples (10 µg) were
subjected to 12% SDS-PAGE. Samples were either Coomasie-stained (left) or were transferred to nitrocellulose and immunoblotted
with T7
Tag antibodies (right). This is a representative
of an experiment carried out at least three
times.
Figure 2:
Interactions of JM and CT-peptides with
Shc. A, cytosolic extracts were prepared from CHO cells
overexpressing the IR. Samples (300 µg) were incubated with 10
µg of purified JM, CT
, or buffer alone.
Ni
beads were added, and after extensive washes, the
beads were boiled in 60 µl of Laemmli sample buffer. Samples
(including a 30-µg aliquot of the original total extract) were
resolved by means of 10% SDS-PAGE and immunoblotted with anti-Shc
antibodies. B, same as in A save for the fact that
the extracts were incubated either with native (CT) or
Tyr-phosphorylated CT
(pCT). C, quantitation of
the intensity of the bands, corresponding to the three Shc isoforms
shown in A. Results of three independent experiments are
presented as mean ± S.D. D, quantitation of the
intensity of the bands, corresponding to the three Shc isoforms shown
in B. Results of five independent experiments are presented as
mean ± S.D. E, same as in A save for the fact
that extracts from control or insulin-treated cells were incubated with
CT
.
Figure 3:
Interactions of JM and
CT
with IRS-1. A, cytosolic extracts were
prepared from CHO cells overexpressing the IR. Samples (300 µg)
were incubated with 10 µg of purified JM
,
CT
, or buffer alone. Ni
beads were
added, and after extensive washes, the beads were boiled in 60 µl
of Laemmli sample buffer. Samples (including a 30-µg aliquot of the
original total extract) were resolved by means of 7.5% SDS-PAGE and
immunoblotted with anti-IRS-1 antibodies. B, quantitation of
the intensity of the bands, corresponding to IRS-1 shown in A.
Results of three independent experiments are presented as mean ±
S.D. C, same as in A save for the fact that extracts
were derived from control or insulin-treated
cells.
Figure 4:
Binding of Tyr-phosphorylated IRS-1 to
JM. A, cytosolic extracts were prepared from
insulin-treated CHO cells overexpressing the IR. The extracts were
incubated with 10 µg of purified JM
and were processed
as described in Fig. 3. Samples (30 µg) of total cell
extracts and the material eluted from the immobilized JM
were resolved by means of 7.5% SDS-PAGE and immunoblotted with
anti-IRS-1 or anti-P-Tyr (
-pY) antibodies. B,
quantitation of the intensity of the bands, corresponding to the
content of the IRS-1 protein and the content of the Tyr-phosphorylated
IRS-1 shown in A. Results of two independent experiments are
presented as mean ± S.D.
Figure 5:
Binding of WT and mutated IRS-1
(IRS-1) to JM
. A, cytosolic
extracts were prepared from insulin-treated CHO cells overexpressing WT
or a mutated form of IRS-1 (IRS-1
). The extracts were
incubated with 10 µg of purified JM
and were processed
as described in Fig. 3. Samples (20 µg) of total cell
extracts and the material eluted from the immobilized JM
were resolved by means of 7.5% SDS-PAGE and immunoblotted with
IRS-1 antibodies. B, quantitation of the intensity of the
bands corresponding to WT and mutated IRS-1 shown in A.
Results of four independent experiments are presented as mean ±
S.D.
In the present study, we demonstrate that the JM and CT regions, within the cytoplasmic portion of the insulin receptor, are functional independent entities that contain sufficient structural information to enable independent and direct binding of effector molecules.
The JM region most likely serves as a binding site for
IRS-1, since IRS-1 preferentially interacts with the JM peptide and
exhibits no significant binding to the CT peptide. Binding of IRS-1
presumably involves the NPEY motif present in this domain. Support for
this notion is provided by the fact that (i) mutation of
Tyr, within this motif, or deletion of 12 amino including
this motif (38) impedes Tyr phosphorylation of IRS-1 by the
insulin receptor kinase; (ii) IGF-1 and the IL-4 receptors, which
contain LX
NPXYXS motifs, also
phosphorylate IRS-1(11, 12) ; (iii) a fusion protein
between GST and amino acids 424-561 of the IL-4 receptor, named
P3 (which includes the NPXY motif), binds IRS-1(12) ;
(iv) mutations of either Asn
, Pro
, or
Tyr
abolish IRS-1 interactions with IR in the yeast
two-hybrid system(39) ; (v) a Y960F mutant of IR fails to bind
the isolated PTB domain of IRS-1 (40) .
Our results indicate
that the JM region, in its non-phosphorylated form, is sufficient to
promote interactions with IRS-1. According to our model these
interactions depend upon the presence of an intact NPEY motif per
se, and might take place even when Tyr is not
phosphorylated. Phosphorylation of the latter, most likely potentiates
the interactions, but is not an absolute prerequisite. Three lines of
evidence support this model. First, a number of studies from different
laboratories indicate that Tyr
is poorly phosphorylated in vivo(9, 41, 42, 43) ,
although the JM region is a major site for insulin-stimulated Ser
phosphorylation(44) . Second, there is no direct evidence that
Tyr
is indeed phosphorylated when IR is expressed in the
yeast two-hybrid system (39) under conditions that promote
interaction with IRS-1. Third, the P3 GST-fusion peptide (vide
supra), which apparently contains a non-phosphorylated
NPXY motif, interacts with IRS-1(12) .
Recent
studies have implicated a phosphopeptide (15 amino acids long)
surrounding the NPEY motif of IR as a key contributor to the
interactions of the JM region of IR with the PTB domains of IRS-1 and
Shc(40) . The ID for the interactions of this
phosphopeptide with IRS-1 is
170 µM, while the
nonphosphorylated peptide exhibits only negligible binding (ID
> 1 mM). Since we show a significant binding of
nonphosphorylated JM
to IRS-1 at a peptide concentration
of 1 µM, it suggests that extending the size of the
peptide from 15 to 41 amino acids, drastically increases, about
1000-fold, the affinity of the nonphosphorylated JM region to IRS-1. It
therefore appears that sequences outside of the 15-amino acid peptide
significantly contribute to the interactions of the JM region with the
PTB domain of IRS-1 and these interactions take place even when a
nonphosphorylated Tyr residue is present within the NPXY
motif.
The role of the PH domain of IRS-1 in mediating interactions
with the IR was also evaluated. We have previously shown that a mutated
(Pro
-Pro
) form of IRS-1 whose PH domain
was partially deleted (IRS-1
) undergoes significantly
reduced insulin-dependent tyrosine phosphorylation in vivo,
compared with wild-type (WT) IRS-1. In contrast, both WT IRS-1 and IRS-1
undergo comparable
insulin-dependent Tyr phosphorylation in vitro when incubated
with partially purified insulin receptor kinase(22) . These
findings suggest that the overall structure of IRS-1 is not altered by
deletion of its PH domain and that this domain is presumably not
involved in direct interactions between IRS-1 and the IR. The present
study supports this notion since we demonstrate that partial deletion
of the PH region does not impair the capacity of IRS-1 to interact with
the isolated JM region. These findings are consistent with a model in
which the PH region mediates protein-lipid interactions and serves to
dock IRS-1 in close proximity to the receptor, while the actual
protein-protein interactions with the JM region, are mediated by the
PTB domain of IRS-1(40, 45) .
The interactions of Shc with the IR are slightly more complex. Shc derived from untreated or from insulin-treated cells interacts with the CT peptide to similar extents. This suggests that Tyr-phosphorylated Shc isoforms interact with the CT peptide to the same extent as the nonphosphorylated forms; alternatively, the fraction of Tyr-phosphorylated Shc could be so low that it does not affect the overall pattern of Shc binding. We favor the latter possibility since preliminary studies indicated that Tyr-phosphorylated Shc fails to interact with the CT region.
Interaction of Shc with the CT region are enhanced about 2-fold when
the CT region is subjected to in vitro Tyr phosphorylation by
a partially purified insulin receptor kinase. Two autophosphorylation
sites at Tyr and Tyr
are located at this
region, and one of them (Tyr
) is placed in a context of
YXXM motif, a potential binding site for SH2
domains(46) . The SH2 domain of Shc could therefore interact
with the CT region of IR, presumably through binding to the region that
includes Tyr
. Involvement of the CT region of the IR in
the interactions with Shc is also implicated by studies indicating that
an IR mutant, in which 82 amino acids at the carboxyl-terminal end were
deleted (IR
), fails to promote Shc but not IRS-1
phosphorylation(30) . Similarly, deletion of the SH2 domain of
Shc reduces its interaction with the IR more than 50% as assayed by the
yeast two-hybrid system(45) . It should be noted, however, that
the isolated Shc SH2-domain fails to interact with IR in the yeast
two-hybrid system, suggesting that the native conformation of the whole
Shc protein is required for optimal Shc binding.
Shc interacts, albeit to a very low extent, with the nonphosphorylated JM region of IR. Since the isolated JM region fails to undergo significant in vitro Tyr phosphorylation, we were unable to determine whether Tyr phosphorylation of this domain potentiates these interactions. Previous studies implicated the PTB domain of Shc in mediating interactions with NPXY motifs present in the JM region of IR(40) . Mutation of the Tyr residue within the NPXY motif of IR severely impairs Shc phosphorylation in cultured cells (30) and impedes interactions of Shc with either the IR (45) or the IGF-1 receptor (47) in the yeast two-hybrid system. Hence, the low extent of Shc binding to the nonphosphorylated JM region could be attributed to the fact that the Shc PTB domain interacts exclusively with Tyr-phosphorylated NPXY motifs(23, 24, 31, 40, 48) .
The low affinity of the Shc PTB domain for the nonphosphorylated JM region could be compensated by the interactions of Shc SH2 domains with the nonphosphorylated CT region of IR. A similar phenomenon was seen when the interactions of Shc with the EGFR were studied(24) . Mutations of the highly conserved FLVR sequence in the SH2 domain of Shc reduces the binding of Shc to the EGFR by approximately 90%. In contrast, deletion of the amino-terminal region (including a significant portion of the PTB domain) reduces the binding of Shc to the EGFR by only 50%. These observations suggest that the amino-terminal domain can cooperate with the SH2 domain to promote binding to growth factor receptors (24) .
In summary, although both IRS-1 and Shc interact with IR, significant differences in the nature of these interactions were observed. Shc, but not IRS-1, interact with the CT region, while IRS-1 interacts with a higher affinity with the nonphosphorylated JM region. These differences in binding to the JM region could be accounted for by the fact that PTB domain of Shc interacts with a 5-fold higher affinity with phosphopeptides containing NPEpY motifs(40) , while binding of the PTB domain of IRS-1 is also promoted by JM sequences distal to the NPEY motif. This unique feature of the PTB domain of IRS-1 enables it to selectively bind with a high affinity to the nonphosphorylated JM region of IR. The distinct binding characteristics of IRS-1 and Shc to the different regions of IR could account for the different efficiency of their phosphorylation in vivo, their different sensitivity to the action of kinase inhibitors, and the different biological responses mediated by these effector molecules(49) .
Finally, our results indicate that the interactions of IR with IRS-1, and presumably also with Shc, are regulated by Tyr phosphorylation of the effector proteins. Once IRS-1 is subjected to Tyr phosphorylation, its affinity for the IR is markedly reduced. This phenomenon is not surprising if we consider the fact that effectors like IRS-1 serve as substrates for the insulin receptor kinase. Our findings, therefore, suggest that once IRS-1 is phosphorylated, it translocates away from the receptor into a new subcellular location. This translocation could be part of insulin signal transduction mechanism that is mediated by effector proteins of the insulin receptor kinase.