(Received for publication, April 28, 1995; and in revised form, July 31, 1995)
From the
Using the yeast two-hybrid system, a genetic assay for studying protein-protein interactions, we have examined and compared the interaction of the insulin-like growth factor-I receptor (IGF-IR) and the insulin receptor (IR) with their two known substrates p52Shc and the insulin receptor substrate-1 (IRS-1). We also mapped the specific domains of the IGF-IR and p52Shc participating in these interactions. Our findings can be summarized as follows: (i) the tyrosine kinase activity of the IGF-IR is essential for the interaction with p52Shc and IRS-1, (ii) p52Shc and IRS-1 bind to the IGF-IR in the NPEY-juxtamembrane motif, (iii) contrary to p52Shc, IRS-1 binds also to the major autophosphorylation sites (Tyr-1131, -1135, and -1136) of the IGF-IR, and (iv) the amino-terminal domain of p52Shc is required for its association with the IR and the IGF-IR. We propose that (i) the IGF-IR and the IR share at least in part the same molecular mechanism underlying their interplay with their two substrates, p52Shc and IRS-1, and (ii) IRS-1 interacts with the IGF-IR in a fashion that is different from that used by p52Shc. Finally, our data highlight the crucial role of the juxtamembrane domain in signaling by both the IR and the IGF-IR.
Insulin and insulin-like growth factor-I (IGF-I) ()elicit a wide variety of biological responses after
binding to their respective cell surface receptors(1) . The
insulin receptor (IR) and the IGF-I receptor (IGF-IR) are structurally
related and are composed of two extracellular
-subunits and two
transmembrane
-subunits linked together by disulfide
bonds(2, 3) . Both receptors are members of the
tyrosine kinase receptor family. Binding of the ligand to the
-subunit stimulates the
-subunit intrinsic tyrosine kinase
activity, leading to multisite autophosphorylation of the
-subunit
and tyrosine phosphorylation of cellular substrates. The tyrosine
autophosphorylation sites in the IR and in the IGF-IR are located at
homologous positions in their corresponding domain. A cluster of three
residues (Tyr-1146, -1150, and -1151 in the IR and Tyr-1131, -1135, and
-1136 in the IGF-IR) is localized in the kinase domain. One residue,
Tyr-960 in the IR, which corresponds to the Tyr-950 in the IGF-IR, is
found in the juxtamembrane domain, and two sites (Tyr-1316 and -1322)
are situated in the COOH terminus domain of the IR, whereas in the
IGF-IR, only one (Tyr-1316) is present at this position. Receptor
autophosphorylation is now generally believed to be essential for the
biological activity and the action of the IR and the
IGF-IR(4, 5, 6, 7) .
Known
targets of IR and IGF-IR include a cytosolic 185-kDa protein, termed
IRS-1 (insulin receptor substrate-1)(8) , and cytosolic
proteins of 46, 52, and 66 kDa, termed Shc (Src-homology
2/-collagen) (9, 10, 11) . p46Shc and
p52Shc are both expressed from the same mRNA transcript by alternate
translational initiation sites and thus differ in the extent of their
amino-terminal domain sequences. p66Shc is likely to result from a
distinct transcript. The three Shc proteins contain a carboxyl-terminal
Src homology 2 (SH2) domain, a central glycine/proline-rich region
homologous to the
1 chain of collagen, and an amino-terminal
region containing a recently designated PID domain (for
phosphotyrosine-interacting domain)(12) .
IRS-1 and Shc act as docking molecules linking the IR and the IGF-IR to downstream signaling pathways. Indeed, tyrosine phosphorylation sites in IRS-1 provide binding motifs for several distinct SH2 domain-containing proteins including the p85 regulatory subunit of phosphatidylinositol 3-kinase, the tyrosine-specific phosphatase Syp, and the small adaptor protein Grb-2(13) . By contrast, upon IR and IGF-IR activation tyrosine-phosphorylated Shc proteins appear to bind only to the SH2 domain of Grb-2(14, 15) . Association between Grb-2 and tyrosine-phosphorylated forms of IRS-1 and/or Shc have been directly implicated in the activation of the Ras signaling pathway(14, 16, 17) .
While it is well established that IRS-1 and Shc proteins are direct substrates for the IR and IGF-IR, little is known about the molecular nature of their interplay with these two receptors. However, these interactions appear to involve transient and/or weak binding since, using classical biochemical techniques, it has been difficult to detect in vivo physical association between the receptors and IRS-1 or Shc. Indeed, little or no IRS-1 can be co-immunoprecipitated with activated IR or IGF-IR by using anti-receptor antibodies, and only a small fraction of IR can be detected in anti-IRS-1 immunoprecipitates (18, 19) . Recently, using the yeast two-hybrid system as a genetic assay for detecting protein-protein interactions, O'Neill et al.(20) have revealed an interaction between the IR and IRS-1. In agreement with previous studies(21, 22) , they found that tyrosine 960 of the receptor juxtamembrane domain is involved and that the amino-terminal region of IRS-1 comprising amino acids 160-516 is sufficient for this interaction. Concerning the Shc proteins, it has been reported that these proteins do not co-immunoprecipitate with activated IR or IGF-IR(9, 10, 23) . However, Shc proteins co-immunoprecipitate with other activated tyrosine kinase receptors such as epidermal growth factor receptors, erbB-2/neu, and Trk(11, 15, 24, 25) . Since there is mounting evidence that Shc is a key factor in insulin and IGF-I receptor signaling(10, 26, 27) , unraveling the precise nature of the interplay between Shc and these receptors will certainly contribute to our understanding of insulin and IGF-I action.
The recently developed yeast two-hybrid system has rapidly turned out to be an exquisitely sensitive strategy for studying interacting proteins(28) . Therefore, we have used this technology to examine interactions between the two receptors and their substrates p52Shc and IRS-1. In addition, we have mapped the specific domains of the IGF-IR and p52Shc participating in these interactions.
We have examined the interaction of the IGF-IR and IR with
their substrates, i.e. p52Shc and IRS-1, by using the yeast
two-hybrid system of Fields and Song(36) . In our study, the
first hybrid is a fusion between the DNA binding domain of LexA and the
cytoplasmic domain of the IR or the IGF-IR (LexA-IR or
LexA-IGF-IR
). The second hybrid is a fusion between the
transcriptional activation domain of Gal4 and the full-length sequence
of p52Shc (GAD-Shc) or IRS-1 (GAD-IRS-1). Both of these hybrid proteins
are expressed in a yeast Saccharomyces cerevisiae strain. If
the LexA receptor hybrid interacts with the GAD substrate hybrid, a
functional transcription factor is created, leading to the
transcription of reporter genes. As reporter strain, we used the L40
strain, which possesses two reporter genes, HIS3 and LacZ, containing upstream LexA binding sites(30) .
Activation of LexA-LacZ and LexA-His3 genes can be
easily monitored by the production of
-galactosidase and growth in
the absence of histidine, respectively.
First we verified that the
two LexA receptor (IR and IGF-IR) fusions expressed in L40 were
incapable by themselves, or in combination with an unrelated GAD fusion
protein, of activating the expression of the two reporter genes (data
not shown). We next investigated the interaction of LexA-IR and
LexA-IGF-IR
with GAD-Shc and GAD-IRS-1 in our two-hybrid assay.
We found that coexpression of the LexA receptor construct together with
GAD-Shc or GAD-IRS-1 in L40 resulted in an interaction detected by both
the expression of
-galactosidase and the growth in absence of
histidine. We conclude that p52Shc interacts specifically with both the
IR and the IGF-IR, and as recently shown(20) , IRS-1 associates
with the IR but also with the IGF-IR (data not shown).
Figure 1:
Quantitative
analysis of the interaction of Shc and IRS-1 with different mutated
IGF-IR forms. Left panel, schematic representation of the
LexA-IGF-IR construct and its derivatives tested in the
two-hybrid system. WT corresponds to the wild-type IGF-IR
-subunit (amino acids 933-1337) fused to the DNA binding domain of
LexA. K1003T contains a point mutation in the ATP binding site
(Lys-1003 mutated to Thr).
1290-1319 is a mutated form with
amino acids 1290-1319 deleted.
85 has a carboxyl-terminal
deletion of the last 85 amino acids.
947-950 is a mutated
form with the NPEY motif deleted. Y950F has the Tyr-950 mutated to Phe.
N947A/P948A contains two point mutations at Asn-947 and Pro-948. The
juxtamembrane domain of the
-subunit is shown as a shadedbox, the tyrosine kinase domain as an openbox, and the COOH-terminal domain as a hatchedbox. Right panel, determination of the
-galactosidase activity in transformed yeast cells. The reporter
strain L40 was cotransformed with the indicated panel of LexA-IGF-IR
constructs and either the plasmid encoding GAD-Shc or the plasmid
encoding GAD-IRS-1. Transformants were isolated on selective plates.
The
-galactosidase activities in cell lysates were measured using
the substrate o-nitrophenyl-
-D-galactopyranoside. The
indicated activities were expressed as Miller's units (35) and were the average (±S.E.) calculated from
samples prepared from five independent transformants. Similar results
were obtained by analyzing growth on His
plates.
The NPEY
motif, located in the immediate juxtamembrane domain of the IGF-IR, is
also found in the corresponding domain of the IR. Therefore, we mutated
within this motif the Tyr-960 to Phe in the LexA-IR hybrid to
test whether this mutation would also affect the interaction with
p52Shc. We found that similar to the observation made with IGF-IR,
mutation of Tyr-960 in LexA-IR
prevented its interaction with
GAD-Shc (data not shown). This mutant also failed to interact with
GAD-IRS-1, which is consistent with the report of O'Neill et
al.(20) . We interpret our data to mean that for both the
IR and IGF-IR, mutation of the corresponding tyrosine within the NPEY
motif receptor eliminates interaction with both IRS-1 and p52Shc. From
this we conclude that both p52Shc and IRS-1 bind the same conserved
region of the two receptors. Further, our data obtained with the
LexA-IGF-IR
K1003T mutant would suggest that receptor
autophosphorylation of Tyr-950 is crucial for these interactions.
It has been proposed that the juxtamembrane domain of both the IGF-IR and the IR is the site of interaction with IRS-1. Indeed, mutation of Tyr-960 in the IR and of Tyr-950 in the IGF-IR has been shown to eliminate IRS-1 phosphorylation in intact cells, without significantly affecting the tyrosine kinase activity of the mutated receptors(21, 22, 38) . Further, the biological actions of insulin and of IGF-I were severely impaired in cells expressing these mutated receptors. Our present demonstration that Tyr-960 of the IR and Tyr-950 of the IGF-IR are not only the sites of interaction with IRS-1 but also the sites of interaction with p52Shc provides a key insight. Indeed, our data allow us to suggest that the profound alteration of the biological responses observed in these previous studies is likely to be due to the fact that the tyrosine phosphorylation of Shc proteins is also impaired.
Figure 2:
Quantitative analysis of the interaction
of IGF-IR and IR with Shc deletion mutants. Left panel,
schematic representation of the GAD-Shc(1-473) and its
derivatives tested in the two-hybrid system. Fragments of Shc
corresponding to the indicated residues were obtained and expressed as
a fusion protein with the Gal4 activation domain. The amino-terminal
domain of Shc is shown as a shadedbox, the collagen
domain as an openbox, and the SH2 domain as a hatchedbox. MET +1 and MET +2 correspond
to the two translation initiation sites. Right panel,
determination of the -galactosidase activity in transformed yeast
cells. The reporter strain L40 was cotransformed with plasmid encoding
the indicated panel of GAD-Shc constructs and either the plasmid
encoding LexA-IGF-IR
or the plasmid encoding LexA-IR
. The
-galactosidase activities in cell lysates were measured using the
substrate o-nitrophenyl-
-D-galactopyranoside and
were calculated according to Miller(35) . Values represent the
average (±S.E.) of five independent transformants. Similar
results were obtained by analyzing growth on His
plates.
After our manuscript was submitted, a report by Gustafson et al.(43) appeared in which the two-hybrid system was used to characterize the interaction between the IR and Shc. The findings obtained in this study are totally in agreement with our conclusion concerning the interaction between these two molecules.
In summary, using the two-hybrid system we have shown that p52Shc and IRS-1 bind to tyrosine-phosphorylated IGF-IR. Further, we found that the amino-terminal domain of p52Shc binds to the IGF-IR and to the IR in the juxtamembrane NPEY motif, and that IRS-1 and Shc bind this same motif in the two receptors. However, in contrast to Shc, IRS-1 shows no interaction with different IGF-IR forms mutated on the principal Tyr-autophosphorylation sites, suggesting that interaction between the IGF-IR and IRS-1 requires at least two major determinants on the receptor: a first one located in the juxtamembrane region and a second one in the kinase domain. Our conclusions have been inferred from observations made with the yeast two-hybrid system, and we can not exclude the possibility that the interactions detected are indirect and require intermediates within the yeast nucleus. One could also argue that the relevant environment of the cytoplasm of mammalian cells is different from the yeast nucleus. Despite this possibility, the yeast two-hybrid system is clearly the most efficient method allowing the detection of interaction between the IR or the IGF-IR and their two identified substrates, Shc and IRS-1.
Both Shc and IRS-1 have been implicated in mediation of insulin- or IGF-I-induced activation of Ras and of the mitogen-activated protein kinase cascade(14, 16, 17) . In response to insulin or IGF-I stimulation, tyrosine-phosphorylated Shc (or IRS-1) binds to the adaptor Grb-2, which forms a complex with Sos, a guanine nucleotide exchange factor of Ras. These events couple Shc to Sos or IRS-1 to Sos. Based on our results it is tempting to propose a model in which the interaction of Shc (or IRS-1) with the immediate juxtamembrane domain of the IGF-IR and the IR results in the targeting of the complex Grb-2/Sos near the membrane where Ras is localized. Hence, the interaction of Shc and IRS-1 with the juxtamembrane domains of the two receptors could represent an anchoring and pulling step in the membrane recruitment of Shc-Grb-2-Sos and IRS-1-Grb-2-Sos complexes to Ras.
Finally, our demonstration that for both the IGF-IR and the IR the two central proteins in signaling, IRS-1 and Shc, use at least in part the same receptor domain highlights the essential role of the juxtamembrane domain in receptor functioning.