(Received for publication, March 23, 1995; and in revised form, July 7, 1995)
From the
The wild type insulin-like growth factor I (IGF-I) receptor has
both mitogenic and transforming activities. We have examined the effect
of point mutations at tyrosine residues 1250 and 1251 on these two
properties of the receptor. For this purpose, we stably transfected
plasmids expressing mutant and wild type receptors into R cells, which are 3T3-like cells, derived from mouse embryos with
a targeted disruption of the IGF-I receptor genes, and therefore devoid
of endogenous IGF-I receptors. A tyrosine to phenylalanine mutation of
either the 1250 or 1251 residue, or both, has no effect on the ability
of the receptor to transmit a mitogenic signal. However, the tyrosine
1251 mutant receptor and the double mutant have lost the ability to
transform R
cells (colony formation in soft agar),
even when the receptors are expressed at very high levels, while the
Y1250F mutant is fully transforming. These experiments show that the
1251 tyrosine residue is required for the transforming activity of the
IGF-I receptor.
Overexpression and constitutive activation of the insulin-like
growth factor I receptor (IGF-IR) ()in a variety of cell
types leads to ligand-dependent growth in serum-free medium and to the
establishment of a transformed phenotype (Kaleko et al., 1990;
McCubrey et al., 1991; Liu et al., 1993a; Sell et
al., 1994; Coppola et al., 1994). Conversely, 3T3-like
mouse embryo cells with a targeted disruption of the IGF-IR genes (Liu et al., 1993b; Baker et al., 1993) are refractory to
transformation by SV40 large T antigen, an activated Ras or a
combination of both, that readily transform cells from wild type
littermate embryos or other 3T3-like cells (Sell et al., 1993,
1994). The important role of the IGF-IR in the establishment and
maintenance of the transformed phenotype is also supported by other
findings, indicating that antisense oligodeoxynucleotides or antisense
expression plasmids against IGF-II (Christophori et al.,
1994), IGF-I (Trojan et al., 1992, 1993), or the IGF-I
receptor (Sell et al., 1993; Baserga et al., 1994;
Resnicoff et al., 1994a and 1994b), antibodies to the IGF-IR
(Arteaga, 1992; Kalebic et al., 1992), and dominant negative
mutants of the IGF-IR (Prager et al., 1994; Li et
al., 1994), can all reverse the transformed phenotype, inhibit
tumorigenesis, and induce loss of the metastatic phenotype (Long et
al., 1995).
Our laboratory has been investigating the role that
various tyrosine residues of the IGF-IR have on two of its properties,
mitogenic activity and ability to transform mouse embryo cells, using
R cells (Sell et al., 1993, 1994; Coppola et al., 1994) established by a 3T3-like protocol from mouse
embryos with a targeted disruption of the IGF-IR genes (Liu et al. 1993b, Baker et al. 1993). The absence of an endogenous
background of IGF-IRs in these cells facilitates a mutational analysis.
R
cells grow in 10% serum, albeit at a reduced rate
in comparison with cells from wild type littermate embryos (W cells),
fail to grow in serum-free medium (SFM) supplemented with the growth
factors that sustain the growth of other 3T3-like cells (Sell et
al., 1994; Valentinis et al., 1994), and, as mentioned
above, cannot be transformed by SV40 T antigen and/or an activated and
overexpressed Ras. The growth phenotype of R
cells,
including their resistance to transformation, returns to normal (i.e. like W cells or other 3T3-like cells) when they are
stably transfected with a plasmid expressing a wild type human IGF-IR
cDNA (Ullrich et al., 1986) but not when transfected with a
human receptor with a point mutation at the ATP-binding site (Sell et al., 1994; Coppola et al., 1994). Liu et al. (1993a) have previously reported that C-terminal truncations of a
constitutively activated Gag-IGF-IR hybrid construct affected its
transforming activity. This was confirmed in a more rigorous way and
extended by our own findings that a human IGF-IR lacking the C-terminal
108 amino acids (Surmacz et al. 1995) and an overexpressed
insulin receptor (see below) cannot transform R
cells. We therefore investigated in the present paper the
mitogenic and transforming activities of the IGF-IR with point
mutations at tyrosine residues 1250 and 1251 that are absent in the IR
(Ullrich et al., 1986). Our principal aim was to determine
whether these two tyrosine residues are necessary for mitogenesis and
transformation of R
cells. A secondary aim was to
determine other properties of the mutant receptors, such as ability to
phosphorylate cellular substrates in vivo.
We show that a
receptor with a mutation at Tyr-1251, but not at Tyr-1250, cannot
transform R cells, while both mutant receptors are
fully mitogenic. Several indications suggest that this preservation of
a mitogenic response with loss of transforming activity is not simply
due to a quantitative deficit of the Y1251F mutant receptor. The
identification of a receptor mutant that is fully mitogenic but
nontransforming opens the possibility of identifying new transformation
pathways that can be separated from the mitogenic pathways.
The first PCR was performed using a pBluescript SK IGF-IR (a pBluescript SK phagemid (Stratagene) in which an XbaI-BamHI fragment including the whole IGF-IR cDNA was inserted) as a template, a 5`-primer (mutagenic primer), and a 3`-primer as follows. 5`-primers (mutagenic primers) were 5`-GGCTTtCGcGAGGTCTCCTTCTTCTACAG-3` (1250F), 5`-GGCTTtCGcGAGGTCTCCTTCTACTTCAG-3` (1251F), and 5`-GGCTTtCGcGAGGTCTCCTTCTTCTTCAG-3` (1250F/1251F); the 3`-primer was 5`-GTAAAACGACGGCCAGT-3` (M13-20 primer, whose sequence is in the pBluescript SK).
The first 5`-nucleotide of the mutagenic primers followed a thymine residue in the template sequence to avoid problems with template-independent incorporation of an adenine at the 3`-termini of the amplified products. All three primers were designed to create a NruI site with silent mutations. The mutated sites in the codon corresponding to amino acid position 1250 or 1251, are underlined, while the silent mutations are shown in lowercase type. This first PCR product contains a unique BamHI site located 6 base pairs past the stop codon of the IGF-IR cDNA. The second PCR was performed using the same template to the first PCR, a 5`-primer, and the first PCR product as a 3`-primer (an antisense strand actually serves as a 3`-primer). The 5`-primer was designed to locate beyond the unique HindIII site as follows: 5`-ACAGTGAACGAGGCCGCAAG-3`.
The second PCR product was subcloned into a TA cloning vector, pCRII (Invitrogen), and the correct mutation was confirmed by digestion with NruI and by dideoxy sequencing (not shown). A wild type HindIII-BamHI fragment of a pBluescript SK Sal-Bam IGF-IR (a pBluescript SK phagemid containing a SalI-BamHI fragment of the whole IGF-IR cDNA) was replaced by the mutant HindIII-BamHI fragment from the pCRII containing the second PCR product, and a SalI-NotI fragment including the mutated, whole IGF-IR cDNA was subcloned into a XhoI-NotI site of a pBPV expression vector (Pharmacia Biotech Inc.) to generate the pBPV Y1250F, pBPV Y1251F, and pBPV Y1250F/Y1251F expression plasmids.
A cell sorting method by flow
cytometry was used to establish cell lines expressing the Y1250F,
Y1251F, or Y1250F/Y1251F mutant as follows: R cells
were co-transfected with a pBPV Y1250F, pBPV Y1251F, or pBPV
Y1250F/Y1251F plasmid and a pPDV6
plasmid, which
encodes the puromycin resistance gene (de la Luna et al.,
1988), and selected with puromycin (Coppola et al., 1994). The
resultant clones were passaged a few times. After the cells were
trypsinized and washed with phosphate-buffered saline, they were
incubated with anti-IGF-IR antibody (1:10) (Oncogene Science) at 4
°C for 20 min. After washing with phosphate-buffered saline, the
cells were incubated with anti-mouse IgG conjugated with fluorescein
isothiocyanate (1:50) (Oncogene Science) at 4 °C for 20 min. The
cells were washed with phosphate-buffered saline, the intensity
distribution of the fluorescence was analyzed with flow cytometry
(Coulter), and the upper 5% of the population was sorted under sterile
conditions. The sorted cells were washed and allowed to grow in the
incubator. The receptor number was determined by Scatchard analysis as
described below.
To generate R/IR clones,
R
cells were co-transfected with an expression
plasmid encoding a human IR cDNA (pBPV-IR, a kind gift of Dr. Derek
LeRoith, National Institutes of Health) and the plasmid encoding the
puromycin resistance gene (see above). After selection in 2.5 µg/ml
of puromycin, several clones overexpressing the IR were identified on a
single point binding assay with
I-insulin (DuPont NEN).
To determine a possible dominant negative effect of the mutant receptors, C6 rat glioblastoma cells (Trojan et al., 1993; Resnicoff et al., 1994a) that form colonies in soft agar were co-transfected with the mutant receptors and the plasmid for neo-resistance, After selection, the number of IGF-IRs in several clones was determined by Scatchard analysis, as described below.
The same procedure was used to determine receptor number in
R cells stably transfected with the plasmid
expressing the IR, except that the ligand was
I-insulin
(0.18 nM (1 ng/ml)), the amount of unlabeled insulin varied
from 0 to 7 nM, and nonspecific binding was determined with
0.2 µM unlabeled insulin.
Figure 1:
Scatchard analysis of cell lines with
mutant IGF-I receptors. R cells were transfected with
plasmids expressing human IGF-I receptor cDNAs with the indicated
mutations at Tyr-1250, Tyr-1251, or both. Cell lines were selected and
analyzed as described under ``Materials and Methods.''
, Y1250F;
, Y1251F;
,
Y1250F/Y1251F.
Figure 2:
Growth of R-derived
cells in IGF-I. The various cell lines were plated in
serum-supplemented medium overnight, then transferred to serum-free
medium supplemented solely with IGF-I (50 ng/ml). The cell number was
determined 48 h. after changing growth medium to serum-free medium plus
IGF-I. The cell lines are described under
``Results''.
Figure 3:
Growth in soft agar of R
cells and their derivatives. Microphotographs of soft agar assays are
shown. A, R
cells; B, R
cells; C, Y1250F cells; D, Y1251F cells. The bar in panelA is 200
µm.
Figure 4:
Autophosphorylation of the IGF-I receptors
and Shc phosphorylation in the various cell lines. A, whole
cell lysates were immunoblotted with a phosphotyrosine antibody. The upperarrow indicates the p185 protein band (putative
IRS-1); the lowerarrow indicates the subunit
of the IGF-I receptor. B, lysates were immunoprecipitated with
an anti-Shc antibody and stained with a phosphotyrosine antibody. The arrow indicates 52-kDa Shc. The aspecific band is the IgG used
in the procedure. Oddnumberedlanes are
unstimulated cells; evennumberedlanes are
cells stimulated for 5 min with IGF-I (20 ng/ml). Lanes1 and 2, R
cells; lanes3 and 4, Y1250F cells; lanes5 and 6, Y1251F cells; lanes7 and 8,
Y1250F/Y1251F cells.
In Fig. 4B, we have examined Shc phosphorylation. In
Y1250F cells Shc is phosphorylated (lane4) at least
as efficiently as in R cells, whereas Shc
phosphorylation is slightly decreased in Y1251F cells (lanes5 and 6) and in the double mutant cells (lanes7 and 8). However, it is clear that
even the Y1251F mutant can phosphorylate Shc, although perhaps less
efficiently than the Y1250F mutant (the cell lines with the mutant
receptors have approximately the same number of receptors (Table 2)).
In these experiments, our main goal was to determine whether the two tyrosine residues at 1250 and 1251 of the IGF-IR are required for mitogenesis and transformation. The results indicate that 1) both tyrosines can be mutated (to Phe), without affecting the ability of the IGF-IR to induce IGF-I-mediated cell proliferation; and 2) a Y1251F mutation severely impairs the transforming activity of the IGF-IR, while a Y1250F mutation does not. Secondary findings include the fact that 1) there is no gross alteration in the extent of autophosphorylation of the mutant receptors or the tyrosyl phosphorylation of the p185 protein (the mutant receptors are fully mitogenic); and 2) the Y1251F mutant is somewhat less efficient in phosphorylating Shc than the Y1250F mutant after IGF-I stimulation.
The most important finding in the present paper is that the Y1251F mutant, overexpressed, is functional enough to transmit an IGF-I-mediated mitogenic signal but, even at very high levels of receptor expression, does not support transformation, as measured by colony formation in soft agar. A mutation at Tyr-1250 does not affect the transforming ability of the IGF-IR.
A number of studies have investigated the mitogenicity of the IGF-IR (Lammers et al., 1989; Kato et al., 1993, Sasaoka et al., 1994; Kato et al. 1994), but until recently, very few studies had addressed its role in transformation (see the Introduction). In the case of transformation, other IGF-IR mutants have already been shown to have lost the ability to induce IGF-I-mediated mitogenesis and to transform cells, for instance, a triple tyrosine mutant at Tyr-1131, -1135, -1136 (Li et al. 1994), and the Y950F mutant (Miura and Baserga, 1995), indicating that these two domains are required both for cell proliferation and for transformation. Our present results clearly show that neither Tyr-1250 nor Tyr-1251 can be enumerated among the domains of the IGF-IR necessary for mitogenesis, since, at the receptor levels we have investigated, both mutant receptors are fully mitogenic. However, the Y1251F mutant (but not the Y1250F mutant) has a markedly decreased transforming activity. An important point is whether the failure of the Y1251F mutant to transform mouse embryo cells is due to a qualitative or a quantitative deficit. In the latter case, one could think that a receptor that is weakly active may be sufficient for transmission of a mitogenic signal but not adequate for the establishment of the transformed phenotype; alternatively, the Tyr-1251 residue may be specifically required for transformation, although in association with other domains of the receptor that are needed for the mitogenic signal. Two observations argue against the first alternative: 1) the Y1251F mutant is nontransforming at receptor levels that are 19-fold the levels of wild type receptor sufficient for the establishment and maintenance of the transformed phenotype (Pietrzkowski et al., 1992a, 1992b; Coppola et al., 1994); 2) the Y1251F mutant acts as a dominant negative in a cell type that has 500,000 endogenous IGF-IRs. It would be difficult to explain how the additional input from a receptor transmitting a weak but otherwise normal signal could reverse the signal of a wild type receptor. Indeed, the Y1250F mutant receptor not only does not inhibit soft agar growth of C6 cells but actually augments it, as one would expect from an additive effect.
The mechanism is, of course, the next important question. Not surprisingly, our experiments do not show gross differences in tyrosyl phosphorylation of the IGF-IR or IRS-1, which are involved in insulin and IGF-I-mediated mitogenesis (Waters et al.(1993), Rose et al.(1994), Yamauchi and Pessin(1994), and see review by White and Kahn(1994)). We say not surprisingly, because both mutant receptors are fully mitogenic, and, therefore, there is no apriori reason why the known mitogenic pathways of the IGF-IR should be affected. For the same reason, we have not explored in this paper the pathways downstream of IRS-1 and Shc. The difference between the wild type receptor and the Y1251F mutant receptor is in their ability to confer a transformed phenotype (colony formation in soft agar). However, soft agar assays are done in 10% serum, which also activates other growth factor receptors that use the Ras pathway, and this precludes a simple analysis at this point. The same criticism can be applied to an explanation involving Shc, which is known to be a transforming protein (Pelicci et al., 1992; Skolnik et al., 1993; White and Kahn, 1994). Indeed, the Y1251F mutant receptor seems to be somewhat less efficient in its ability to phosphorylate Shc, at least in comparison with the Y1250F mutant (Fig. 4), which makes this explanation a still viable one, especially since we obtained the same results with the C terminus truncated receptor (Surmacz et al., 1995). But again, Shc phosphorylation was determined in serum-free medium supplemented with IGF-I, whereas the transformation assay is done in 10% serum, which contains other growth factors capable of activating Shc (White and Kahn, 1994). Also one cannot ignore some recent reports that there is some kind of balance between IRS-1 and Shc that is important for the stimulation of DNA synthesis by the IR (Yamauchi and Pessin, 1994), and that a transmembrane mutant of the v-ros oncogene that had lost its transforming activity still retained its ability to phosphorylate Shc (Zong and Wang, 1994).
Indeed, the fact that a combination of SV 40 T antigen and an
activated, overexpressed Ras fail to transform R-cells (Sell et al. 1994, Surmacz et al. 1995), clearly indicates that one of
the transforming pathways of the IGF-IR is Ras-independent. While there
is no question that the Ras pathway is required for transformation (see
for instance McCormick(1993) and Medema and Bos (1993)), another
pathway that the IGF-IR does not share with the platelet-derived growth
factor and epidermal growth factor receptors has to be hypothesized, a
conclusion supported by other independent findings (Falco et
al., 1988; Aaronson, 1991; Matuoka et al., 1993;
Silvennoinen et al., 1993). In previous papers (Coppola et
al. 1994, DeAngelis et al. 1995), we showed that an
overexpressed epidermal growth factor or platelet-derived growth factor
receptor cannot transform R cells, although they are
capable of transforming wild type cells from littermate mouse embryos.
The requirement for a functional IGF-IR for transformation seems to be
true also in the case of the IR (this paper), despite the fact of the
extensive homology between the two receptors (Ullrich et al.,
1986) and the sharing of signal-transducing pathways (for a review, see
Tavaré and Siddle(1993) and White and
Kahn(1994)). Again, these findings indicate that the IGF-IR has at
least another transforming pathway, which it does not share with the
other three receptors.
Our experiments do not tell us whether tyrosine phosphorylation is actually necessary for the transforming activity of the IGF-IR. We have avoided addressing this question at this point, and we prefer to state that a Tyr to Phe mutation at 1251 (but not at Tyr-1250) almost completely abolishes the transforming activity of the IGF-IR, leaving unresolved whether this is due to the lack of Tyr-1251 phosphorylation or to a conformational change. Neither do our experiments say that Tyr-1251 is the exclusive depository of the information for a transforming signal. What they say is that a functional Tyr-1251 is required for the transforming ability of the receptor.
The secondary findings have been reported here mainly to indicate that certain functions of the mutant receptors are not grossly impaired, as in the case of the ATP-binding site mutant (Kato et al., 1993), which has lost most of its functions and is neither mitogenic nor transforming (Kato et al., 1993; Sell et al., 1994; Coppola et al., 1994).
In conclusion, our experiments, while confirming the importance of the IGF-IR in the establishment and maintenance of transformation, have shown that tyrosine residues 1250 and 1251 are not necessary for IGF-I-mediated mitogenesis but that tyrosine 1251 is required for IGF-IR-dependent transformation. The dissociation of mitogenesis and transformation at the level of the receptor itself opens intriguing possibilities about the roles of the signal-transducing pathways in the two processes, regardless of whether the dissociation is qualitative, quantitative, or due to a prolongation of the stimulus (Marshall, 1995). Another obvious question to be addressed is whether the transforming activity of the IGF-IR correlates with its ability to protect cells from apoptosis (Harrington et al., 1994; Sell et al. 1995).