Modulating effects of the extracellular sequence of the human insulinlike growth factor I receptor on its transforming and tumorigenic potential.

We reported previously that an N-terminally truncated insulinlike growth factor I receptor (IGFR) fused to avian sarcoma virus UR2 gag p19 had a greater transforming potential than did the native IGFR, but it failed to cause tumors in vivo. To investigate whether the 36 amino acids (aa) of the IGFR extracellular (EC) sequence in the gag-IGFR fusion protein encoded by the retrovirus UIGFR have a modulatory effect on the biological and biochemical properties of the protein, four mutants, NM1, NM2, NM3, and NM4 of the EC sequence were constructed. NM1 lacks the entire 36 aa residues; NM2 lacks the N-terminal 16 aa residues (aa 870 to 885), including two potential N-linked glycosylation sites of the EC sequence; NM3 contains a deletion of the C-terminal 20 aa residues (aa 886 to 905) of the EC sequence; and NM4 contains N-to-Q substitutions at both N-linked glycosylation sites. NM1 was the strongest of the four mutants in promoting anchorage-independent growth of transfected chicken embryo fibroblasts, while NM2 and NM4 had weaker transforming potential than did the original UIGFR virus. Only NM1 and NM3 were able to induce sarcomas in chickens. The four NM mutant-transformed cells expressed the expected proteins with comparable steady-state levels. The in vitro tyrosine kinase activity of P53NM1 was about fourfold higher than that of the parental P57-75UIGFR, whereas NM2 and NM4 proteins exhibited four- to fivefold-lower kinase activities. Despite lacking the IGFR EC sequence, P53NM1 formed covalent dimers similar to those formed by the parental P57-75UIGFR. Increased phosphatidylinositol (PI) 3-kinase activity was found to be associated with the mutant IGFR proteins. Among NM4 proteins. Elevated tyrosine phosphorylation of cellular proteins of 35, 120, 140, 160, and 170 kDa was detected in all mutant IGFR-transformed cells. We conclude that the EC 36-aa sequence of IGFR in the gag-IGFR fusion protein exerts intricate modulatory effects on the protein's transforming and tumorigenic potential. The 20 aa residues immediately upstream of the transmembrane domain have an inhibitory effect on the tumorigenic potential of gag-IGFR, whereas N-linked glycosylation within the EC sequence appears to have a positive effect on the transforming potential of UIGFR. Increased in vitro kinase activity and, to a lesser extent, in vivo tyrosine phosphorylation as well as the elevated association of PI 3-kinase activity with IGFR proteins seem to be correlated with the transforming potential of IGFR mutant proteins.     

Enhancement of transforming potential of human insulinlike growth factor 1 receptor by N-terminal truncation and fusion to avian sarcoma virus UR2 gag sequence.

The human insulinlike growth factor 1 (hIGF-1) receptor (hIGFR) is a transmembrane protein tyrosine kinase (PTK) molecule which shares high sequence homology in the PTK domain with the insulin receptor and, to a lesser degree, the ros transforming protein of avian sarcoma virus UR2. To assess the transforming potential of hIGFR, we introduced the intact and altered hIGFR into chicken embryo fibroblasts (CEF). The full-length hIGFR cDNA (fIGFR) was cloned into a UR2 retroviral vector, replacing the original oncogene v-ros. fIGFR was able to promote the growth of CEF in soft agar and cause morphological alteration in the absence of added hIGF-1 to medium containing 11% calf and 1% chicken serum. The transforming ability of hIGFR was not further increased in the presence of 10 nM exogenous hIGF-1. The 180-kDa protein precursor of hIGFR was synthesized and processed into alpha and beta subunits. The overexpressed hIGFR in CEF bound hIGF-1 with high affinity (Kd = 5.4 x 10(-9) M) and responded to ligand stimulation with increased tyrosine autophosphorylation. The cDNA sequence coding for part of the beta subunit of hIGFR, including 36 amino acids of the extracellular domain and the entire transmembrane and cytoplasmic domains, was fused to the 5' portion of the gag gene in the UR2 vector to form an avian retrovirus. The resulting virus, named UIGFR, was able to induce morphological transformation and promote colony formation of CEF with a stronger potency than did fIGFR. The UIGFR genome encodes a membrane-associated, glycosylated gag-IGFR fusion protein. The specific tyrosine phosphorylation of the mature form of the fusion protein, P75, is sixfold higher in vitro and threefold higher in vivo than that of the native IGFR beta subunit, P95. In conclusion, overexpression of the native or an altered hIGFR can induce transformation of CEF with the gag-IGFR fusion protein possessing enhanced transforming potential, which is consistent with its increased in vitro and in vivo tyrosine phosphorylation.     

Molecular and biochemical bases for activation of the transforming potential of the proto-oncogene c-ros.

The transforming gene of avian sarcoma virus UR2, v-ros, encodes a receptor-like protein tyrosine kinase and differs from its proto-oncogene, c-ros, in its 5' truncation and fusion to viral gag, a three-amino-acid (aa) insertion in the transmembrane (TM) domain, and changes in the carboxyl region. To explore the basis for activation of the c-ros transforming potential, various c-ros retroviral vectors containing those changes were constructed and studied for their biological and biochemical properties. Ufcros codes for the full-length c-ros protein of 2,311 aa, Uppcros has 1,661-aa internal deletion in the extracellular domain, CCros contains the 3' c-ros cDNA fused 150 aa upstream of the TM domain to the UR2 gag, CVros is the same as CCros except that the 3' region is replaced by that of v-ros, and VCros is the same as CCros except that the 5' region is replaced by that of v-ros. The Ufcros, Uppcros, CCros, and CVros are inactive in transforming chicken embryo fibroblasts, whereas VCros is as potent as UR2 in cell-transforming and tumorigenic activities. Upon passages of CCros and CVros viruses, the additional extracellular sequence in comparison with that of v-ros was delected; concurrently, both viruses (named CC5d and CV5d, respectively) attained moderate transforming activity, albeit significantly lower than that of UR2 or VCros. The native c-ros protein has a very low protein tyrosine kinase activity, whereas the ppcros protein is constitutively activated in kinase activity. The inability of CCros and CVros to transform chicken embryo fibroblasts is consistent with the inefficient membrane association, instability, and low kinase activity of their encoded proteins. The CC5d and CV5d proteins are indistinguishable in kinase activity, membrane association, and stability from the v-ros protein. The reduced transforming potency of CC5d and CV5d proteins can be attributed only to their differential substrate interaction, notably the failure to phosphorylate a 88-kDa protein. We conclude that the 5' rather than the 3' modification of c-ros is essential for its oncogenic activation; the sequence upstream of the TM domain has a negative effect on the transforming activity of CCros and CVros and needs to be deleted to activate their biological activity.     

The unique insert of cellular and viral fms protein tyrosine kinase domains is dispensable for enzymatic and transforming activities.

The receptors for colony stimulating factor-1 (CSF-1), platelet derived growth factor and the c-kit protein tyrosine kinase (PTK) contain within their catalytic domains a stretch of 60-100 residues, largely unrelated in sequence, with no counterpart in other PTKs. Of the 64 amino acids within this kinase insert, 58 were deleted from the mouse CSF-1 receptor by oligonucleotide-directed mutagenesis. The mutant CSF-1 receptor was not markedly affected in its kinase activity, post-translational processing or its ability to induce autocrine transformation of NIH 3T3 mouse fibroblasts. Similarly, retention of kinase and transforming activities were observed following deletion of part or all of the kinase insert from the v-fms oncoprotein. The c- and v-fms kinase inserts were probed using monoclonal and polyclonal antibodies and were found to be highly antigenic. Two monoclonal antibodies raised to the v-fms cytoplasmic domain both recognized epitopes within the insert, and bound enzymatically active v-fms glycoproteins. These results indicate that the fms kinase insert is located on the surface of the protein and folds separately from the rest of the catalytic domain, but is not required for the biological activity of fms PTKs ectopically expressed in mouse fibroblasts. The insert may therefore play a specific function in cells such as monocytes and trophoblasts that normally express the CSF-1 receptor.     

The transforming potential of the c-erbB-2 protein is regulated by its autophosphorylation at the carboxyl-terminal domain.

The mutant c-erbB-2 protein with Glu instead of Val-659 exhibited transforming activity in NIH 3T3 cells. This protein showed enhanced tyrosine kinase activity in vitro and enhanced autophosphorylation at Tyr-1248 located proximal to the carboxyl terminus. Enhanced tyrosine phosphorylation of several cellular proteins was detected in cells expressing the Glu-659 c-erbB-2 protein. Introduction of an additional mutation at the ATP-binding site (Lys-753 to Met) of this protein resulted in abolition of its transforming ability. These data indicate that the transforming potential of c-erbB-2 is closely correlated with elevated tyrosine kinase activity of the gene product. To investigate the role of autophosphorylation in cell transformation, we introduced an additional mutation at the autophosphorylation site of the Glu-659 c-erbB-2 protein (Tyr-1248 to Phe). This mutant protein exhibited lower tyrosine kinase activity and lower transforming activity. On the other hand, when the carboxyl-terminal 230 amino acid residues were deleted from the c-erbB-2 protein, the tyrosine kinase activity and cell-transforming activity of the protein were enhanced. Thus, the carboxyl-terminal domain, which contains the major autophosphorylation site, Tyr-1248, may regulate cellular transformation negatively and autophosphorylation may eliminate this negative regulation.     

Activation of the Insulin-like Growth Factor Type 1 Receptor by Deletion of Amino Acids 870–905

We have created a deletion mutant of the insulin-like growth factor type 1 receptor (IGF-1 R) which lacks the 36 amino acids (aa) immediately N-terminal to the transmembrane domain (Δ870–905 IGF-1 R). This region has been reported to have a negative effect on the transforming potential of an avian sarcoma virus gag-IGF-1 R fusion protein. We have sought to determine whether this region plays a similar role in the intact IGF-1 R. Analysis of the tyrosine kinase activity of the Δ870–905 IGF-1 R shows that the mutant receptor is autophosphorylated without IGF-1 stimulation, indicating that the tyrosine kinase domain is constitutively active. In addition, processing of the receptor is decreased, resulting in accumulation of a high molecular weight proreceptor containing both α and β-subunits. A well-characterized substrate of the IGF-1 R, IRS-1, is constitutively phosphorylated by the Δ870–905 IGF-1 R and phosphoinositide (PI) 3-kinase activity, which is normally activated by the phosphorylation of IRS-1 following IGF-1 stimulation, is increased even in the absence of IGF-1. A second intracellular signal pathway normally activated by IGF-1, the MAP kinase pathway, showed no increase in activity in the absence of IGF-1. The Δ870–905 IGF-1 R promoted cell proliferation only in the presence of IGF-1. We conclude that this deletion increases the basal activity of the IGF-1 receptor tyrosine kinase and activates PI 3-kinase, but is unable to stimulate MAP kinase in the absence of ligand. These results confirm those seen in the gag-IGF-1 R fusion protein and indicate that aa 870–905 exert a negative effect on the tyrosine kinase domain of the β-subunit of the IGF-1 R.     

Reversible tyrosine phosphorylation/dephosphorylation of proline-directed protein kinase FA/glycogen synthase kinase-3α in A431 cells

Modulation of protein kinase FA /glycogen synthase kinase-3α (kinase FA /GSK-3α) by reversible tyrosine phosphorylation/dephosphorylation was investigated. In addition to genistein, other protein tyrosine kinase (PTK) inhibitors, such as tyrphostin A47 and B42, also could induce tyrosine dephosphorylation and inactivation of kinase FA /GSK-3α in A431 cells, and this process was found to be reversible. Pretreatment of the cells with 100 μM orthovanadate, a protein tyrosine phosphatase (PTP) inhibitor, could diminish significantly the effects of PTK inhibitors on both enzyme activity and phosphotyrosine content of the kinase, suggesting that the PTK inhibitors induced tyrosine dephosphorylation/inactivation of this kinase is mediated by orthovanadate-sensitive PTP(s) in A431 cells. Moreover, the phosphotyrosine moiety of kinase FA /GSK-3α was found to be highly turned over in resting cells. Interestingly, we found that the less active, tyrosine-dephosphorylated form of kinase FA /GSK-3α immunoprecipitated from genistein-treated cells was able to reactivate partially with concomitant rephosphorylation of tyrosine residue in vitro. Taken together, these findings demonstrate that tyrosine phosphorylation and concomitant activation of kinase FA /GSK-3α can be carried out both in vitro and in vivo and an in vivo phosphatase activity may function in antagonism to PTK activation of kinase FA /GSK-3α. J. Cell. Physiol. 171:95–103, 1997. © 1997 Wiley-Liss, Inc.     

Critical and distinct roles of amino- and carboxyl-terminal sequences in regulation of the biological activity of the Chp atypical Rho GTPase

Chp (Cdc42 homologous protein) shares significant sequence and functional identity with the human Cdc42 small GTPase, and like Cdc42, promotes formation of filopodia and activates the p21-activated kinase serine/threonine kinase. However, unlike Cdc42, Chp contains unique amino- and carboxyl-terminal extensions. Here we determined whether Chp, like Cdc42, can promote growth transformation and evaluated the role of the amino- and carboxyl-terminal sequences in Chp function. Surprisingly, we found that a GTPase-deficient mutant of Chp exhibited low transforming activity but that deletion of the amino terminus of Chp greatly enhanced its transforming activity. Thus, the amino terminus may serve as a negative regulator of Chp function. The carboxyl terminus of Cdc42 contains a CAAX (where C is cysteine, A is aliphatic amino acid, X is terminal amino acid) tetrapeptide sequence that signals for the posttranslational modification critical for Cdc42 membrane association and biological function. Although Chp lacks aCAAXmotif, we found that Chp showed carboxyl terminus-dependent localization to the plasma membrane and to endosomes. Furthermore, an intact carboxyl terminus was required for Chp transforming activity. However, treatment with inhibitors of protein palmitoylation, but not prenylation, caused Chp to mislocalize to the cytoplasm. Thus, Chp depends on palmitoylation, rather than isoprenylation, for membrane association and function. In summary, Chp is implicated in cell transformation, and the unique amino and carboxyl termini of Chp represent atypical mechanisms of regulation of Rho GTPase function.     

Inhibition of insulin-dependent lipogenesis and anti-lipolysis by protein tyrosine kinase inhibitors.

Protein tyrosine kinase (PTK) blockers which competitively inhibit the kinase activity of insulin receptors were synthesized and their properties examined. The best insulin receptor kinase (IRK) inhibitors possess either one hydroxyphenyl ring and two carboxyl groups or two phenyl rings and one carboxyl group. All the inhibitors, except tBoc-tyrosine aminomalonate, effectively block the IRK-catalyzed phosphorylation of exogenous substrate, but only partially block receptor autophosphorylation. These PTK blockers inhibit the insulin induced [14C]glucose assimilation into lipids (lipogenesis), but fail to inhibit the anti-lipolytic effect of the hormone. Only tBocTyr-aminomalonate was found to inhibit all the effects of insulin measured: insulin-stimulated phosphorylation of exogenous substrate, IRK autophosphorylation, insulin-dependent lipogenesis and the insulin-dependent anti-lipolytic effect. This inhibitor is the first blocker which is reported to block insulin-dependent anti-lipolysis. The inhibitors examined are devoid of general adverse effects since they have no effect on insulin-independent lipolysis, on [U14C]fructose assimilation or on (-)isoproterenol-stimulated lipolysis. These studies suggest that insulin-dependent lipogenesis and anti-lipolysis may be mediated by two distinguishable signalling pathways. This study also suggests that PTK inhibitors may become useful tools in the investigation of the signalling pathways of PTKs.     

Differential requirement for Rho family GTPases in an oncogenic insulin-like growth factor-I receptor-induced cell transformation

Insulin-like growth factor I receptor (IGFR) plays an important role in cell growth and transformation. We dissected the downstream signaling pathways of an oncogenic variant of IGFR, Gag-IGFR, called NM1. Loss of function mutants of NM1, Phe-1136 and dS2, that retain kinase activity but are attenuated in their transforming ability were used to identify signaling pathways that are important for transformation of NIH 3T3 cells. MAPK, phospholipase C gamma, and Stat3 were activated to the same extent by NM1 and its two mutants, suggesting that activation of these pathways, individually or in combination, was not sufficient for NM1-induced cell transformation. The mutant dS2 has decreased IRS-1 phosphorylation levels and IRS-1-associated phosphatidylinositol 3'-kinase activity, suggesting that this impairment may be in part responsible for the defectiveness of dS2. We show that Rho family members, RhoA, Rac1, and Cdc42 are activated by NM1, and this activation, particularly RhoA and Cdc42, is attenuated in both mutants of NM1. Dominant negative mutants of Rho, Rac, and Cdc42 inhibited NM1-induced cell transformation, as measured by focus and colony forming ability. Dominant negative Rho most potently inhibited the focus forming activity, whereas Cdc42 was most effective in inhibiting the colony forming ability of NM1-expressing cells. Conversely, constitutively activated (ca) Rho is more effective than ca Rac or ca Cdc42 in rescuing the focus forming ability of the mutants. By contrast, ca Cdc42 is most effective in rescuing the colony forming ability of both mutants.     

Receptor protein tyrosine phosphatase alpha participates in the m1 muscarinic acetylcholine receptor-dependent regulation of Kv1.2 channel activity.

The phosphorylation state of a given tyrosine residue is determined by both protein tyrosine kinase (PTK) and protein tyrosine phosphatase (PTP) activities. However, little is known about the functional interaction of these opposing activities at the level of an identified effector molecule. G protein-coupled receptors (GPCRs), including the m1 muscarinic acetylcholine receptor (mAChR), regulate a tyrosine kinase activity that phosphorylates and suppresses current generated by the Kv1.2 potassium channel. We examined the possibility that PTPs also participate in this signaling pathway since the tyrosine phosphatase inhibitor vanadate increases the extent of both Kv1.2 phosphorylation and suppression. We show that an endogenous transmembrane tyrosine phosphatase, receptor tyrosine phosphatase alpha (RPTPalpha), becomes tyrosine phosphorylated and co-immunoprecipitates with Kv1.2 in a manner dependent on m1 receptor activation. The N- and C-termini of Kv1.2 are shown to bind RPTPalpha in vitro. Overexpression of RPTPalpha in Xenopus oocytes increases resting Kv1.2 current. Biochemical and electrophysiological analysis reveals that recruiting RPTPalpha to Kv1.2 functionally reverses the tyrosine kinase-induced phosphorylation and suppression of Kv1.2 current in mammalian cells. Taken together, these results identify RPTPalpha as a new target of m1 mAChR signaling and reveal a novel regulatory mechanism whereby GPCR-mediated suppression of a potassium channel depends on the coordinate and parallel regulation of PTK and PTP activities.     

Requirement for receptor-intrinsic tyrosine kinase activities during ligand-induced membrane ruffling of KB cells. Essential sites of src-related growth factor receptor kinases

We have prepared site-specific antibodies toward human insulin, insulin-like growth factor-I, and epidermal growth factor receptors with chemically synthesized peptides derived from the cDNA-predicted amino acid sequences of these receptors. Two classes of antibodies were produced toward each receptor: one toward the carboxyl termini and the other against the kinase domains containing sequences homologous to the tyrosyl phosphorylation site of the product of src gene (pp60v-src). Both classes of antibodies specifically immunoprecipitated the appropriate 125I-ligand-receptor complexes and [35S]methionine-labeled receptors with almost equal potencies. Antibodies toward the kinase domains inhibited both autophosphorylation and tyrosine kinase activity of the corresponding receptors in a cell-free system, whereas antibodies toward the carboxyl termini did not. Microinjection of the kinase-inhibitory antibodies into the cytoplasm of human epidermoid carcinoma KB cells blocked the ability of the corresponding ligand to induce membrane ruffling. In contrast, these inhibitory antibodies did not block the ability of noncorresponding ligands to induce the same response. Furthermore, control immunoglobulin and antibodies toward the carboxyl termini did not block this biological response. These results support a role for the tyrosine-specific protein kinase activities of these growth factor receptors in mediating their biological effects and suggest that the regions homologous to the tyrosyl phosphorylation site of pp60v-src are important for these kinase activities both in cell-free and intact cell systems.     

Features of the pp60v-src carboxyl terminus that are required for transformation.

Analysis of the biological and biochemical activities of pp60recombinant-src proteins encoded by 12 carboxyl-terminal mutants showed that a wide family of alternate src carboxyl termini permit complete transforming and kinase activities. src proteins having carboxyl termini which are up to 10 amino acids longer than that of pp60c-src (17 amino acids longer than that of pp60v-src) still permit transformation. Transformation-positive mutations preserve leucine-516, a residue which is highly conserved in protein-tyrosine kinase sequences; removal causes in vivo protein instability. Successive deletion mutants show that this residue is at the boundary of a region required for kinase activity. pp60src which is truncated just outside this point still transforms cells and binds both pp50 and pp90 cellular proteins.     

A protein tyrosine phosphatase inhibitor, sodium orthovanadate, causes parthenogenetic activation of pig oocytes via an increase in protein tyrosine kinase activity.

This study was conducted to determine whether a protein tyrosine kinase (PTK) activity is involved in the initiation of the events that occur at fertilization in pig oocytes. After maturation for 47 h, a 7-h treatment of oocytes with 1 mM sodium orthovanadate, which is an inhibitor of protein tyrosine phosphatase, caused more than 90% pronuclear formation, cortical granule exocytosis, and a decrease in mitogen-activated protein kinase activity. Immunoblotting with an antibody specific for phosphotyrosine showed at least three proteins whose phosphotyrosine contents were significantly increased upon treatment of oocytes with 1 mM sodium orthovanadate. Preincubation of pig oocytes with 50 microM tyrphostin 47, a specific PTK inhibitor, completely blocked the ability of sodium orthovanadate to trigger activation events. In addition, when oocytes were pretreated with the calcium-chelating agent BAPTA-AM, sodium orthovanadate-stimulated pronuclear formation was significantly (P < 0.01) reduced (94.0% vs. 43.1%). These results suggest that PTK may be involved in pig oocyte activation in a calcium-dependent manner and that the stimulation of tyrosine kinase is able to signal a series of intracellular changes that lead to the activation events associated with fertilization.     

The application of the polymerase chain reaction to cloning members of the protein tyrosine kinase family

Degenerate oligodeoxyribonucleotide (oligo) primers derived from amino acid (aa) sequence motifs held in common between all members of the protein tyrosine kinase (PTK) family were used to prime the amplification of PTK-related sequences from a variety of murine cDNA sources, including the haemopoietic cell lines, FDC-P1 and WEHI-3B D+, peritoneal macrophages and whole brain. Several parameters, such as the length (short, i.e., less than 20 nucleotides (nt) vs. long, i.e., greater than 30 nt) and degeneracy (i.e., moderately degenerate vs. highly degenerate) of the oligo primers and the temperature of the extension phase of the reaction, were examined. The data from these analyses suggest that the most effective type of primer in this application of the polymerase chain reaction is a short, moderately degenerate oligo such as that which might be derived from the small patches of aa sequence homology that are frequently found to be held in common among members of protein families. In addition to a number of previously described PTK sequences, a novel mammalian PTK-related sequence was uncovered.     

Protein Tyrosine Kinase-Mediated Potentiation of Currents from Cloned NMDA Receptors

Abstract: Although serine/threonine phosphorylation has been more commonly recognized as a mechanism to modulate the function of ion channels and receptors, tyrosine phosphorylation is under increasing scrutiny. An important subtype of glutamate receptor, the NMDA receptor, is shown to be regulated by insulin via protein tyrosine kinase (PTK). NMDA currents through cloned receptors are potentiated by insulin in a subunit-specific manner. The insulin-mediated potentiation of NMDA current is diminished by inhibitors of PTKs. At least one exogenous cytosolic PTK, pp60^{c- src} , is also able to potentiate NMDA current. Because later application of PTK inhibitors can reverse the seemingly stable insulin-mediated potentiation of NMDA current, it appears that tyrosine residues responsible for potentiation are continually rephosphorylated by some long-term PTK activity that was induced via insulin treatment.     

The pathway mediating insulin's effects on pyruvate dehydrogenase bypasses the insulin receptor tyrosine kinase

The effect of insulin on pyruvate dehydrogenase activity was examined in two different cell types that over expressed either normal or defective human insulin receptors, RAT 1 embryonic fibroblasts and Chinese hamster ovary (CHO) cells. Insulin stimulated pyruvate dehydrogenase activity in cells that expressed normal insulin receptors (RAT 1 HIRc, and CHO-WT and CHO-T cells), or receptors in which lysine 1018 in the ATP-binding site of the tyrosine kinase domain was exchanged for alanine (RAT 1 A/K1018 and CHO-mut cells). For both rat and hamster cell lines, the insulin dose-response curves from cells that expressed the mutant receptors were identical to those from the appropriate controls that over expressed the normal insulin receptors. Insulin failed to stimulate pyruvate dehydrogenase activity in CHO-delta cells, which expressed a mutant human insulin receptor that was truncated by 112 amino acids at the carboxyl terminal of the beta chain. Control studies verified that all the cells used in this study exhibited the expected phenotypes with respect to the number of insulin receptors which they expressed, insulin-stimulated tyrosine kinase activity, and the biological consequences of inactivating the insulin receptor tyrosine kinase. These findings show that the insulin receptor tyrosine kinase does not play an obligatory role in the insulin signaling pathway that stimulates pyruvate dehydrogenase activity.