the response to insulin-like growth factor I was essen- tially lost. ... volved in the insulin induction of c-fos and a second, early growth response gene (Egr-I),' ...
Vol. 266, No. 1, Issue of January 5, pp. 455-460,1991 Printed in lJ S.A.
THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1991 by The American Society for Biochemistry and Molecular Biology, Inc.
Cellular Expression of Mutant Insulin Receptors Interferes with the Rapid Transcriptional Response to Both Insulinand Insulin-like Growth Factor I* (Received for Publication, July 20, 1990)
Deborah J. StumpoS and Perry J. Blackshears From the Howard HughesMedical Institute Laboratories, and the Section of Diabetes and Metabolism, Division of Endocrinology, Metabolism, and Genetics, Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710
We examined the expression of the proto-oncogene @-subunit is activated.Activation of this tyrosine kinase regene, Egr-1, in sults in autophosphorylationof the @-subunitof the receptor Rat 1 fibroblasts expressing high levels of normal or (4) as well as phosphorylation of exogenous peptides and mutated human insulin receptors (McClain, D. A., Mae- proteins (5). Several studies have suggested that the protein gawa, H., Lee, J., Dull, T. J., Ullrich, A., and Olefsky, tyrosine kinase activity of the insulin receptor is necessary J. M. (1987) J. Biol. Chem. 262, 14663-14671). In for insulin action (6-9). cells expressing large numbers of normal human insuInsulin is known to both increase (10-19) and decrease (20lin receptors (HIRc-B cells), insulin (20.7 nM) stimu- 24) the levels of specific mRNAs within targetcells. We have lated the rapid accumulation of mRNAs for bothgenes. demonstrated that insulin rapidly and transiently stimulated This response was blunted, but not lost, in cells ex- the accumulation of mRNA for the proto-oncogene c-fos in pressing large numbers of humaninsulinreceptors missing 4 3 amino acidsat the carboxyl terminus of the 3T3-Ll adipocytes, apparently acting through itsown recep&subunit. In contrast, the insulin response was com- tor (25). In the present study, we examined whether or not is inpletely absent in cells expressing large numbers of the insulin receptor protein tyrosine kinase activity receptors that contained a mutation at the ATP-bind- volved in the insulin induction of c-fos and a second, early growth response gene (Egr-I),’ which displays c-fos-like ining site that destroyed intrinsic protein tyrosine kinase duction kinetics (26, 27). To do this, we utilized four cell lines activity (A/K 1018-B cells). This mutation also suppressed the modest transcriptional response to insulin derived from Rat1fibroblasts thatexpressed varying numbers (6,28-32). The parental 1cells. The transcrip- of normal or mutant insulin receptors that occurred in the parental Rat tional response to serum was normal in theA/K 1018- line of Rat 1 fibroblasts was not transfected and contained B cells, evenafter protein kinase C depletion; however, 1700 endogenous rat insulin receptors. The first derivative the response to insulin-like growth factor I was essen- line, HIRc-B, hadbeen transfected with a plasmid containing tially lost. These studies suggest that overexpression normal human insulin receptorsequences and expressed 1.25 of a kinase-deficient insulin receptor can suppressthe x lo6 normal human insulin receptors per cell. Two other transcriptional response to both insulin and insulinlines, A/K 1018-B and HIRACT-P, expressed two different likegrowthfactor I that is ordinarilytransduced types of mutant human insulin receptors. A/K 1018-B cells through endogenous insulin and insulin-like growth expressed 2.2 x lo5 humaninsulinreceptorsper cell; the factor I receptors, respectively. Competition for sharedreceptors contain a single amino acid change at position 1018 substrates of these related receptor kinasesis a poten- in the tyrosine kinase domainof the @-subunit. The lysine at tial mechanism for this effect. position 1018 that is necessaryfor ATP binding has been changed to an alanine. The second mutant cell line, HIRACTP, expressed 2.5 X lo5 receptors per cell; these receptors are Insulin is a major anabolic hormone that influences a wide missing the 43 carboxyl-terminal amino acids of the insulin array of metabolicprocesses (1).Theseinfluences include receptor. short-term effects on membrane transport (e.g. glucose and EXPERIMENTALPROCEDURES amino acids) as well as long-term effects on DNA and RNA Cell Culture-Rat 1, HIRc-B, A/K 1018-B, and HIRACT-P cells synthesis. Themolecular mechanisms by which insulin elicits its pleiotropic effects in mammalian cells are not fully under- (6, 28-32) were a generous gift from Drs. D. A. McClain and J. M. stood. Theinitialstepininsulinaction is the binding of Olefsky, University of California, San Diego and Veterans AdminisMedical Center, San Diego and from A. Ullricb, Genentech, insulin to its cell surface receptor, a glycoprotein consisting tration South San Francisco. Rat1 fibroblasts were grown in minimal essenof two extracellular a-subunits and two transmembrane @- tial medium (with Earle’s salts) supplemented with 10% (v/v) fetal subunits (2, 3). Upon insulin binding to the a-subunit, an calf serum. The mutant cell lines were maintained in F-l2/Dulbecco’s modified Eagle’s medium (1:l) deficient in hypoxanthine and thymiintrinsic protein tyrosine kinase activity contained within the c-fos and the early growth response
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore he hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate thisfact. $ Associate of the Howard HughesMedical Institute. I Investigator of the Howard Hughes Medical Institute. To whom correspondenceshould be addressed Box 3897, DukeUniversity Medical Center, Durham, NC 27710.
dine and supplemented with 10% (v/v) fetal calf serum. All media (Gihco Laboratories) contained 2 mM glutamine, 100 units/ml penicillin, and 100 pg/ml streptomycin. Prior to inducing c-fos or Egr-1, the cells were grown to confluence andpretreated for 16h with serum-free Dulbecco’s modified Eagle’s medium containing 1%(w/v)
’
The abbreviations used are: Egr-1, early growth response gene; IGF-I, insulin-like growth factor I; PMA, phorbol 12-myristate 13acetate; CHO, Chinese hamster ovary cells.
455
456
Kinase-deficient Insulin Receptors and C-fos Expression
bovine serum albumin (crystallized and lyophilized) (Sigma) as described (33). When indicated, cells were pretreated overnight with 16 pM phorbol 12-myristate13-acetate (PMA) (Sigma) to make them protein kinase C-deficient as described (3.7). RNA Isolation and Northern AnaIuvsis-For gel blot analysis, total cellular RNA was isolated and analyzed onformaldehyde-agarose gels as described (34) with the following modifications. The formaldehyde& agarose gels were transferred directly to Nytran(Schleicher Schuell) in 10 X SSC (0.15 M sodium chloride and 15 mM sodium citrate) after staining withacridine orange and destaining.The baked blots were prehybridized for 24 h followed by hyhridization for 24 h with '"P-labeled cDNA restriction fragments (IO" cpm/ml). Northern blots were first hybridized with a '"P-labeled I-kilobase PstI fragment of pfos-1 (25) and then stripped by placing in 0.1 X SSC, 0.1% sodium dodecyl sulfate that was then brought to 100 "C and allowed to cool to 70 "C. The blots were then hyhridized witha "'P labeled 3.1kilohase EcoRI fragment of OC3.1, Egr-1 clone 191 (26, 27). &-I clone 191 was a generous gift from V. 1'. Sukhatme, Howard Hughes Medical Institute, University of Chicago. RESULTS
28s-
18s-
28s-
18s-
Dose Response of Insulin-stimulated c-fos and Egr-1 mRNA Accumulation-We (25, 35) and others (36) have shown previously that insulin can induce c-fos expression in cell lines that express a significant number of insulin receptors. We examined the ability of insulin to stimulate c-fos in four rat ~ & I C O d d d + - ~ O d o O I C ~ fibroblast cell lines expressing various numbers of normal and " mutanthumaninsulinreceptors.Inconfluent,serum-deA / K 1018-8 HIRACT-P prived Rat 1 fibroblasts, insulin (0.7-70 nM) marginally stimFIG. 1. Dose response of insulin-stimulated c-fos mRNA aculated c-fos mRNA accumulation. This small stimulation is cumulation in Rat 1, HIRc-€3, A/K 1018-€3.and HIHACT-P probably attributable to the low level of endogenous rat in- cells. Confluent, serum-deprived Rat 1 , HIRc-H. A/K 1018-H,or sulin receptors in thesecells (approximately 1700/cell). In the HIRACT-P fibroblasts were treated for 45 min with water alone ( 0 ) HIRc-B cells, which express a large number of normal human or the indicated nanomolar concentrations of insulin (Squihh-Novo. insulinreceptors,insulinstimulatedanincrease in c-fos Princeton, Nd). Total cellular RNA was isolated and 15-pg samples mRNA levels to a much greater extent than was seen in the were fractionated on a 1.2%formaldehyde-agarose gel and transferred to Nytran. The Northern blot was hybridized with 5 X 10" cpm of Rat 1 cells. Induction was evident at aninsulin concentration "P-labeled pfos-1 cDNA and then subjected to autoradiography. The of 0.07 nM with maximum induction occurring a t 7 nM (Fig. markers indicate the positions of the major rihosomal RNA species 1).There was no observed induction of c-fos in response to on the stained gel. insulin in the A/K 1018-B cells, supporting the view that fos and Egr-1 seen in the parental Rat 1 cells. Of several insulin receptor protein tyrosine kinase activity is required for insulin to stimulate c-fos expression. In addition, the smallpossible explanations for this effect, we considered the posstimulation of c-fos seen in insulin-treated Rat 1 fibroblasts sibilitiesthatthemutanthumaninsulinreceptordimers was noticeably absent in these cells. Finally, we observed an could be recombining with the normal rat receptorn[j dimers induction of c-fos in HIRACT-P cells, but only a t 7 and 70 to form inactive hybrid receptors or that the mutant receptors nM concentrations of insulin. Thelevels of c-fos mRNA were might be bindingtoand possibly sequesteringimportant cellular substrates of, or cofactors for, the insulin receptor not as great as those seen in HIRc-B cells but were significantly greater than those seen in untransfected Rat 1 fibro- protein kinase. T o study this phenomenon further, we perblasts. formed studies in the samecells with two other agents known T o examine whether this effect of insulin was confined to to act in part through activation of receptor protein tyrosine c-fos stimulation, we looked at the ability of insulin to stim- kinase activities: serum, presumably acting through the plateulate another gene, Egr-1, which is rapidly and transiently let-derivedgrowth factorreceptor,andinsulin-like growth induced by serum, PMA, and insulin (26, 27)in a manner factor I (IGF-I). The studies with serum in particular were very similar to thatobserved for c-fos (25). As was seen for c- performed in protein kinase C-depletedcells, in the hope that fos, insulin stimulated Egr-1 mRNA levels in HIRc-B cells this response would be more likely to resemble the relatively (Fig. 2). Induction was first observed with an insulin concen- "pure" tyrosine kinase response elicited by the activated insulin receptor. tration of 0.07 nM and was maximal a t 7 nM. There was a Induction of c-fos and Egr-1 mRNA in Normal and Protein small induction in Rat 1 cells, and no induction was observed in A/K 1018-B or HIRACT-P cells. As in the case of c-fos, Kinase C-deficient HIRc-R and A/K 1018-RCells-We have c-fos induction in R protein the expression of the mutant receptors in the A/K 1018-B shown that insulin stimulated cells completely inhibited the insulin induction of Egr-1, in kinase C-independent fashion and that the growth factorcontrast to the modest induction seen in the parental Rat 1 stimulated increase in c-fosmRNAaccumulation occurred cells. via both protein kinase C-dependent and -independent pathIn both the Rat 1 and HIRc-B cells, insulin induction of c- ways (25). We therefore examined theeffect of serum, insulin, fos expression displayed kinetics similar to those seen in other and (IGF-I) on c-fos and Egr-1 mRNA accumulation in norcells, i.e. mRNA levels were maximal by 30 minandhad mal and protein kinase C-deficient HIRc-I3 and A/K 1018-H decreased almost tobasal levels by 120 min (data not shown). cells. We havepreviously of reportedthatpreincubation The most striking finding of these initial studies was that mouse fibroblasts with 16 p~ PMA for 16 h resulted in the expression of the mutant insulin receptors in the A/K 1018- almost complete disappearanceof protein kinaseC from these B cells completely repressed the modest induction of both c- cells (25, 33, 34). Treatment of normal and protein kinase CIC
kc,+
Kinase-deficient Insulin Rot 1
Receptors and C-fos Expression
HIRc-B
457 B. Egr-1
A. Fos HlRc-0
A l K 1018%
HIRC-B
"
C
285
-
185
-
18s-
PMA
C
oe-8 PMA
" "
c s c s c s c s
28s-
AIK
"
C P ? h % C P M A
" "
c s c s c s c s
2115
-
FIG. 4. The effect of preincubation with PMA on the serum stimulation of c-fos and Egr-1 mRNA in HIRc-R and A/K 1018-B cells. In A. confluent, serum-deprived HIRE-R and A/K 1018-B fibroblasts were pretreated as described in the legend to Fig. 3 and subsequently treated with water alone ( C ) or 10% (v/v) fetal calf serum ( S ) for 45 min. RNA was isolated and 15-pg samples were fractionated, transferred to Nytran, and hybridizedwith pfos-1 as described in the legend to Fig. 1. In E, fibroblasts were treated as in A except the Northern blot was hybridized with Egr-1 as described in the legend to Fig. 2.
18s-
28s
A. Fos c O b o o r ,
HIRc-B
hg6h
C
o o o o c ~ o o o o c ~ I
1018-8
c
PMA
00.1 7 0 07 7
E. HIRc-B/Egr-l
c
HIRc-8
1018-8
C
PMA
PMA
"
0077 0 0 7 7
20s-
FIG.3. The effect of preincubation with PMA on the insulin stimulation of c-fos and Egr-1 mRNA in HIRc-B cells. In A, confluent, serum-deprived HIRc-B fibroblasts were pretreated with 0.1% (v/v) dimethyl sulfoxide ( C ) or 16 p M PMA for 16h and subsequentlyincubated for 45 minwith wateralone (O), 0.7 nM insulin, or 7 nM insulin. RNA was isolated, fractionated (15 pg/lane), transferred to Nytran, and hybridized with pfos-1 as described in the legend to Fig. 1. In H , fibroblasts were treated as in A except the Northern blot was hybridized with Qr-I cDNA as described in the legend to Fig. 2.
deficient HIRc-B cells with insulin resulted in an equivalent increase of both c-fos (Fig. 3A) and Egr-1 (Fig. 3 R ) mRNA levels. Fetal calf serum treatment of confluent, serum-deprived HIRc-B cells resulted in the stimulation of c-fos (Fig. 4A) and Egr-1 (Fig. 4R) mRNA. The effect of serum was partially blocked in protein kinase C-deficient HIRc-B cells, indicating that serum was exerting its effects through both protein kinase C-dependent and -independent mechanisms. However, in contrast to the effect of insulin, serum was able t o induce both c-fos and Egr-I expression to a normal extent
A/K
PMI
C
1018-8 PMA
" "
c s I c s I c s I c s I
I FlI;LP- ~-
HIRACT-P
cumulation in Rat 1, HIRc-B, A/K 1018-B, and HIRACT-P cells. Confluent,serum-deprived Rat 1, HIRE-B,A/K 1018-B, or HIRACT-P fibroblasts were treated for 45 min as described in the legend to Fig. 1. Total RNA was isolated, fractionated (15 pg/lane), transferred to Nytran, andhybridized with 5 X loficpm of R'P-labeled Egr- I cDNA.
"
A/K C
c s I c s I c s I c s I
FIG. 2. Dose respone of insulin-stimulated Egr-1 mRNAac-
A. HIRc-B/Fos
PMA
" "
I
1
A/K
B. Eqr-1
285-
10s-
-
285-
I . * ' " v T
w w P+ 1s-
FIG. 5. The effect of serum and insulin-like growth factor I on c-fos and Egr-1 mRNA expression in control and PMApretreated HIRc-B and A/K 1018-E3 cells. In A, confluent. serum-deprived HIRc-B and A/K 1018-R fibroblasLq were pretreated as described in the legend to Fig. 3 and subsequently treated for 45 min with 20.3 P M acetic acid ( C ) , 10% (v/v) fetal calf serum ( S ) , or 70 nM IGF-I (I) (Amgen Biologicals, Thousand Oaks, CA)in 20.3 JIM acetic acid. RNA was isolated, fractionated (15 ,&lane), transferred to Nytran, and hybridized with pfos-1 as descrihed in the legend to Fig. 1. In R, fibroblasts were treated as in A except the Northern blot was hybridized with fi;gr-l as described in the legend to Fig. 2.
in A/K 1018-B cells, and this stimulationwas not affected by PMA preincubation of the cells (Figs. 4, A and H and 5, A and B ) . A/K 1018-B, HIRc-B, and Rat 1 cells have been shown to express a similar number of IGF-I receptors (1.2-1.5 X lo"/ cell) (30). IGF-IbindingandIGF-Istimulation of hexose transport are unaffected by the expression of the A/K 1018B receptors (30). However, the expression of the A/K 1018-R kinase-deficient receptors inhibited IGF-I mediated mitogenesis and IGF-I-stimulated phosphorylation of two endogenous substrates, pp220 and pp170 (32). We examined whether or not IGF-I was capable of stimulating c-fos and Egr-1mRNA in HIRc-B and A/K 1018-B cells. Treatment of confluent, serum-deprived HIRc-Bcells with 70 nM IGF-I resulted in an accumulation of c-fos (Fig. 5A) and Egr-1 (Fig. 5H) mRNA. In contrast to these results, IGF-I had noeffect on the levels of c-fos (Fig. 5 A ) or Egr-1 (Fig. 5R)mRNAs in A/K 1018-R cells. Thus, as in the case of insulin stimulation, thepresence of the mutant insulin receptorscompletely blocked the effect of IGF-I on expressionof the two genes. However, in contrast to the lack of effect of insulin and IGF-I on c-fos and Egr-1 induction in the A/K 1018-B cells,fetal calf serum was capable of stimulating both c-fos and Egr-I expression to the
458
Kinase-deficient Receptors Insulin and
C-fos Expression
same extent in HIRc-B and A/K 1018-B cells (Figs. 4 and 5). Chinesehamster ovarycells ( 7 , 8) with respect, toinsulin Dose Response of IGF-I-stimulated c-fos and Egr-I mRNA stimulation of hexose transport, glycogen synthase activity, Accumulation-Insulin and IGF-I bind to distincthigh affin- and mitogenesis. This inhibition of normal insulin action in ity receptors, although both hormones can bind to the recep- the kinase-deficient A/K 1018-R cells could be due to intertors for the other ligand at high concentrations (37). The ference with the expression of the normal endogenous recepinitial experiments with IGF-I (Fig. 5) were carried out with tors, abnormal combination with the normal endogenous t d 70 nM, a concentration that is capable of binding to the insulin receptor dimers, or inhibition at any of the postbinding steps receptor. T o rule out the possibility that IGF-I was inducing in insulin action. Maegawa et al. (30) investigated the mechgene expression only by cross-reacting with the insulin recep- anism of this inhibition and demonstrated that, in the kinasetor, we examined the effect of varying concentrationsof IGF- deficient A/K 1018-B cells, theendogenousRat 1 insulin I on c-fos and Egr-1 mRNA accumulation in Rat 1, HIRc-B, receptors were present. to the same extent as in untransfected and A/K 1018-B cells. IGF-I at 7 nM stimulated induction of Rat 1 cells. Theendogenousreceptors possessed tyrosine c-fos (Fig. 6A) and Egr-I (Fig. 6R) in both Rat 1 and HIRc-B kinase activity and underwent autophosphoryltion in vitro cells, indicating that IGF-Iwas acting through its own recep- and in vivo. These studies suggest that both the biosynthesis tors at this concentration. However, a t a concentration of 7 of the endogenous rat receptors and their subsequent assemnM IGF-I, there was a negligible effect in the A/K 1018-B bly into mature (Y& receptors are normal in the A/K 1018-R cells. Thus, a direct comparison of the c-fos and Egr-1 mRNAcells. These authors alsoshowed that IGF-I binding and IGFaccumulation caused by IGF-I in Rat 1 and A/K 1018-B cells I-stimulated hexose transport were similar in Rat 1 and A/K showed that expression of the mutant insulin receptors not 1018-R cells(30),indicating that the endogenous IGF-I receponlyinhibitedinsulinbutalsoIGF-Istimulation of gene tor was functioning normally in the A/K 1018-R cells. Howexpression through the normal endogenous rat IGF-I recep- ever, in contrast to these normal responses, IGF-I stimulated tors. mitogenesis wasinhibited by thepresence of thekinasedeficient A/K 1018-B receptors. DISCUSSION Since the expression, autophosphorylation, and kinase acwere normal The major finding of this studyis that high level expression tivity of the endogenous rat insulin receptors of the kinase-deficientA/K 1018-B insulin receptor inhibited and IGF-I binding appeared tobe normal in the A/K 1018-H both insulin and IGF-I stimulation of gene expression me- cells, Maegawa and co-workers (30, 32) investigated whether diated through theirrespective endogenous rat receptors. The or not any of the intermediate stepsin insulin or IGF-I action inhibitory effect on insulin action hasbeen reported by other were inhibited in these cells. They observed that in Rat 1 cells two substrates, pp170 and pp220, were phosphorylated investigators studying mutant insulin receptors defective with ATP binding expressed in Rat 1 fibroblasts (6, 30, 32) and in in a dose-dependent fashion in response to both insulin and IGF-I; however, in A/K 1018-€3 cells, there was little, if any, or IGF-I-dependent increase in pp170 or insulin-dependent A. Fos pp220 phosphorylation. Thus, one possible explanation for Rat I HIRc-B A/K 1018-8 the inhibition of insulin and IGF-I action seen in A/K 1018“ 7 B cells is thatthekinase-deficientreceptorspreventthe o ko co .o c e o$ o b yo o c e o ~ ~ 2 C e endogenousreceptors from interacting normallywith their endogenous phosphoprotein substrates. Since IGF-I was still capable of stimulating hexose uptake in A/K 1018-R cells, it 28s would appear that neither the phosphorylation of ppl70 nor pp220 is involved in the mediationof IGF-1’s effects on hexose transport. 18sThe blunted transcriptional response to insulin and IGF-I in the A/K 1018-R cells seen in the present study was not mimicked by serum, a complex mixture of agonists that acts on fibroblasts to activate protein tyrosine kinase receptors as B. Egr-1 C. Even in the protein kinase Cwell as protein kinase Rat 1 HIRc-8 A/K 1018-8 1 depleted cells, the gene induction response toserum was similar in the Rat 1, HIRc-B, andA/K 1018-R cells, suggesting that the mutant insulin receptors expressed in the A/K 1018-B cellsdo not interferewith at least someof the receptor tyrosine kinases activatedby serum. The precise mechanism by which the kinase-deficient A/K 1018-B insulin receptors inhibitendogenous insulin and IGF18sI receptor function is unknown, but several possibilities exist. One possibility is that receptor aggregation might be important for insulinandIGF-Iactionandsubsequent protein phosphorylation, as has been suggested for insulin (38)and FIG.6. Dose response of ICF-I-stimulated c-fos and Egr-1 epidermal growth factorreceptors (39). Inactive aggregates mRNA accumulation inRat 1. HIRc-B. and A/K 1018-Bcells. could form upon insulin or IGF-I bindingbetween endogenous In A, confluent,serum-deprived Hat 1, HIHc-R,and A/K 1018-R ratreceptors(insulin or IGF-I)and kinase-deficient A/K fihrohlasts were treated for 45 min with 20.3 p~ acetic acid alone ( 0 ) 1018-R receptors. Another possibility is that inactive hybrid or the indicated nanomolar concentrations of IGF-I in acetic acid. receptors could form between endogenous rat insulin or IGFRNA was isolated and 15-pg samples were fractionated, transferred I receptors and mutant A/K 1018-Rreceptors. This is u n to Nytran, and hyhridized with pfos-I as descrihed in the legend to likely, based on data reported by Maegawa ct al. (80). They Fig. 1. In R. fihrohlasts were treated as in A except the Northern hlot was hyhridized with Kgr-1 as descrihed in the legend to Fig. 2. showed that when ahuman-specificantibody was used to
Kinase-deficient Insulin
Expression C-fos Receptors and
459
The present study was designed to determine whether or immunoprecipitate transfected human receptors from A/K 1018-B cell extracts, a small amount of phosphorylated p- not insulin receptor protein tyrosine kinase activity was inof tworapidly transcribed subunit was consistently found. This was not due to cross- volved in the insulin induction reactivity between the rat receptor and the human antibody genes, c-fos and Egr-1. Our results show that the normal of c-fos since no phosphorylated @-subunit was immunoprecipitated insulin receptor is necessary for the insulin induction from Rat 1 cell extracts. The phosphorylated receptor could and Egr-1 and that eliminationof the tyrosine kinase activity represent a small degree of A/K 1018-Breceptor phosphoryl- of the insulin receptorwas sufficient to inhibit the induction ation (perhaps on serine or threonine residues) or heterotet- by insulin. We also observed that expression of the kinase1018-B cells inhibitedthe rameric receptors that are human-rat hybrids. If these hybrids deficientreceptorsintheA/K do exist, they representa small percentage of total rat recep- ability of insulin to stimulate c-fos and Egr-1 mRNA accumulation through the endogenous rat receptor. Further extors (approximately10% of the rat receptors can be accounted for by the material precipitated from A/K 1018-B cells with amination of the inhibitory effect of the A/K 1018-B insulin the human specific antibody), and thus, mixing of receptor receptor showed that the abilityof IGF-I t o induce c-fos and cup dimers does not seem to occur to a great degree during Egr-1 was also blocked but that serum actionwas unaffected. receptor synthesis and assembly (30). A third possibility is Previous studies (30, 32) have suggested that the inhibition that the kinase-deficient receptors compete with the endoge- by the mutant receptorsof normal insulin and IGF-I actions was posnous insulin and IGF-I receptors fora limited number of through endogenous rat insulin and IGF-I receptors cellular substrates. The substrates may bind or associatewith sibly due to competitionbetween the mutant insulin receptors limited the mutant receptors and therebybecome unavailable to the and the endogenous insulin and IGF-I receptors afor normal rat receptors. The fact that insulin- and IGF-I-me- number of cellular substrates. Sincec-fos expression is closely diated phosphorylation of pp170 and pp220 was inhibited in associated with cell growth, it ispossible that the inabilityof insulin and IGF-I to stimulatemitogenesis in the A/K 1018A/K 1018-B cells supports this possibility (30, 32). A second finding of the present study is that the presence B cells (32) might be due to the fact that c-fos expression is also not induced by insulin or IGF-I in thesecells. of normal insulin receptors containing tyrosine kinase activity is required for the induction of c-fos and Egr-1 in response to insulin. Previous studies have shown that the protein tyrosine Acknowledgments-We are very gratefultoDrs.DonMcClain, Jerrold Olefsky, and Axel Ullrich for the Rat 1, HIRc-B, A/K 1018kinase activityof the insulin receptor or the immediate prodB, and HIRACT-P cells and to Dr. Vikas Sukhatme for the Egr-I uct of that reaction, the autophosphorylated receptor, is re- cDNA. We also thank Jane Tuttlefor technical assistance. quired for mediatingthe effects of insulinon S6 kinase activation, hexose uptake, glycogen synthetaseactivation, REFERENCES endogenous and exogenous protein substrate phosphoryla1. Rosen, 0. M. (1987) Science 237, 1452-1458 tion, and thymidine incorporation into DNA (6-8, 30). We 2. Freychet, P., Roth, J., and Neville, D. M., Jr. (1971) Proc. Natl. have previously demonstrated that the intact insulin receptor Acad. S C ~U. . S. A. 68, 1833-1837 3. Bergeron, J. J. M., Cmz, J., Kahn, M. N., and Posner, B. I. (1985) is necessary for the insulin induction of c-fos expression in Annu. Reo. Physiol. 47, 383-403 Chinese hamsterovary (CHO) cells (35). In these studies,we 4. Kasuga, M., Karlsson, F. A,, and Kahn, C. R. (1982) Science usedCHO cells stably transfected with either the normal 215, 185-187 human insulin receptor (CH0.T) or a truncated human in5. Rosen, 0. M., Herrera, R., Olowe, Y., Petruzzeli, L.M.. and Cobb, sulin receptor that lacked the 112 carboxyl-terminal amino M. (1983) Proc. Natl. Acad. sei. U. S. A. 80,3237-3240 acids (CH0.T-t) (9). Induction of c-fos by insulin wasob6. McClain, D. A., Maegawa, H., Lee, J., Dull, T. J.,Ullrich, A., and Olefsky, J. M. (1987) J . Biol. C h m . 262, 14663-14671 served in CH0.T cells but not in CH0.T-t cells. Ellis et al. (9) have shown that, in CH0.T-t cells that express the trun- 7. Chou, C. K., Dull, T. J., Russell, D. S., Gherzi, R., Lebwohl, D., Ullrich, A,, and Rosen, 0.M. (1987) J. Biol. Chem. 262,1842cated insulin receptor, there is no evidence of autophospho1847 rylation of the insulin receptor, exogenous substrate phos8. Ebina, Y., Araki, E., Taira, M., Shimada, F., Mori, M., Craik, C. phorylation, or insulin-stimulatedhexose uptake. In the presS., Siddle, K., Pierce, S. B., Roth, R. A., and Rutter, W . J. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 704-708 ent study, we examined theeffect of insulin incells containing 9. Ellis, L., Clauser, E., Morgan, D. O., Edery, M., Roth, R. A., and a n intact human insulin receptor that was normal or conRutter, W. J . (1986) Cell 45, 721-732 tained a conservative lysine to alanine substitution that de10. Alexander, M., Curtis, G., Avruch, J., and Goodman, H. M. (1985) stroyed the ATP-binding siteof the receptor and thusmade J. Biol. Chem. 260, 11978-11985 it tyrosine kinase-deficient. These data support our previous 11. Alexander,M., and Goodman, H. M. (1986) in Mechanisms of results and also more precisely correlate the lack of insulin Insulin Action (Belfrage, P., Donner, J., and Stralfors, P., eds) Vol. 7, pp. 395-404, Elsevier Science Publishers B. V., Amsterresponse to the absence of insulin receptor tyrosine kinase dam activity. 12. Bolander, F. F., Jr., Nicholas, K. R., van Wyk, J. J., and Topper, Insulin was still capableof stimulating c-fos and, toa lesser Y. J. (1981) Proc. Notl. Acad. Sci. U. S. A. 78, 5682-5684 extent, Egr-1 expression in the HIRACT-Pcells, eventhough 13. Stumpo, D. J., and Kletzien, R. F. (1984) Eur. J. Eiochem. 144, 43 the expressed receptors are missing the carboxyl-terminal 497-502 amino acids (28). These receptors still exhibit insulin-stimu- 14. Dani, C., Grimaldi, P., and Ailhaud, G . (1986) in Mechanisms of Insulin Action (Belfrage, P., Donner, J., and Stralfors, P., eds) lated tyrosine kinase activity (28, 29), although they appear Vol. 7, pp. 383-394, Elsevier Science Publishers B. V., Amstert o be defective in mediating insulin stimulation of glucose dam uptake and glycogen synthase activity (29). These results are 15. Flaim, K. E., Hutson, S. M., Lloyd, C. E., Taylor, J. M., Shiman, incontrasttothoseobtainedforinsulin-stimulated c-fos R., and Jefferson,L. S. (1985) Am. J.Physiol. 249, E447-E453 expression in CH0.T-tcells (35); in these cells, the expressed 16. Evans, M. I., and McKnight, G. S. (1984) Endocrinology 115, 368-377 receptors are more severely truncated, lacking the 112 carboxyl-terminal amino acids (9). This truncation is sufficient 17. Blackshear, P. J., Nemenoff, R. A., Hovis, J . G., Halsey, D. L., Stumpo, D. J., and Huang, J.-K.(1987) Mol. Endocrinol. 1,44t o completely destroy the intrinsic tyrosine kinase activity of 52 the mutant receptorsin the CH0.T-tcells (9), as well as their 18. Messina, J . L., Hamlin, J., Azizkahn, J., and Larner, J. (1985) ability to respond to insulin with c-fos induction (35). Biochem. Biophys. Res. Commun. 133, 1168-1174
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