RESUMO
The insulin receptor is a ligand-activated tyrosine kinase that phosphorylates its major substrate protein, insulin receptor substrate 1 (IRS1), at multiple sites. Tyrosine-phosphorylated IRS1 then serves as a docking/effector protein for at least four Src homology 2 (SH2)-domain proteins involved in signal transduction. This initial step in signalling distinguishes the insulin receptor from other receptor tyrosine kinases, which directly bind several SH2-domain proteins, and establishes IRS1 as a founding member of a group of proteins whose function is to link activated tyrosine kinases to SH2-domain proteins.
RESUMO
Transferrin receptors in detergent extracts of subcellular membrane fractions prepared from 3T3-L1 adipocytes were measured by a binding assay. There was a small but significant increase (1.2-fold) in the amount of receptor in a crude plasma membrane fraction and a 40% decrease in the number of transferrin receptors in microsomal membranes prepared from insulin-treated cells, when compared with corresponding fractions from control cells. Intracellular vesicles containing insulin-responsive glucose transporters (GT) have been isolated by immunoadsorption from the microsomal fraction (Biber, J. W., and G. E. Lienhard. 1986. J. Biol. Chem. 261:16180-16184). All of the transferrin receptors in this fraction were localized in these vesicles; however, because the GT vesicles contain approximately 30-fold fewer transferrin receptors than GT, on the average only one vesicle in three contains a transferrin receptor. The binding of 125I-pentamannose 6-phosphate BSA to 3T3-L1 adipocytes at 4 degrees C was used to monitor surface insulin-like growth factor II (IGF-II)/mannose 6-phosphate receptors. Exposure of cells to insulin at 37 degrees C for 5 min resulted in a 2.5-4.5-fold increase in surface receptors. There was a corresponding 20% decrease in the amount of IGF-II receptors in the microsomal membranes prepared from insulin-treated cells, as assayed by immunoblotting. Moreover, the IGF-II receptors and GT were located in the same intracellular vesicles, since antibodies to the carboxyterminal peptide of either protein immunoadsorbed vesicles containing 70-95% of both proteins initially present in the microsomal fraction. In conjunction with other studies, these results indicate that in 3T3-L1 adipocytes, three membrane proteins (the GT, the transferrin receptor, and the IGF-II receptor) respond similarly to insulin, by redistributing to the surface from intracellular compartment(s) in which they are colocalized.
Assuntos
Tecido Adiposo/metabolismo , Fator de Crescimento Insulin-Like II/metabolismo , Proteínas de Transporte de Monossacarídeos/metabolismo , Receptores de Superfície Celular/metabolismo , Receptores da Transferrina/metabolismo , Somatomedinas/metabolismo , Animais , Células Cultivadas , Insulina/farmacologia , Cinética , Camundongos , Receptores de Superfície Celular/isolamento & purificação , Receptores de Somatomedina , Receptores da Transferrina/efeitos dos fármacos , Frações Subcelulares/metabolismoRESUMO
A semiquantitative method using immunocytochemistry on ultrathin cryosections and the protein A-gold technique was performed to study the effect of insulin on the cellular distribution of the glucose transporters in cultured 3T3-L1 adipocytes. In basal cells a substantial portion of the label was present in a tubulovesicular structure at the trans side of the Golgi apparatus, likely to represent the trans-Golgi reticulum, and in small vesicles present in the cytoplasm. Treatment with insulin induced a rapid translocation of transporters from the tubulovesicular structure to the plasma membrane. The transporter labeling of the plasma membrane increased three-fold and that of the tubulovesicular structure decreased by half. There was no effect of insulin on the degree of label in the small cytoplasmic vesicles. Removal of insulin from stimulated cells rapidly reversed the distribution of transporters to that seen in basal cells. This study thus provides the first morphological evidence for the occurrence of transporter translocation in insulin action and identifies for the first time the intracellular location of the responsive transporters.
Assuntos
Membrana Celular/metabolismo , Grânulos Citoplasmáticos/metabolismo , Insulina/farmacologia , Proteínas de Transporte de Monossacarídeos/metabolismo , Tecido Adiposo/citologia , Animais , Transporte Biológico/efeitos dos fármacos , Compartimento Celular/efeitos dos fármacos , Linhagem Celular , Imuno-Histoquímica , Membranas Intracelulares/metabolismo , Camundongos , Microscopia EletrônicaRESUMO
Antibodies specific for the insulin-regulatable glucose transporter (GLUT 4) were used to immunolocalize this protein in brown adipose tissue from basal- and insulin-treated rats. Cryosections of fixed tissue were incubated with antibodies, which were subsequently labeled with Protein A/gold and examined by EM. Antibodies against albumin and cathepsin D were also used with gold particles of different sizes to identify early and late endosomes, respectively. Under basal conditions 99% of the GLUT 4 labeling was located within the cell. Labeling was predominantly in the trans-Golgi reticulum and tubulo-vesicular structures elsewhere in the cytoplasm. In insulin-stimulated cells approximately 40% of the GLUT 4 labeling was at the cell surface, where it was randomly distributed, except for occasional clustering in coated pits. Moreover, after insulin treatment, GLUT 4 was also enriched in early endosomes. We conclude that translocation of GLUT 4 to the cell surface is the major mechanism by which insulin increases glucose transport. In addition, these results suggest that in the presence of insulin GLUT 4 recycles from the cell surface, probably via the coated pit-endosome pathway that has been characterized for cell surface receptors, and also that insulin causes the redistribution of GLUT 4 by stimulating exocytosis from GLUT 4-containing tubulo-vesicular structures, rather than by slowing endocytosis of GLUT 4.
Assuntos
Tecido Adiposo Marrom/metabolismo , Insulina/farmacologia , Proteínas de Transporte de Monossacarídeos/metabolismo , Animais , Membrana Celular/metabolismo , Endocitose , Exocitose , Membranas Intracelulares/metabolismo , Masculino , Microscopia Eletrônica , Proteínas de Transporte de Monossacarídeos/imunologia , Ratos , Ratos EndogâmicosRESUMO
The application of transition-state theory to enzymatic catalysis provides an approach to understanding enzymatic catalysis in terms of the factors that determine the strength of binding of ligands to proteins. The prediction that the transition state should bind to the enzyme much more tightly than the substrate is supported by the experimental results with stable analogs of transition states. Transition-state analogs have great potential for use in understanding enzymatic catalysis and in inhibiting enzymes. Because of their potency and specificity as enzyme inhibitors, some of them may become very useful chemotherapeutic agents.
Assuntos
Enzimas/metabolismo , Sítios de Ligação , Catálise , Transferência de Energia , Inibidores Enzimáticos , Cinética , Ligação ProteicaRESUMO
The amino acid sequence of the glucose transport protein from human HepG2 hepatoma cells was deduced from analysis of a complementary DNA clone. Structural analysis of the purified human erythrocyte glucose transporter by fast atom bombardment mapping and gas phase Edman degradation confirmed the identity of the clone and demonstrated that the HepG2 and erythrocyte transporters are highly homologous and may be identical. The protein lacks a cleavable amino-terminal signal sequence. Analysis of the primary structure suggests the presence of 12 membrane-spanning domains. Several of these may form amphipathic alpha helices and contain abundant hydroxyl and amide side chains that could participate in glucose binding or line a transmembrane pore through which the sugar moves. The amino terminus, carboxyl terminus, and a highly hydrophilic domain in the center of the protein are all predicted to lie on the cytoplasmic face. Messenger RNA species homologous to HepG2 glucose transporter messenger RNA were detected in K562 leukemic cells, HT29 colon adenocarcinoma cells, and human kidney tissue.
Assuntos
Proteínas de Transporte , Glucose/metabolismo , Proteínas de Membrana , Sequência de Aminoácidos , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Membrana Celular/ultraestrutura , Clonagem Molecular , DNA/genética , Eritrócitos/metabolismo , Humanos , Neoplasias Hepáticas Experimentais/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Peso Molecular , Proteínas de Transporte de Monossacarídeos , Conformação Proteica , RNA Mensageiro/genética , Distribuição TecidualRESUMO
The possibility that the stimulation of hexose transport in human fibroblasts by phorbol myristate acetate (PMA), insulin, platelet-derived growth factor (PDGF) or epidermal growth factor (EGF) is associated with phosphorylation of the glucose transporter has been investigated. The time and concentration dependencies of the stimulation of transport by these agents under conditions identical to those used for phosphorylation were determined. Each agent, when used at the concentration that resulted in the maximal increase in transport rate, elicited this effect within 30 min of exposure. The extent of stimulation ranged from 15 to 70%. For determination of phosphorylation of the glucose transporter, fibroblasts were incubated for 16 h with [32P]Pi and exposed to the agonist for 30 min; the transporter was then isolated from a detergent lysate of the cells by immunoprecipitation with a monoclonal antibody. Under these conditions, there was no phosphorylation of transporter in basal cells and only PMA caused detectable incorporation of phosphate into the transporter. Thus, it is unlikely that the stimulation of glucose transport by insulin, PDGF and EGF involve transporter phosphorylation.
Assuntos
Fibroblastos/metabolismo , Substâncias de Crescimento/farmacologia , Proteínas de Transporte de Monossacarídeos/metabolismo , Fosfoproteínas/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Fator de Crescimento Epidérmico/farmacologia , Fibroblastos/efeitos dos fármacos , Humanos , Insulina/farmacologia , Cinética , Fosforilação , Fator de Crescimento Derivado de Plaquetas/farmacologiaRESUMO
In the course of searching for proteins that interact with protein kinase B in 3T3-L1 adipocytes, we isolated a 105 kDa protein from 3T3-L1 adipocytes. Peptides sequenced from the protein were found to be present in several expressed sequence tags. A cDNA containing one of these expressed sequence tags was sequenced and appears to contain the entire coding region. Computer analysis revealed a potential protein kinase domain at the N-terminus; however, the first subdomain and several invariant residues characteristic of protein kinases are absent. An antibody was raised against a peptide from the 105 kDa protein. By immunoblotting, it was found that the protein was widely expressed in mouse tissues, and concentrated in the cytosol and low density microsome fractions of 3T3-L1 adipocytes.
Assuntos
Adipócitos/metabolismo , Fragmentos de Peptídeos/genética , Proteínas Serina-Treonina Quinases , Proteínas/genética , Proteínas Proto-Oncogênicas/química , Células 3T3 , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Citosol/enzimologia , DNA Complementar/química , Etiquetas de Sequências Expressas , Camundongos , Microssomos/enzimologia , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Proteínas/química , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-aktRESUMO
There is evidence that the atypical protein kinases C (PKC(lambda), PKC(zeta)) participate in signaling from the insulin receptor to cause the translocation of glucose transporters from an intracellular location to the plasma membrane in adipocytes. In order to search for downstream effectors of these PKCs, we identified the proteins that were immunoprecipitated by an antibody against PKC(lambda/zeta) from lysates of 3T3-L1 adipocytes through peptide sequencing by mass spectrometry. The data show that PKC(lambda) is the major atypical PKC in these cells. Moreover, an oligomeric complex consisting of alpha- and gamma-adducin, which are cytoskeletal proteins, coimmunoprecipitated with PKC(lambda). Association of the adducins with PKC(lambda) was further indicated by the finding that the adducins coimmunoprecipitated proportionally with PKC(lambda) in repeated rounds of immunoprecipitation. Such an association is consistent with literature reports that the adducins contain a single major site for PKC phosphorylation in their carboxy termini. Using antibody against the phospho form of this site for immunoblotting, we found that insulin caused little or no increase in the phosphorylation of this site on the adducins in a whole cell lysate or on the small portion of the adducins that coimmunoprecipitated with PKC(lambda). PKC(lambda) and the adducins were located in both the cytosol and subcellular membranous fractions. The binding of PKC(lambda) to adducin may function to localize PKC(lambda) in 3T3-L1 adipocytes.
Assuntos
Adipócitos/metabolismo , Proteínas de Ligação a Calmodulina/metabolismo , Proteína Quinase C/metabolismo , Células 3T3 , Sequência de Aminoácidos , Animais , Proteínas de Ligação a Calmodulina/química , Eletroforese em Gel de Poliacrilamida , Insulina/farmacologia , Isoenzimas , Camundongos , Dados de Sequência Molecular , Fosforilação , Testes de Precipitina , Proteína Quinase C/química , Proteínas/análise , Ratos , Frações Subcelulares/metabolismoRESUMO
Treatment of intact human erythrocytes with trypsin had no effect upon either the rate of hexose transport or the binding of cytochalasin B to the transport system. In contrast, proteolysis of inside-out vesicles prepared from human erythrocyte membranes inactivated both hexose transport and cytochalasin B binding. When purified hexose transporter, reconstituted into phospholipid vesicles of undetermined size, was treated with trypsin, approx. 50% of the cytochalasin B binding activity was lost. This loss correlated with a decrease in the amount of the transporter polypeptide, as assayed by gel electrophoresis. These results show that the orientation of the transporter can be established through trypsin treatment in conjunction with cytochalasin B binding. Small unilamellar vesicles containing transporter were prepared by sonication of larger species and by a cycle of cholate solubilization and removal of the detergent. In the former case, the transporter orients almost randomly, whereas in the latter approx. 75% of the transporters have the cytoplasmic domain external.
Assuntos
Membrana Eritrocítica/metabolismo , Eritrócitos/metabolismo , Hexoses/sangue , Transporte Biológico , Glicemia/metabolismo , Citocalasina B/sangue , Membrana Eritrocítica/efeitos dos fármacos , Membrana Eritrocítica/ultraestrutura , Congelamento , Humanos , Ligação Proteica , Sorbose/metabolismo , Tripsina/farmacologiaRESUMO
The cytochalasin B binding component of the human erythrocyte monosaccharide transport system has been purified. The preparation appears to contain one major protein with an apparent polypeptide chain molecular weight of 55,000 and about 0.4 binding sites per chain. Cytochalasin B binds to the reconstituted preparation with a dissociation constant of 1.3.10(-7) M, a value which is similar to that reported for the transport system in the intact erythrocyte.
Assuntos
Proteínas de Transporte/sangue , Citocalasina B/sangue , Membrana Eritrocítica/metabolismo , Eritrócitos/metabolismo , Proteínas de Membrana/sangue , Monossacarídeos/sangue , Transporte Biológico , Proteínas de Transporte/isolamento & purificação , Humanos , Cinética , Proteínas de Membrana/isolamento & purificação , Ligação ProteicaRESUMO
The glucose transporter from human erythrocytes is a heterogeneously glycosylated protein that runs as a very broad band of average apparent Mr 55 000 upon sodium dodecyl sulfate polyacrylamide gel electrophoresis. When the purified preparation of transporter, solubilized in Triton X-100, was treated with endoglycosidase F, much of it ran as a sharp band of Mr 46 000 upon electrophoresis. Moreover, endoglycosidase F released 80% of the radioactivity in a preparation of the transporter labeled in its oligosaccharides with galactose oxidase and tritiated borohydride, and almost none of the remaining radioactivity was located in the Mr 46 000 band. These results suggest that endoglycosidase F can release virtually all of the carbohydrate linked to the transporter polypeptide. A quantitative analysis of the gels was complicated by partial aggregation of polypeptides that occurs due to prolonged incubation in Triton X-100, but at least 65% of the protein in the preparation of purified transporter is the 46 kDa polypeptide. The extracellular domain of the transporter is very resistant to proteolysis; no cleavage occurred upon treatment of intact erythrocytes with seven different proteases at high concentration.
Assuntos
Proteínas de Transporte/sangue , Eritrócitos/análise , Glicosídeo Hidrolases/metabolismo , Oligossacarídeos/metabolismo , Proteínas de Transporte/isolamento & purificação , Eletroforese em Gel de Poliacrilamida , Humanos , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidase , Peso Molecular , Proteínas de Transporte de Monossacarídeos , Octoxinol , PolietilenoglicóisRESUMO
Insulin elicits the tyrosine phosphorylation of one or more proteins of 160-185 kDa in many cell types. Peptide sequences, obtained from this protein purified from mouse 3T3-L1 adipocytes (pp160), were used as the basis for cloning its cDNA, pp160 is highly homologous to the insulin receptor substrate 1, previously cloned from rat liver. Thus, this component of the insulin signaling pathway is the same in adipocytes and in liver.
Assuntos
Tecido Adiposo/metabolismo , Insulina/farmacologia , Fosfoproteínas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Proteínas Substratos do Receptor de Insulina , Fígado/metabolismo , Camundongos , Dados de Sequência MolecularRESUMO
Our previously described immunoadsorption method for the isolation of vesicles containing the insulin-responsive intracellular glucose transporters from 3T3-L1 adipocytes has been improved in two ways. First, the minimal number of g minutes required to sediment the plasma membranes from the cell homogenate has been determined and, as a result, the supernatant used for immunoadsorption in the new procedure contained twice as much of the intracellular transporters. Second, the immunoadsorption has been performed with affinity-purified antibodies directed against the carboxy terminal peptide of the transporter, rather than against the entire protein. 10(7) cells (10 mg protein) yielded about 12 micrograms of vesicular protein and 11 micrograms of vesicular phospholipid. The transporter constituted 3% of the protein in the vesicles; this amount equates to approx. eight copies of the transporter per 50 nm vesicle. The polypeptide composition of the vesicles was determined by gel electrophoresis and protein staining. Major components, other than the glucose transporter, are polypeptides of Mr 270,000, 245,000, 165,000 and 115,000. The vesicles contained several phosphoproteins; the major ones have a Mr of 245,000, 190,000, 115,000 and 25,000. Insulin treatment of adipocytes did not significantly change the phosphoprotein composition of the vesicles. The vesicles were not enriched in the Golgi marker enzyme, galactosyltransferase. The cellular content of the marker for the trans-Golgi reticulum, sialyltransferase, was too low to detect.
Assuntos
Tecido Adiposo/metabolismo , Membrana Celular/metabolismo , Insulina/farmacologia , Proteínas de Transporte de Monossacarídeos/metabolismo , Tecido Adiposo/efeitos dos fármacos , Animais , Membrana Celular/efeitos dos fármacos , Células Cultivadas , Cinética , Lipídeos de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Peso Molecular , Fosfatos/metabolismo , Fosfolipídeos/metabolismo , Fosfoproteínas/isolamento & purificação , Radioisótopos de FósforoRESUMO
The release of adipsin, a serine proteinase with complement factor D activity, from 3T3-L1 adipocytes was measured by quantitative immunoblotting. This protein is secreted constitutively from 3T3-L1 adipocytes, and there is a 2-fold increase in the amount of adipsin released from cells treated with insulin for 1 to 10 min. Longer exposure to insulin had no further effect on the rate of adipsin release. Adipsin does not appear to be anchored by a glycosylphosphatidylinositol moiety, since adipsin which was been released with Triton X-114 from an intracellular membrane fraction partitions into the aqueous phase. Using a previously described procedure for the isolation of vesicles containing the insulin-responsive intracellular glucose transporters (GT vesicles), we show here that these GT vesicles contain an insulin-responsive pool of adipsin. Thus, insulin stimulates the secretion of a soluble protein, adipsin, as well as translocation to the plasma membrane of integral membrane proteins, including the glucose transporter, the transferrin receptors, and the insulin-like growth factor II receptor.
Assuntos
Tecido Adiposo/metabolismo , Insulina/farmacologia , Animais , Compartimento Celular/efeitos dos fármacos , Linhagem Celular , Fator D do Complemento , Membranas Intracelulares/metabolismo , Camundongos , Peso Molecular , Proteínas de Transporte de Monossacarídeos/metabolismo , Taxa Secretória/efeitos dos fármacos , Serina Endopeptidases , SolubilidadeRESUMO
The glucose transporter in 3T3-L1 adipocytes has been identified as a polypeptide of average Mr 51000 by means of its reaction with antibodies raised against the purified human erythrocyte glucose transporter and by photolabeling with [3H]cytochalasin B. The finding that the antibodies immunoprecipitated the photolabeled polypeptide demonstrated that both methods detected the same polypeptide. The 3T3-L1 adipocyte glucose transporter has been partially purified. The main steps in the purification procedure were the preparation of salt-washed cellular membranes, Triton X-100 solubilization, and immunoaffinity chromatography on affinity-purified antibodies against the human erythrocyte transporter. A simple method of affinity purification of these antibodies, which consists of adsorption from serum onto protein-depleted erythrocyte membranes and release with acid, and an assay for the 3T3-L1 adipocyte transporter polypeptide, which employs immunoblotting, have been developed.
Assuntos
Tecido Adiposo/análise , Proteínas de Transporte de Monossacarídeos/isolamento & purificação , Tecido Adiposo/citologia , Animais , Linhagem Celular , Membrana Celular/análise , Cromatografia de Afinidade , Insulina/farmacologia , Camundongos , Proteínas de Transporte de Monossacarídeos/análise , Proteínas de Transporte de Monossacarídeos/imunologiaRESUMO
In the course of our examination of proteins associated with the GLUT4-containing vesicles of rat adipocytes we have identified a new 22 kDa member of the family of endoplasmic reticulum (ER) proteins known as reticulons. The protein, which we refer to as vp20, was purified from a preparation of GLUT4-containing vesicles of rat adipocytes, and tryptic peptides were micro-sequenced. From this information a cDNA encoding a single open reading frame for a protein of 22 kDa was cloned. This protein is homologous to known members of the reticulon protein family. vp20 has two hydrophobic stretches of about 35 amino acids that could be membrane spanning domains and an ER retention motif at its carboxy-terminus. vp20 was most abundant in the high density microsome fraction of adipocytes, which is the fraction most enriched in ER. Only a small fraction of vp20 was present in the GLUT4 vesicle population, and that fraction appears to be due to ER vesicles that were non-specifically bound to the adsorbent. Analysis of tissue distribution of vp20 in rats revealed that it is concentrated in muscle, fat and the brain.
Assuntos
Adipócitos/metabolismo , Proteínas de Membrana/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Encéfalo/metabolismo , Clonagem Molecular , DNA Complementar/química , Proteínas de Membrana/química , Proteínas de Membrana/isolamento & purificação , Microssomos/metabolismo , Dados de Sequência Molecular , Músculos/metabolismo , Proteínas da Mielina , Proteínas Nogo , RatosRESUMO
In the course of characterizing proteins present in a preparation of vesicles from rat adipocytes containing glucose transporters, we examined a protein that migrated at 115 kDa upon SDS gel electrophoresis (designated vp115). Sequences of tryptic peptides were obtained, and from this information the cDNA for rat vp115 was cloned. The cDNA encodes an open reading frame for a protein of 121 kDa. Computer-aided sequence analysis predicted that vp115 has a potential membrane-inserted or membrane-spanning domain toward its amino terminus, followed by five C2 domains. Immunoblotting revealed that vp115 was not actually a component of the glucose transporter-containing vesicles, was most abundant in the plasma membranes and high density microsome fractions of rat adipocytes, and was expressed in all the major rat tissues.
Assuntos
Adipócitos/metabolismo , Proteínas de Membrana/genética , Adipócitos/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Immunoblotting , Proteínas de Membrana/análise , Proteínas de Membrana/química , Dados de Sequência Molecular , Ratos , Homologia de Sequência , Frações Subcelulares/metabolismoRESUMO
To survey and compare the signaling pathways from the insulin and insulin-like growth factor-I (IGF-I) receptors in undifferentiated and differentiated muscle cells, we examined the phosphotyrosine (Ptyr)-containing polypeptides elicited in L6 and Sol8 myoblasts and myotubes by the combination of insulin and IGF-I. These polypeptides were detected by immunoblotting with antibodies against Ptyr. In the L6 myoblasts and myotubes and the Sol8 myoblasts, Ptyr polypeptides of approximately 240, 175, 115, 100, 41, and 37 kilodaltons (kDa) appeared in response to insulin-IGF-I. With the Sol8 myotubes, the 240-, 175-, and 37-kDa Ptyr polypeptides were detected in basal cells, and only the Ptyr content of the 175-kDa one increased in response to insulin-IGF-I. The polypeptides of 175, 41, and 37 kDa were tentatively identified as the insulin receptor substrate 1 (IRS1) and extracellular signal-regulated kinases 1 and 2 (ERK1 and -2), respectively, by immunoblotting with antibodies specific for these proteins, and the 115- and 100-kDa polypeptides are probably the beta-subunits of the insulin and IGF-I receptors. The amounts of IRS1, ERK1, and ERK2 were roughly the same in the L6 and Sol8 myoblasts and myotubes. Thus, differentiation of the myoblasts to myotubes was not accompanied by the detectable appearance of new insulin-IGF-I-elicited Ptyr polypeptides or marked changes in the amounts of known participants in their signaling pathways.