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Structural and functional homologies in the receptors for insulin and the insulin-like growth factors
Cell, Vol. 31, 8-l
0, November
1982,
Copyright
0 1982
by MIT
Structural and Functional Homologies in the Receptors for Insulin and the Insulin-Like Growth Factors Michael P. Czech Department of Biochemistry University of Massachusetts 55 Lake Avenue North Worcester, Massachusetts
Medical
Center
01605
One of the fascinating problems in the field of peptide hormone action is the coordination of cellular responses to insulin and the two related peptides, insulin-like growth factor I and II (IGF-I and IGF-II). The peptide structures of the three ligands are quite similar in that they share considerable amino acid sequence homology and the similar positioning of disulfide bonds within their structures (Rinderknecht and Humbel, JBC 253, 2769-2776, 1978; Rinderknecht and Humbel, FEBS Lett. 89, 283-286, 1978). An interesting distinction between insulin and IGFs is the retention by the latter of the connecting peptide structural element that is lost upon conversion of proinsulin to insulin. Thus the insulin-like growth factor polypeptides are single chains, while insulin contains two disulfide-linked polypeptides. IGF-I and IGF-II are secreted by the liver, although other, as yet unknown tissues may also contribute significantly to their production. IGF-I production by the liver is regulated quite strictly by pituitary growth hormone and may play a key role in mediating the growth effects of this hormone. In contrast, IGF-II production does not depend on growth hormone, and its precise physiological role is not understood. Insulin secretion by the /? cells of the pancreas is highly sensitive to circulating substrates such as glucose and amino acids. Thus the source and regulation of circulating levels of this family of peptides appear to be quite different. It should be noted that the terms somatomedins, multiplicationstimulating activity and nonsuppressible insulin-like activity have also been used to define polypeptides similar or identical to the insulin-like growth factors (Giordano et al., eds. Somatomedins and Growth. Proceedings of the Serono Symposia, 23, Academic Press, pp. l-95, 1979; Rechler et al., J. Supramol. Struct. 75, 253-256, 1981). The biological actions of insulin, IGF-I and IGF-II on target tissues can be divided into two types: those that involve the rapid modulation of membrane-transport systems or enzyme activities, and those that are longer in onset and appear to involve regulation of transcription or DNA replication. Experiments performed over the last decade with the purified peptides produced descriptive results that illustrate two basic and rather paradoxical elements of insulin and IGF action. First, studies on the binding of the ‘251-labeled peptides to cells and membranes indicated that distinct receptor species were present that exhibited cross-reactivity for the various ligands. For example, the insulin receptor in human placenta that bound insulin with high affinity bound IGF-II with lower affin-
ity, and IGF-I with lowest affinity. Yet careful analysis of the results of these studies showed a large number of differences among the tissues studied in the relative binding affinities of the three ligands to the putative receptors. This suggested the possibility that a large number of heterogeneous receptor structures might account for the great diversity of binding characteristics observed (Rechler et al., Endocrinology 707, 1451-1459, 1980). Second, low physiological concentrations of insulin but not of IGF-I or IGF-II were normally observed to modulate the rapid cellular responses, such as increased transport activities in target tissues. High concentrations of the IGFs were required to elicit these responses. In contrast, low concentrations of IGF-I and IGF-II were observed to stimulate DNA replication and cell proliferation, while high concentrations of insulin were able to mimic these effects. These data were consistent with the hypothesis that the growth factor receptors mediated cell proliferation and insulin receptors mediated the rapid cellular responses. Yet critical exceptions arose; extremely low concentrations of insulin could activate cell division, as in the rat hepatoma H35 cell line, and the IGFs could rapidly activate transport as in rat soleus muscle. Significant clarification of these issues has been achieved by the direct identification and characterization of specific receptor structures present in cell membranes that interact with insulin and the IGFs. It is now clear that cellular components that regulate and mediate the biological responses of insulin, IGF-I and IGF-II share several levels of structural and functional homologies and are highly integrated. The insulin receptor defined as the structure exhibiting highest affinity for insulin in all tissues studied has been extensively characterized by affinity purification, photoaffinity labeling, affinity crosslinking and immunoaffinity purification. Results generated with affinity crosslinking techniques and with purified receptor led us (Massague et al., PNAS 77, 7137-7141, 1980; Czech et al., TIBS 6, 222-225, 1981; Massague et al., JBC 257, 6729-6738, 1982) and others (Jacobs et al., JBC 255, 6937-6940, 1980; Jacobs and Cuatrecasas, Endocrine Rev. 2, 251-263, 1981) to propose, independently, a model of the minimum structure of the insulin receptor subunit that is consistent with virtually all data available. This model postulated the immunoglobulin-like structure (,f?-S-S-a)-S-S(a-S-S-p), in which LYis a glycoprotein subunit with an apparent molecular weight of 125,000 and /3 is a glycoprotein subunit with an apparent molecular weight of 90,000. This symmetrical, disulfide-linked heterotetrameric structure probably binds more than one insulin molecule, based on results obtained by immunoprecipitation of the “51-insulin-receptor complex. The disulfide or disulfides that link the two (cu-S-S-/3) receptor halves are denoted as class I
Cdl 9
disulfides, and are significantly more sensitive to dissociation by exogenous reductant than the class II disulfides that link 01subunits top subunits. The insulin receptor structure, like other receptors, is quite sensitive to proteolytic nicking during membrane preparation and purification. This can lead to the observation of subunit fragments of lower molecular weight. In particular, the receptor forms (,&-,/I-P-+SS-(a-S-S-/?) and (PI-S-S-a)-S-S-(a-S-S--p,) result from a proteolytic nick in ,& at approximately the center of its amino acid sequence. The application of photoaffinity and affinity crosslinking methodology, developed for analysis of the insulin receptor structure, to the IGF system led to the discovery of an IGF receptor structurally homologous to the insulin receptor (Bhaumick et al., PNAS 78, 4279-4283, 1981 ; Chernausek et al., Biochemistry 27, 7345-7350, 1981; Kasuga et al., PNAS 79, 1864-l 868, 1982; Massague and Czech, JBC 257, 5038-5045, 1982). This IGF receptor, with the apparent structure (/3-S-S-&S-S-(0-S-S-P), contains subunits of molecular weight and physical properties essentially identical, as determined on dodecyl sulfate gels, to the insulin receptor structure. Class I and class II disulfides similar to those present in the insulin receptor were also detected. Analysis of the inhibition of affinity labeling of the IGF receptor by unlabeled insulin, IGF-I and IGF-II allowed the conclusion that in all tissues studied it exhibits the following relative affinities for ligands: IGF-I > IGF-II > insulin. We denote this cellular component as the type I IGF receptor. How can we account for the divergent affinities of the insulin and type I IGF receptors, given their structural similarity? One possibility is that the two receptors are protein products of the same gene segments, but that posttranslational modifications alter their affinities for ligands. An alternative view is that partially homologous but distinct genes encode these receptors or certain variable protein domains of the receptors. It is not yet possible to distinguish between these postulates, but a series of recent observations indicates that the cellular expression of these two receptor structures may be highly coordinated. In three different cellular models of differentiation or mutation, appearance or disappearance of both receptor types occurs in parallel. Thus, during differentiation of the 3T3-Ll fibroblast to the adipocyte-like morphology, expression of both insulin receptor and type I IGF receptor structures is increased severalfold. Similarly, skin fibroblasts from two different human mutants with extreme insulin resistance that show altered insulin receptor characteristics also exhibit parallel defects in the type I IGF receptor, as detected by binding studies or affinity labeling techniques (Van Obberghen et al., J. Clin. Invest. 68, 1356-l 365, 1982). A second and divergent IGF receptor, denoted as the type II IGF receptor, is visualized by affinity label-
ing studies and migrates on dodecyl sulfate gels with an apparent molecular weight of approximately 250,000, without detectable disulfide-linked subunits (Massague and Czech, op. cit.). It should be noted that a variety of other receptor systems for peptide growth factors that have been identified recently, including those for nerve growth factor, platelet-derived growth factor, epidermal growth factor and transforming growth factor, also lack structural homology to the insulin and type I receptors. The type II IGF receptor species in all tissues studied consistently binds IGF-II with higher affinity than IGF-I, and has very little or no affinity for insulin. In the course of investigation with over 30 different cell types in our laboratory, only the characteristic polypeptides associated with the insulin receptor and the type I and II IGF receptor structures have been consistently detected and specifically labeled by affinity crosslinking methodology. It should be noted that the heterotetrameric insulin and type I IGF receptors and the single-chain type II IGF receptor may contain other, noncovalently linked subunits that are not detectable by the methods employed in the above studies. Can the three receptor structures identified be responsible for all of the complex ‘251-insulin and ‘251IGF binding and ligand competition patterns observed in diverse tissues? The evidence supports the view that the receptor species identified can account for both the binding and biological actions of the three peptide hormones. The complex and heterogeneous binding kinetics observed among different systems appears to relate to two factors. First, the absence or presence, as well as the number, of each of these receptor species varies markedly among cell types. Certain cells, such as the classic targets for insulin action, hepatocytes and adipocytes, contain no type I IGF receptor species. Others, such as the chicken embryo fibroblast or Erlich-Lettre carcinoma cell line, contain large amounts of the type I structure, but vanishingly low amounts of insulin receptor and no detectable type II IGF receptor. Second, the three individual receptor structures exhibit marked heterogeneity in their relative affinities for ligands in the different tissues. Thus the type II IGF receptor in human fibroblasts exhibits a relatively lower affinity for IGF-I than the type II IGF receptor in liver cells; the type I IGF receptor in chicken embryo fibroblasts exhibits markedly higher affinity for IGF-II than this receptor in Erlich-Lettre strain E cells. This heterogeneity in binding kinetics of the three receptor types among different tissues must result from the association and influence of other membrane components that differ among tissues, or from a structural heterogeneity in the receptor molecules themselves. Consistent with the latter interpretation are detectable differences in apparent molecular weights of the type II IGF receptor from various sources. The combination of these two factors-the number and types of the
Cell IO
receptor structures present, and the distinct pattern of ligand affinities displayed by each of the receptor types in disparate tissues-accounts for the overall binding kinetics observed in individual tissues. The identification of the three insulin and IGF receptor types has also clarified the relationships between the receptors and their biological actions. The structurally homologous heterotetrameric receptor structures for both insulin and IGF-I appear capable of activating the rapid transport and enzyme changes normally associated with insulin action when they are present in an appropriately responsive tissue-that is, skeletal muscle. In contrast, the type II IGF receptor is impotent in catalyzing these responses. Thus the IGFs do not activate at low concentrations the rapid insulin-like effects in adipocytes and hepatocytes, because these cells have only insulin and type II IGF receptor types and lack the type I receptor structure. This conclusion is satisfying because homologous receptor structures might be expected to couple to identical or homologous cellular signaling mechanisms. Unfortunately, the extension of this concept to the slow-onset growth effects does not seem to hold, and illustrates an unsolved, intriguing complexity. While many cell types in culture appear to respond to type I IGF receptor stimulation with DNA replication, the insulin receptor structure appears impotent in mediating this response in most cell types (King et al., J. Clin. Invest. 66, 130-140, 1980). In human fibroblasts, for example, insulin at high concentrations acts through the type I IGF receptor species, not its own receptor, to promote proliferation. An exception to this is the H35 hepatoma cell line, which is devoid of the type I IGF receptor and unequivocally responds to insulin receptor perturbation with increased DNA replication and cell division (Koontz et al., Science 27 7, 947-949, 1981). This difference in insulin receptor function among cell lines is unclear. The type II IGF receptor presumably mediates a cell proliferative response, but this has not been unequivocally documented to date. A striking feature of the relationship among the receptors for insulin and the IGFs is a potent biological action executed by the insulin receptor on the type II IGF receptor. The binding of insulin to its high-affinity receptor markedly increases, by up to tenfold, the affinity of the type II receptor for its ligand in at least two cell types (Schoenle et al., Diabetologia 73, 243-249, 1977; Massague et al., JBC, in press). This action of insulin is observed in intact cells but not isolated membrane preparations. In addition, we have recently found that the process of cell disruption that is necessary to prepare membranes itself mimics in-
sulin action to enhance the affinity of the type II receptor. The mechanism by which this response to insulin is mediated is not understood, but the potential physiological implication is apparent. This mechanism may allow the insulin receptor to stimulate rapid transport and enzyme effects directly, while indirectly potentiating the cell-growth response by way of promoting IGF action through the type II receptor. Such modulation of one receptor system by another receptor now appears quite common, and may be of substantial significance. We have not found evidence to suggest a similar effect of the type I IGF receptor on the type II receptor. A key unanswered question about the functions of the insulin and IGF receptors relates to the molecular mechanism or mechanisms they employ to transduce their biological effects. Insulin action has been the most carefully and exhaustively studied, but still remains largely a mystery. The identification of the receptor structures that participate in signal transduction by these peptides and the ability to isolate and characterize their properties under a variety of conditions provide a powerful approach to the problem that has only recently been available. Using antireceptor antibodies to immunoprecipitate the insulin receptor following incubation of 32P-labeled intact cells with or without insulin, Kahn and colleagues demonstrated increased incorporation of labeled phosphate into the 90,000 molecular weight ,l3 subunit (Kasuga et al., Science 275, 185-I 87, 1982). Insulin also markedly stimulated 32P labeling of this receptor subunit in detergent extracts of membranes incubated with y32P-ATP (Kasuga et al., Nature 298, 667-669, 1982). Both serine and tyrosine residues on the receptor were phosphorylated, although the latter were most stimulated by hormone in a cell-free system. The relevance of these observations to receptor function is unknown, but parallel observations have previously been made with the most extensively studied epidermal growth factor receptor system. Furthermore, the association between tyrosine phosphorylation and cellular growth patterns during viral transformations has spurred great interest in these phenomena. The picture that emerges is that the insulin receptor, as well as the epidermal growth factor and probably others, may itself be a protein kinase or is closely associated with a kinase activity in the cellsurface membrane. The central focus of current studies is to ascertain whether this property of the insulin receptor is also shared by the IGF receptor structures, and whether it directly relates to receptor signaling or perhaps other receptor functions, such as desensitization or hormone-induced cellular internalization.
0, November
1982,
Copyright
0 1982
by MIT
Structural and Functional Homologies in the Receptors for Insulin and the Insulin-Like Growth Factors Michael P. Czech Department of Biochemistry University of Massachusetts 55 Lake Avenue North Worcester, Massachusetts
Medical
Center
01605
One of the fascinating problems in the field of peptide hormone action is the coordination of cellular responses to insulin and the two related peptides, insulin-like growth factor I and II (IGF-I and IGF-II). The peptide structures of the three ligands are quite similar in that they share considerable amino acid sequence homology and the similar positioning of disulfide bonds within their structures (Rinderknecht and Humbel, JBC 253, 2769-2776, 1978; Rinderknecht and Humbel, FEBS Lett. 89, 283-286, 1978). An interesting distinction between insulin and IGFs is the retention by the latter of the connecting peptide structural element that is lost upon conversion of proinsulin to insulin. Thus the insulin-like growth factor polypeptides are single chains, while insulin contains two disulfide-linked polypeptides. IGF-I and IGF-II are secreted by the liver, although other, as yet unknown tissues may also contribute significantly to their production. IGF-I production by the liver is regulated quite strictly by pituitary growth hormone and may play a key role in mediating the growth effects of this hormone. In contrast, IGF-II production does not depend on growth hormone, and its precise physiological role is not understood. Insulin secretion by the /? cells of the pancreas is highly sensitive to circulating substrates such as glucose and amino acids. Thus the source and regulation of circulating levels of this family of peptides appear to be quite different. It should be noted that the terms somatomedins, multiplicationstimulating activity and nonsuppressible insulin-like activity have also been used to define polypeptides similar or identical to the insulin-like growth factors (Giordano et al., eds. Somatomedins and Growth. Proceedings of the Serono Symposia, 23, Academic Press, pp. l-95, 1979; Rechler et al., J. Supramol. Struct. 75, 253-256, 1981). The biological actions of insulin, IGF-I and IGF-II on target tissues can be divided into two types: those that involve the rapid modulation of membrane-transport systems or enzyme activities, and those that are longer in onset and appear to involve regulation of transcription or DNA replication. Experiments performed over the last decade with the purified peptides produced descriptive results that illustrate two basic and rather paradoxical elements of insulin and IGF action. First, studies on the binding of the ‘251-labeled peptides to cells and membranes indicated that distinct receptor species were present that exhibited cross-reactivity for the various ligands. For example, the insulin receptor in human placenta that bound insulin with high affinity bound IGF-II with lower affin-
ity, and IGF-I with lowest affinity. Yet careful analysis of the results of these studies showed a large number of differences among the tissues studied in the relative binding affinities of the three ligands to the putative receptors. This suggested the possibility that a large number of heterogeneous receptor structures might account for the great diversity of binding characteristics observed (Rechler et al., Endocrinology 707, 1451-1459, 1980). Second, low physiological concentrations of insulin but not of IGF-I or IGF-II were normally observed to modulate the rapid cellular responses, such as increased transport activities in target tissues. High concentrations of the IGFs were required to elicit these responses. In contrast, low concentrations of IGF-I and IGF-II were observed to stimulate DNA replication and cell proliferation, while high concentrations of insulin were able to mimic these effects. These data were consistent with the hypothesis that the growth factor receptors mediated cell proliferation and insulin receptors mediated the rapid cellular responses. Yet critical exceptions arose; extremely low concentrations of insulin could activate cell division, as in the rat hepatoma H35 cell line, and the IGFs could rapidly activate transport as in rat soleus muscle. Significant clarification of these issues has been achieved by the direct identification and characterization of specific receptor structures present in cell membranes that interact with insulin and the IGFs. It is now clear that cellular components that regulate and mediate the biological responses of insulin, IGF-I and IGF-II share several levels of structural and functional homologies and are highly integrated. The insulin receptor defined as the structure exhibiting highest affinity for insulin in all tissues studied has been extensively characterized by affinity purification, photoaffinity labeling, affinity crosslinking and immunoaffinity purification. Results generated with affinity crosslinking techniques and with purified receptor led us (Massague et al., PNAS 77, 7137-7141, 1980; Czech et al., TIBS 6, 222-225, 1981; Massague et al., JBC 257, 6729-6738, 1982) and others (Jacobs et al., JBC 255, 6937-6940, 1980; Jacobs and Cuatrecasas, Endocrine Rev. 2, 251-263, 1981) to propose, independently, a model of the minimum structure of the insulin receptor subunit that is consistent with virtually all data available. This model postulated the immunoglobulin-like structure (,f?-S-S-a)-S-S(a-S-S-p), in which LYis a glycoprotein subunit with an apparent molecular weight of 125,000 and /3 is a glycoprotein subunit with an apparent molecular weight of 90,000. This symmetrical, disulfide-linked heterotetrameric structure probably binds more than one insulin molecule, based on results obtained by immunoprecipitation of the “51-insulin-receptor complex. The disulfide or disulfides that link the two (cu-S-S-/3) receptor halves are denoted as class I
Cdl 9
disulfides, and are significantly more sensitive to dissociation by exogenous reductant than the class II disulfides that link 01subunits top subunits. The insulin receptor structure, like other receptors, is quite sensitive to proteolytic nicking during membrane preparation and purification. This can lead to the observation of subunit fragments of lower molecular weight. In particular, the receptor forms (,&-,/I-P-+SS-(a-S-S-/?) and (PI-S-S-a)-S-S-(a-S-S--p,) result from a proteolytic nick in ,& at approximately the center of its amino acid sequence. The application of photoaffinity and affinity crosslinking methodology, developed for analysis of the insulin receptor structure, to the IGF system led to the discovery of an IGF receptor structurally homologous to the insulin receptor (Bhaumick et al., PNAS 78, 4279-4283, 1981 ; Chernausek et al., Biochemistry 27, 7345-7350, 1981; Kasuga et al., PNAS 79, 1864-l 868, 1982; Massague and Czech, JBC 257, 5038-5045, 1982). This IGF receptor, with the apparent structure (/3-S-S-&S-S-(0-S-S-P), contains subunits of molecular weight and physical properties essentially identical, as determined on dodecyl sulfate gels, to the insulin receptor structure. Class I and class II disulfides similar to those present in the insulin receptor were also detected. Analysis of the inhibition of affinity labeling of the IGF receptor by unlabeled insulin, IGF-I and IGF-II allowed the conclusion that in all tissues studied it exhibits the following relative affinities for ligands: IGF-I > IGF-II > insulin. We denote this cellular component as the type I IGF receptor. How can we account for the divergent affinities of the insulin and type I IGF receptors, given their structural similarity? One possibility is that the two receptors are protein products of the same gene segments, but that posttranslational modifications alter their affinities for ligands. An alternative view is that partially homologous but distinct genes encode these receptors or certain variable protein domains of the receptors. It is not yet possible to distinguish between these postulates, but a series of recent observations indicates that the cellular expression of these two receptor structures may be highly coordinated. In three different cellular models of differentiation or mutation, appearance or disappearance of both receptor types occurs in parallel. Thus, during differentiation of the 3T3-Ll fibroblast to the adipocyte-like morphology, expression of both insulin receptor and type I IGF receptor structures is increased severalfold. Similarly, skin fibroblasts from two different human mutants with extreme insulin resistance that show altered insulin receptor characteristics also exhibit parallel defects in the type I IGF receptor, as detected by binding studies or affinity labeling techniques (Van Obberghen et al., J. Clin. Invest. 68, 1356-l 365, 1982). A second and divergent IGF receptor, denoted as the type II IGF receptor, is visualized by affinity label-
ing studies and migrates on dodecyl sulfate gels with an apparent molecular weight of approximately 250,000, without detectable disulfide-linked subunits (Massague and Czech, op. cit.). It should be noted that a variety of other receptor systems for peptide growth factors that have been identified recently, including those for nerve growth factor, platelet-derived growth factor, epidermal growth factor and transforming growth factor, also lack structural homology to the insulin and type I receptors. The type II IGF receptor species in all tissues studied consistently binds IGF-II with higher affinity than IGF-I, and has very little or no affinity for insulin. In the course of investigation with over 30 different cell types in our laboratory, only the characteristic polypeptides associated with the insulin receptor and the type I and II IGF receptor structures have been consistently detected and specifically labeled by affinity crosslinking methodology. It should be noted that the heterotetrameric insulin and type I IGF receptors and the single-chain type II IGF receptor may contain other, noncovalently linked subunits that are not detectable by the methods employed in the above studies. Can the three receptor structures identified be responsible for all of the complex ‘251-insulin and ‘251IGF binding and ligand competition patterns observed in diverse tissues? The evidence supports the view that the receptor species identified can account for both the binding and biological actions of the three peptide hormones. The complex and heterogeneous binding kinetics observed among different systems appears to relate to two factors. First, the absence or presence, as well as the number, of each of these receptor species varies markedly among cell types. Certain cells, such as the classic targets for insulin action, hepatocytes and adipocytes, contain no type I IGF receptor species. Others, such as the chicken embryo fibroblast or Erlich-Lettre carcinoma cell line, contain large amounts of the type I structure, but vanishingly low amounts of insulin receptor and no detectable type II IGF receptor. Second, the three individual receptor structures exhibit marked heterogeneity in their relative affinities for ligands in the different tissues. Thus the type II IGF receptor in human fibroblasts exhibits a relatively lower affinity for IGF-I than the type II IGF receptor in liver cells; the type I IGF receptor in chicken embryo fibroblasts exhibits markedly higher affinity for IGF-II than this receptor in Erlich-Lettre strain E cells. This heterogeneity in binding kinetics of the three receptor types among different tissues must result from the association and influence of other membrane components that differ among tissues, or from a structural heterogeneity in the receptor molecules themselves. Consistent with the latter interpretation are detectable differences in apparent molecular weights of the type II IGF receptor from various sources. The combination of these two factors-the number and types of the
Cell IO
receptor structures present, and the distinct pattern of ligand affinities displayed by each of the receptor types in disparate tissues-accounts for the overall binding kinetics observed in individual tissues. The identification of the three insulin and IGF receptor types has also clarified the relationships between the receptors and their biological actions. The structurally homologous heterotetrameric receptor structures for both insulin and IGF-I appear capable of activating the rapid transport and enzyme changes normally associated with insulin action when they are present in an appropriately responsive tissue-that is, skeletal muscle. In contrast, the type II IGF receptor is impotent in catalyzing these responses. Thus the IGFs do not activate at low concentrations the rapid insulin-like effects in adipocytes and hepatocytes, because these cells have only insulin and type II IGF receptor types and lack the type I receptor structure. This conclusion is satisfying because homologous receptor structures might be expected to couple to identical or homologous cellular signaling mechanisms. Unfortunately, the extension of this concept to the slow-onset growth effects does not seem to hold, and illustrates an unsolved, intriguing complexity. While many cell types in culture appear to respond to type I IGF receptor stimulation with DNA replication, the insulin receptor structure appears impotent in mediating this response in most cell types (King et al., J. Clin. Invest. 66, 130-140, 1980). In human fibroblasts, for example, insulin at high concentrations acts through the type I IGF receptor species, not its own receptor, to promote proliferation. An exception to this is the H35 hepatoma cell line, which is devoid of the type I IGF receptor and unequivocally responds to insulin receptor perturbation with increased DNA replication and cell division (Koontz et al., Science 27 7, 947-949, 1981). This difference in insulin receptor function among cell lines is unclear. The type II IGF receptor presumably mediates a cell proliferative response, but this has not been unequivocally documented to date. A striking feature of the relationship among the receptors for insulin and the IGFs is a potent biological action executed by the insulin receptor on the type II IGF receptor. The binding of insulin to its high-affinity receptor markedly increases, by up to tenfold, the affinity of the type II receptor for its ligand in at least two cell types (Schoenle et al., Diabetologia 73, 243-249, 1977; Massague et al., JBC, in press). This action of insulin is observed in intact cells but not isolated membrane preparations. In addition, we have recently found that the process of cell disruption that is necessary to prepare membranes itself mimics in-
sulin action to enhance the affinity of the type II receptor. The mechanism by which this response to insulin is mediated is not understood, but the potential physiological implication is apparent. This mechanism may allow the insulin receptor to stimulate rapid transport and enzyme effects directly, while indirectly potentiating the cell-growth response by way of promoting IGF action through the type II receptor. Such modulation of one receptor system by another receptor now appears quite common, and may be of substantial significance. We have not found evidence to suggest a similar effect of the type I IGF receptor on the type II receptor. A key unanswered question about the functions of the insulin and IGF receptors relates to the molecular mechanism or mechanisms they employ to transduce their biological effects. Insulin action has been the most carefully and exhaustively studied, but still remains largely a mystery. The identification of the receptor structures that participate in signal transduction by these peptides and the ability to isolate and characterize their properties under a variety of conditions provide a powerful approach to the problem that has only recently been available. Using antireceptor antibodies to immunoprecipitate the insulin receptor following incubation of 32P-labeled intact cells with or without insulin, Kahn and colleagues demonstrated increased incorporation of labeled phosphate into the 90,000 molecular weight ,l3 subunit (Kasuga et al., Science 275, 185-I 87, 1982). Insulin also markedly stimulated 32P labeling of this receptor subunit in detergent extracts of membranes incubated with y32P-ATP (Kasuga et al., Nature 298, 667-669, 1982). Both serine and tyrosine residues on the receptor were phosphorylated, although the latter were most stimulated by hormone in a cell-free system. The relevance of these observations to receptor function is unknown, but parallel observations have previously been made with the most extensively studied epidermal growth factor receptor system. Furthermore, the association between tyrosine phosphorylation and cellular growth patterns during viral transformations has spurred great interest in these phenomena. The picture that emerges is that the insulin receptor, as well as the epidermal growth factor and probably others, may itself be a protein kinase or is closely associated with a kinase activity in the cellsurface membrane. The central focus of current studies is to ascertain whether this property of the insulin receptor is also shared by the IGF receptor structures, and whether it directly relates to receptor signaling or perhaps other receptor functions, such as desensitization or hormone-induced cellular internalization.