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The insulin-like growth factors and their binding proteins
Comp. Biochem. PhysioL Vol. 91B, No. 2, pp. 229-235, 1988
0305-0491/88 $3.00+ 0.00 © 1988 Pergamon Press plc
Printed in Great Britain
MINI-REVIEW THE I N S U L I N - L I K E G R O W T H F A C T O R S A N D THEIR B I N D I N G PROTEINS ROBERT C. BAXTER
Department of Endocrinology, Royal Prince Alfred Hospital, Camperdown, NSW 2050, Australia (Tel: (02)516-6111) (Received 15 October 1987)
Abstract--1. This review provides a brief overview of the structure of the insulin-like growth factors (IGFs or somatomedins), their mRNA and genes; the regulation and sites of production of these peptides; their binding and actions in target tissues; and the structure and biological role of their binding proteins. 2. Molecular cloning techniques have allowed the prediction of precursor forms of IGF-I and IGF-II, have provided tools to study the regulation of the synthesis and translation of IGF mRNAs, and have recently yielded the primary sequence of the IGF-I receptor, supplementing other rapidly-accumulating structural data. 3. Several of the IGF binding proteins have also been purified, and initial structural studies performed. 4. The increased knowledge of the structures of the IGFs, their receptors and binding proteins should now permit rapid progress in understanding the physiology and functions of these proteins.
STRUCTURE OF THE INSULIN-LIKEGROWTH
INTRODUCTION
FACTORS, THEIR mRNA AND GENES
Peptides of the insulin-like growth factor (IGF) family resemble insulin both in their structure and in many of their actions (Froesch and Zapf, 1985). Originally termed somatomedins because they were defined as mediators of the somatogenic actions of growth hormone (Daughaday et al., 1972), this family was once thought to contain many different peptides, including those designated somatomedins A, B and C, basic somatomedin, non-suppressible insulin-like activity (NSILA) and multiplicationstimulating activity (MSA). Purification and structural determination has revealed that somatomedins A and C, basic somatomedin, and NS1LA-I are the same peptide (now termed IGF-I), while NSILA-II and MSA are the human and rat analogues of another peptide (now termed IGF-II) (Baxter, 1986). Although structural variants of IGF-I and I G F - I I have been described recently, there is no evidence for the existence of any other IGFs, despite repeated suggestions of a foetal form. The first part of this review will briefly discuss the properties of these peptides, their regulation, and their binding to, and actions upon, the many known I G F target tissues. As IGFs are always found in the circulation and in cell culture media bound to carrier proteins, which may play an important role in the delivery of the peptides to their sites of action, these proteins, their production and actions will also be discussed. Because of the rapid increase in knowledge in these areas, it is impossible to reference every primary research paper relevant to these topics. Many more important references may be found within the bibliographies of the cited articles.
The complete structures of IGF-I and IGF-II were first elucidated by Rinderknecht and Humbel (1978a, 1978b). Both are single chain polypeptides, IGF-I with 70 amino acids (molecular mass 7.65 kD) and IGF-II with 67 (molecular mass 7.47 kD). They have over 60% homology with each other, and are also highly homologous with insulin (or its precursor proinsulin), having regions which correspond to the A and B insulin chains, joined by two disulphide bridges, and a connecting peptide corresponding to proinsulin C-peptide, though quite different in sequence. The IGFs differ from proinsulin in that their C-peptides cannot be cleaved away, and in having short extensions, termed the D-peptide, at the carboxy-terminus of the A-region (Fig. 1). Several groups of investigators have recently cloned cDNAs coding for precursor forms of human and rodent IGF-I and I G F - I I (e.g. Dull et al., 1984; Jansen et al., 1985; Bell et al., 1986). As illustrated in Fig. 1, deduced sequences of the correponding preprohormones indicate signal peptides of about 25 amino acids at the amino-terminus of the B-region, and further carboxy-terminal extensions in the prohormones which are 85-90 amino acids long for IGF-II, and somewhat shorter for IGF-I. This carboxy-terminal extension has been termed the Eregion (Dull et al., 1984). In Northern hybridisation studies, human and rodent cDNA probes hybridise with several different rat IGF-I m R N A species, of which the predominant forms appear to be 7.0-8.0, 4.6--4.7, 1.7-2.1 and 1.0-1.2 kilobases (kb) in length (Lund et al., 1986;
229
ROBERT C. BAXTER
230
HumanlGF-I
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A-
D
29-12 - 24-
C-
5
oIGW-I I- --11 B - C - A -
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70 a a 7.65 k D a
O []
25-
-35
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l
c
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l
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HumanlGF-ll
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PreprolGF-II~
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D
8-
5
22-
67 a a 7.47kDa
I B- C- A- D []
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180 a a
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- 89
Fig. 1. Schematic structures of IGF-1, IGF-II and their precursors. The letters A to E represent the 5 regions of the prohormones; "Pre" indicates the amino-terminal signal peptide. The number below each region denotes its length in amino acid residues; the total length in amino acids (aa) and molecular mass in kilodaltons are indicated on the right. The diagram on the left shows the relationship of regions A D within the mature polypeptides, with disulphide bonds indicated.
Roberts et al., 1986). IGF-II m R N A species have been identified in 3.4-4.0, 2.2, 1.6-1.75 and 1.1-1.2 kb forms, as well as some other minor species (Brown et al., 1986; Lund et al., 1986). How these multiple mRNAs arise is not yet understood. Sequence analysis of human IGF-I and I G F - I I genes has revealed that both have a discontinuous structure. The IGF-I gene contains five exons spanning a region of at least 45 kb, and can generate at least two different IGF-I mRNAs by alternative processing of the primary transcript. The I G F - I I gene consists of at least seven exons spanning more than 16kb (de Pagter-Holthuizen et al., 1987; Rotwein et al., 1986); its primary product also appears able to give rise to at least two different mRNAs (Jansen et al., 1985). Recent gene localisation studies indicate that the human I G F - I I gene is on the short arm of chromosome 11, contiguous with the insulin gene, while the gene for IGF-I is on chromosome 12, which is evolutionarily related to chromosome 11 (Brissenden et al., 1984; Tricoli et al., 1984). The proximity of the insulin and I G F - I I genes on chromosome 11, and the similarities between the chromosomes bearing the IGF-I and I G F - I I genes, all suggest a close ancestral relationship between the genes for the three peptides, consistent with the close structural relationship seen between the peptides themselves. CONTROL OF INSULIN-LIKE GROWTH FACTOR PRODUCTION
Although IGFs may be produced by many tissues of the body (D'Ercole et al., 1984), in the adult rat 7.5 kb IGF-I m R N A is up to 50 times more abundant in the liver than in other tissues (Lund et al., 1986), and hepatic IGF-I production has been shown to account for most or all of the circulating levels of the peptide (Scott et al., 1985a). In contrast, I G F - I I m R N A in the adult rat is expressed predominantly in the brain (Lund et al., 1986), and circulating IGF-II levels are virtually undetectable. In foetal and neonatal rats, a different picture is seen, with IGF-II m R N A abundant in liver and many other tissues (though low in brain), consistent with a role for this
peptide in foetal growth (Brown et al., 1986; Lund et al., 1986). IGF-I m R N A is also widespread in foetal tissues, but at very low levels compared to that seen in adult liver. It is not, however, clear whether these patterns of tissue and age distribution of I G F mRNAs are always reflected in the secretory patterns of the peptides. It is also important to note that the patterns seen in the rat may be quite different in other species; for example, circulating I G F - I I levels, while low to undetectable in the adult rat, are very high (600/~g/L) in the adult human (Baxter, 1986). One of the definitive characteristics of IGF-I is its regulation by growth hormone (GH) (Clemmons and Van Wyk, 1984). The close relationship between the secretion of GH, which has a pulsatile secretory pattern in most species, and circulating IGF-I levels (Baxter et al., 1983; Bercu et al., 1986), forms the basis of the use of I G F - I measurement as an indicator of GH secretory status. The regulation of IGFs by GH has been recognised since the demonstration 30 years ago that "sulphation factor" (the factor in serum which stimulated proteoglycan synthesis in cartilage, now known to be IGFs) was absent in the serum of hypophysectomised rats (Salmon and Daughaday, 1957). More recent studies have shown the direct stimulation of I G F production, measured by radioimmunoassay, in cultured hepatocytes treated with GH in vitro, or isolated from rats treated with GH in vivo (Scott et al., 1985b). The other major factors which regulate IGF-I levels are age and nutritional status (Clemmons and Van Wyk, 1984). IGF-I is low in the neonate, rises through the prepubertal years to peak values during puberty, then slowly declines with increasing age (Hall and Sara, 1983). The peripubertal rise, although apparently associated with increased sex steroid production, is in fact seen in rats even if they are castrated prepubertally, indicating that some more basic regulatory mechanism, perhaps at the hypothalamie level, is responsible (Handelsman et al., 1987). The role of nutritional status in the regulation of IGF-I production has also been studied extensively, since malnutrition is often associated with growth retardation. Fasting has been shown to cause
Insulin-like growth factors
231
although the function of this process is not yet understood (Oka et al., 1984). It is impossible in a short review to present more than a brief outline of the actions of IGFs on their target tissues. IGF research developed in the 1970s from a synthesis of three separate areas of investigation: studies of factors which stimulated proteoglycan synthesis in cartilage ("sulphation factor"), factors which possessed insulin-like activity which could not be neutralised by anti-insulin antibodies ("non-suppressible insulin-like activity"), and factors which stimulated cell division ("multiplicationstimulating activity"). All of these activities, and many more, are in fact possessed by both IGFs. Expressed more generally, IGF actions fall into three classes: metabolic, mitogenic, and differentiative. The metabolic activities (principally anabolic) include insulin-like actions such as stimulation of glucose oxidation, glycogen synthesis and amino acid transport. In some cell types (notably adipocytes) these functions may be mediated by cross-reaction of the IGFs at insulin receptors, but in other cells, the INSULIN-LIKE GROWTH FACTOR RECEPTORS AND ACTIONS type I IGF receptor appears to be the mediator (Froesch and Zapf, 1985). Insulin-like activity can Like other peptide hormones and growth factors, also be seen in vivo: injection of IGF-I into rats or the IGFs interact with their target cells by binding to humans elicits a hypoglycaemic response similar to specific cell-surface receptors (Rechler and Nissley, that caused by insulin, and with about 6% of the 1985). Despite the structural similarity between IGF- potency of insulin (Zapf et al., 1985; Guler et al., I and IGF-II, their receptors show quite different 1987). The mitogenic activity of the IGFs has been structures (Kasuga et al., 1981; Massagu6 and Czech, demonstrated in many cell types, with DNA, RNA 1982). The receptor with primary specificity for IGF- and protein synthesis, as well as cell proliferation, I resembles the insulin receptor in having a hetero- stimulated by both IGF-I and IGF-II (Baxter, 1986). tetrameric structure consisting of two extracellular ~t In contrast to other cells, the proliferation of lymphochains of approximately 130 kD, linked by disulphide cytes appears to be inhibited by IGF-I (Hunt and bonds to two transmembrane 90-95 kD fl chains in a Eardley, 1986). In BABL/c 3T3 fibroblasts the IGFs fl-ct.ct-fl formation. This receptor, t~rmed the type I have been shown to exert their mitogenic activity in IGF receptor (or IGF-I receptor) to distinguish it from synergism with other peptides such as platelet-derived the structurally different IGF-II receptor, typically growth factor or fibroblast growth factor, which must shows up to 50% cross-reactivity by IGF-II, and first render the cells "competent" before the IGFs can about i% cross-reactivity by insulin. Its complete stimulate DNA synthesis (Stiles et al., 1979). In primary structure has recently been deduced from the general, mitogenic effects have been shown to be due sequence of its cloned cDNA (Ullrich et aL, 1986), to interaction of IGF-I or IGF-II at the IGF-I revealing a precursor polypeptide of 152 kD which receptor, but the IGF-II receptor may have a similar can be cleaved to yield ~ and fl subunits of 80.4 and role in some tissues. Gross somatic growth effects can 70.9 kD respectively. Glycosylation would increase also be seen in vivo, with IGF infusion over several these subunits to 130 and 90 kD. The ~ chains have days increasing tibial length, body weight and other been shown to contain the IGF binding sites, while growth indices (Zapf et al., 1985). the intracellular portions of the fl chains contain a The important role of the IGFs in stimulating cell tyrosine-specific protein kinase which is activated by differentiation and the expression of differentiated IGF-! binding and is capable of autophosphorylating functions has only recently been recognised. As exthe receptor (Le Bon et al., 1986). amples of effects on differentiation, rat IGF-II stimuIn contrast to the type I IGF receptor, the receptor lates neurite formation in chick sensory neurons with primary specificity for IGF-II (the type II IGF (Bothwell, 1983), while both IGF-I and IGF-II proreceptor, or IGF-II receptor) consists of a single mote myoblast differentiation to myotubes (Schmidt polypeptide chain of approximately 220 kD. Al- et al., 1983). A variety of differentiated functions in though early studies showed quite high IGF-I cross- the reproductive system are influenced by IGFs. In reactivity at the IGF-II receptor, the use of peptides granulosa cells, IGF-I acts synergistically with of high purity, or synthesised by recombinant DNA follicle-stimulating hormone to induce receptors for techniques, reveals that IGF-I has 1% or lower luteinising hormone, increase cyclic AMP procross-reactivity, while insulin shows no reactivity at duction, and stimulate steroidogenesis (Adashi et al., all (Baxter et al., 1987a). The type II IGF receptor 1985); comparable effects have also been described has not been demonstrated to possess intrinsic pro- recently in the testis (Lin et al., 1986) and adrenal tein kinase activity, but can itself be phosphorylated cortex (Morera et al., 1986). In the thyroid gland, (Corvera et al., 1986). In fat cells, this receptor is IGF-I synergises with thyroid stimulating hormone in rapidly translocated from an intracellular pool to the stimulating cell proliferation (Tramontano et al., plasma membrane in response to insulin binding, 1986). Thus it appears that full expression of the a rapid fall in circulating IGF-I, which correlates well with nitrogen balance and is reversed on refeeding (Clemmons and Van Wyk, 1984). Similarly to the regulation of IGF-I by GH (Roberts et al., 1986), regulation by nutritional status occurs at least in part at the level of IGF-I gene transcription (Emler and Schalch, 1987). IGF-II is also said to show partial GH dependence, with reduced circulating levels in GH-deficiency. However, in contrast to IGF-I production by various cell types, IGF-II production by foetal fibroblasts is not stimulated by GH, but by placental lactogen (Adams et al., 1983). Since the circulating half-lives of the IGFs are greatly extended by their association with binding proteins (see below), it is possible that the apparent GH-dependence of IGF-II is simply a reflection of the GH-dependence of the major binding protein.
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ROBERT C. BAXTER
activity of some pituitary hormones on their target endocrine tissues (gonads, adrenal, thyroid) may require the concomitant action of IGF-I. °g~ INSULIN-LIKE GROWTH FACTOR BINDING PROTEINS
Unlike most peptide hormones, the insulin-like growth factors are found in the circulation and in cell culture medium associated with one or more binding proteins (Smith, 1984). Two classes of binding proteins have been described, distinct in structure and regulation (Baxter et al., 1986). The GH-dependent binding protein is found predominantly in a 150 kD complex with most or all of the circulating IGFs (Moses et al., 1976; Daughaday et al., 1982), although it has also been described in a variety of other molecular forms (Wilkins and D'Ercole, 1985; Hossenlopp et al., 1986). On acidification this complex yields an acid-stable GH-dependent subunit of approximately 50 kD with one high-affinity binding site for IGF-I or IGF-II (Martin and Baxter, 1986). How this protein forms a 150 kD complex with the IGFs is not known, but it has been suggested that it combines with another subunit which is irreversibly inactivated upon acidification (Furlanetto, 1980). The concentration of the GH-dependent binding subunit in human plasma, determined by a specific radioimmunoassay, is sufficient to account for the sum of IGF-I and IGF-II concentrations (Baxter and Martin, 1986). As shown in Fig. 2 for 64 samples from normal children, the molar concentration of binding protein closely parallels the total IGF concentration. Similarly, in cultured rat hepatocytes, the production rate of IGF-I (the only IGF produced by adult rat liver) is equal to the production rate of binding protein (Scott et al., 1985a). These observations suggest that there might be co-ordination between the production of binding protein and the IGFs. Alternatively, the IGFs might simply have very short half-lives in the circulation or in culture medium unless they are stabilised by binding to the GH-dependent binding protein. This explanation is supported by the observation that IGF-I or IGF-II infused into hypophysectomised rats (which have low or absent GH-dependent binding protein) disappear with a half-life of 10-20 min, whereas the half-life in normal rats is about 4 hr (Zapf et al., 1985). In addition to the GH-dependent IGF binding protein, another protein of about 30 kD, which does not show GH-dependence, has been described. Although it is reported to be inversely dependent on GH (P6voa et al., 1984a), this protein has recently been shown to have a marked diurnal rhythm, which is apparently unrelated to GH secretion (Baxter and Cowell, 1987). A similar or identical protein is present in very high concentrations in amniotic fluid (Drop et al., 1984), where its concentration appears to decrease with advancing foetal maturity (Baxter et al., 1987b). Comparison of the amino-terminal sequence of this protein (P6voa et al., 1984b) with that of a protein isolated from culture medium conditioned by the rat liver-derived cell line BRL-3A (Lyons and Smith, 1986; Mottola et al., 1986) suggests that they are the human and rat homologues of the same protein (Baxter and Martin, 1987). In contrast, adult rat serum, which does not contain
150
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E
OD R
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~~'
/ =
o2
=
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50
100
150
IGF-I+IGFql(nmol/L)
200
Fig. 2. The relationship between the molar concentration of circulating IGF-I plus IGF-II, and the molar concentration of the acid-stable 53 kD GH-dependent IGF binding protein BP-53, in 64 normal children.
detectable concentrations of the BRL-3A protein, has a GH-dependent protein with a 50 kD acid-stable subunit highly homologous to that isolated from human plasma (Baxter and Martin, 1987), ROLE OF THE BINDING PROTEINS
Despite increasing interest in the IGF binding proteins in recent years, their functions are still poorly understood. Since IGFs do not appear to be stored within any tissue, the circulating pool is the only storage form of the peptides. In man, the plasma IGF-I plus IGF-II concentration totals about 800 ~g/L or 100 nmol/L, approximately 1000 times higher than the insulin concentration (Baxter, 1986). With an insulin-like potency more than 5% that of insulin itself (Guler et al., 1987), the IGF pool thus has the potential to contribute 50 times more insulinlike activity than insulin. Clearly this activity is not expressed in vivo, presumably as a result of the binding of the IGFs to binding proteins, preventing the association of the growth factors with their cell-surface receptors. Many studies have demonstrated the inhibitory effects of IGF binding proteins on IGF actions. Preparations from human plasma, human amniotic fluid and rat BRL-3A cells have been shown to be inhibitory, and all types of IGF action may be inhibited, e.g. insulin-like activity in fat cells (Zapf et al., 1979), DNA synthesis in chick embryo fibroblasts (Knauer and Smith, 1980) and proteoglycan synthesis in rabbit chondrocytes (Drop et al., 1979). A small plasma protein isolated on the basis of its IGF inhibitory activity has also been shown to bind IGFs (Kuffer and Herington, 1984), and is immunologically-related to the GH-dependent binding protein (Baxter and Martin, 1986). There is, however, some evidence that association with binding proteins may not always inhibit the activity of the IGFs. Cornell et al. (1987) have demonstrated that some high molecular weight IGF
Insulin-like growth factors complexes from human plasma retain biological activity in rat adipocyte assays for insulin-like activity. Cultured h u m a n fibroblasts also secrete a binding protein of about 35 kD which, rather than inhibiting the interaction of the IGFs with their cell-surface receptors, actually increases cell I G F binding (Clemmons et al., 1986). On the assumption that the fibroblast protein is the same as that isolated from amniotic fluid, Elgin et al. (1987) have studied the effect of purified amniotic fluid binding protein on I G F activity. Surprisingly, whereas impure preparations of this protein are inhibitory, a pure preparation significantly potentiated the effect of I G F - I in stimulating D N A synthesis in porcine smooth muscle cells and chicken, mouse and h u m a n fibroblasts. This study suggests that cell types which produce this protein might be able to enhance their I G F responsiveness in an autocrine manner, and raises many new questions about the function of the binding proteins. CONCLUDING COMMENTS The scope of this short review may give some indication of the pace at which I G F research is progressing. Since the preparation of a previous review by the author (Baxter, 1986), many important advances have been made. For example, much of the information on I G F gene organisation, various m R N A species, and precursor and variant forms of the peptides themselves, has appeared within the past two years. The same period has seen significant progress in the purification and structural analysis of the I G F binding proteins, and in studies of I G F receptors at the molecular level. While advances have also been made in I G F physiology, it is in this area that many of the most important questions remain unanswered. These include the following: What is the role of I G F - I I in the adult (particularly in the brain where I G F - I I gene expression is very high), and what functions are mediated by type II I G F receptors? W h a t are the functional relationships between the two classes of binding protein, and how do they deliver I G F s to their sites of action? The prospect of answering these and many other important questions should make the next few years of I G F research an exciting and challenging period. Acknowledgements--Research grants to the author from the
National Health and Medical Research Council, Australia, are gratefully acknowledged. REFERENCES
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Lin T., Haskell J., Vinson N. and Terracio L. (1986) Characterization of insulin and insulin-like growth factor I receptors of purified Leydig cells and their role in steroidogenesis in primary cultures: a comparative study. Endocrinology 119, 1641-1647. Lund P. K., Moats-Staats B. M., Hynes M. A., Simmons J. G., Jansen M., D'Ercole A. J. and Van Wyk J. J. (1986) Somatomedin-C/insulin-like growth factor-I and insulinlike growth factor-II mRNAs in rat fetal and adult tissues. J. biol. Chem. 261, 14539-14544. Lyons R. M , Smith G. R. (1986) Characterization of multiplication-stimulating activity (MSA) carrier protein. Molec. Cell. Endocr. 45, 263-270. Martin J. L. and Baxter R. C. (1986) Insulin-like growth factor-binding protein from human plasma: purification and characterization. J. biol. Chem. 261, 8754-8760. Massagu6 J. and Czech M. P. (1982) The subunit structure of two distinct receptors for insulin-like growth factors 1 and II and their relationship to the insulin receptor. J. biol. Chem. 257, 5038-5045. Morera A. M. Benahmed M. and Chauvin M. A. (1986) Somatomedin C: a factor of the differentiation of adrenal cortex cells. C. R. Acad. Sci. (III) 303, 581-584. Moses A. C., Nissley S. P., Cohen K. L. and Rechler M. M. (1976) Specific binding of somatomedin-like polypeptide in rat serum depends on growth hormone. Nature 263, 137-140. Mottola C., MacDonald R. G., Brackett J. L., Mole J. E., Anderson J. K. and Czech M. P. (1986) Purification and amino-terminal sequence of an insulin-like growth factor-binding protein secreted by rat liver BRL-3A cells. J. biol. Chem. 261, 11180-11188. Oka Y., Mottola C., Oppenheimer C. L. and Czech M. P. (1984) Insulin activates the appearance of insulin-like growth factor II receptors on the adipocyte cell surface. Proc. hath. Acad. Sci. U.S.A. 81, 4028-4032. P6voa G., Enberg G., Jornvall H. and Hall K. (1984b) Isolation and characterization of a somatomedin-binding protein from mid-term human amniotic fluid. Eur. J. Biochem. 144, 199-204. P6voa G., Roovete A. and Hall K. (1984a) Cross-reaction of serum somatomedin-binding protein in a radioimmunoassay developed for somatomedin-binding protein isolated from human amniotic fluid. Acta Endocr. (Copenh.) 107, 563-570. Rechler M. M. and Nissley S. P. (1985) The nature and regulation of the receptors for insulin-like growth factors. A. Roy. Physiol. 47, 425-442. Rinderknecht E. and Humbel R. E. (1978a) The amino acid sequence of insulin-like growth factor I and its structural homology with proinsulin. J. biol. Chem. 253, 2769-2776. Rinderknecht E. and Humbel R. E. (1978b) Primary structure of human insulin-like growth factor-II. FEBS Lett. 89, 283-286. Roberts C. T. Jr., Brown A. L., Graham D. E., Seelig S., Berry S. Gabbay K. H. and Rechler M. M. (1986) Growth hormone regulates the abundance of insulin-like growth factor I RNA in adult rat liver. J. biol. Chem. 261, 10025-10028. Rotwein P., Pollock K. M., Didier D. K. and Krivi G. G. (1986) Organization and sequence of the human insulin-like growth factor I gene. Alternative RNA processing produces two insulin-like growth factor I precursor peptides. J. biol. Chem. 261, 4828-4832. Salmon W. D. Jr. and Daughaday W. H. (1957) A hormonally-controlled serum factor which stimulated sulfate incorporation by cartilage in vitro. J. lab. clin. Med. 49, 825-836. Schmidt C., Steiner T. and Froesch E. R. (1983) Preferential enhancement of myoblast differentiation by insulin-like growth factors (IGF I and IGF If) in primary cultures of chicken embryonic cells. FEBS Lett. 161, 117-121.
Insulin-like growth factors Scott C. D., Martin J. L. and Baxter R. C. (1985a) Production of insulin-like growth factor-I and its binding protein by adult rat hepatocytes in primary culture. Endocrinology 116, 1094-1101. Scott C. D., Martin J. L. and Baxter R. C. (1985b) Rat hepatocyte insulin-like growth factor-I and binding protein: effect of growth hormone in vitro and in vivo. Endocrinology 116, 1102-1107. Smith G. L. (1984) Somatomedin carrier proteins. Molec. Cell. endocrinol. 34, 83-89. Stiles C. D., Capone G. T., Scher C. D., Antonaides H. N., Van Wyk J. J. and Pledger W. J. (1979) Dual control of cell growth by somatomedins and platelet-derived growth factor. Proc. natn Acad. Sci. U.S.A. 76, 1279-1283. Tramontano D., Cushing G. W., Moses A. C. and Ingbar S. H. (1986) Insulin-like growth factor-I stimulates the growth of rat thyroid cells in culture and synergizes the stimulation of DNA synthesis induced by TSH and Graves'-IgG. Endocrinology 119, 940-942. Tricoli J. V., Rall L. B., Scott J., Bell G. I. and Shows T. B. (1984) Localization of insulin-like growth factor
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genes to human chromosomes 11 and 12. Nature 310, 784-786. Ullrich A., Gray A., Tam A. W., Yang-Feng T., Tsubokawa M,, Collins C., Henzel W. et al. (1986) Insulin-like growth factor I receptor primary structure: comparison with insulin receptor suggests structural determinants that define functional specificity. E M B O J. 5, 2503-2512. Wilkins J. R. and D'Ercole A. J. (1985) Affinity-labeled plasma somatomedin C/insulinlike growth factor I binding protein. J. clin. Invest. 75, 1350-1358. Zapf, J., Schoenle E. and Froesch E. R. (1985) In vivo effects of the insulin-like growth factors (IGFs) in the hypophysectomized rat: comparison with human growth hormone and the possible role of the specific IGF carrier proteins. In Growth Factors in Biology and Medicine (Ciba Foundation Symposium 116), pp. 169-187. Pitman, London. Zapf, J., Schoenle E., Jagers E., Sand I. and Froesch E. R. (1979) Inhibition of the action of non-suppressible insulin-like activity on isolated rat fat cells by binding to its carrier protein. J. clin. Invest. 63, 1077-1084.
0305-0491/88 $3.00+ 0.00 © 1988 Pergamon Press plc
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MINI-REVIEW THE I N S U L I N - L I K E G R O W T H F A C T O R S A N D THEIR B I N D I N G PROTEINS ROBERT C. BAXTER
Department of Endocrinology, Royal Prince Alfred Hospital, Camperdown, NSW 2050, Australia (Tel: (02)516-6111) (Received 15 October 1987)
Abstract--1. This review provides a brief overview of the structure of the insulin-like growth factors (IGFs or somatomedins), their mRNA and genes; the regulation and sites of production of these peptides; their binding and actions in target tissues; and the structure and biological role of their binding proteins. 2. Molecular cloning techniques have allowed the prediction of precursor forms of IGF-I and IGF-II, have provided tools to study the regulation of the synthesis and translation of IGF mRNAs, and have recently yielded the primary sequence of the IGF-I receptor, supplementing other rapidly-accumulating structural data. 3. Several of the IGF binding proteins have also been purified, and initial structural studies performed. 4. The increased knowledge of the structures of the IGFs, their receptors and binding proteins should now permit rapid progress in understanding the physiology and functions of these proteins.
STRUCTURE OF THE INSULIN-LIKEGROWTH
INTRODUCTION
FACTORS, THEIR mRNA AND GENES
Peptides of the insulin-like growth factor (IGF) family resemble insulin both in their structure and in many of their actions (Froesch and Zapf, 1985). Originally termed somatomedins because they were defined as mediators of the somatogenic actions of growth hormone (Daughaday et al., 1972), this family was once thought to contain many different peptides, including those designated somatomedins A, B and C, basic somatomedin, non-suppressible insulin-like activity (NSILA) and multiplicationstimulating activity (MSA). Purification and structural determination has revealed that somatomedins A and C, basic somatomedin, and NS1LA-I are the same peptide (now termed IGF-I), while NSILA-II and MSA are the human and rat analogues of another peptide (now termed IGF-II) (Baxter, 1986). Although structural variants of IGF-I and I G F - I I have been described recently, there is no evidence for the existence of any other IGFs, despite repeated suggestions of a foetal form. The first part of this review will briefly discuss the properties of these peptides, their regulation, and their binding to, and actions upon, the many known I G F target tissues. As IGFs are always found in the circulation and in cell culture media bound to carrier proteins, which may play an important role in the delivery of the peptides to their sites of action, these proteins, their production and actions will also be discussed. Because of the rapid increase in knowledge in these areas, it is impossible to reference every primary research paper relevant to these topics. Many more important references may be found within the bibliographies of the cited articles.
The complete structures of IGF-I and IGF-II were first elucidated by Rinderknecht and Humbel (1978a, 1978b). Both are single chain polypeptides, IGF-I with 70 amino acids (molecular mass 7.65 kD) and IGF-II with 67 (molecular mass 7.47 kD). They have over 60% homology with each other, and are also highly homologous with insulin (or its precursor proinsulin), having regions which correspond to the A and B insulin chains, joined by two disulphide bridges, and a connecting peptide corresponding to proinsulin C-peptide, though quite different in sequence. The IGFs differ from proinsulin in that their C-peptides cannot be cleaved away, and in having short extensions, termed the D-peptide, at the carboxy-terminus of the A-region (Fig. 1). Several groups of investigators have recently cloned cDNAs coding for precursor forms of human and rodent IGF-I and I G F - I I (e.g. Dull et al., 1984; Jansen et al., 1985; Bell et al., 1986). As illustrated in Fig. 1, deduced sequences of the correponding preprohormones indicate signal peptides of about 25 amino acids at the amino-terminus of the B-region, and further carboxy-terminal extensions in the prohormones which are 85-90 amino acids long for IGF-II, and somewhat shorter for IGF-I. This carboxy-terminal extension has been termed the Eregion (Dull et al., 1984). In Northern hybridisation studies, human and rodent cDNA probes hybridise with several different rat IGF-I m R N A species, of which the predominant forms appear to be 7.0-8.0, 4.6--4.7, 1.7-2.1 and 1.0-1.2 kilobases (kb) in length (Lund et al., 1986;
229
ROBERT C. BAXTER
230
HumanlGF-I
[B-
A-
D
29-12 - 24-
C-
5
oIGW-I I- --11 B - C - A -
B
70 a a 7.65 k D a
O []
25-
-35
130aa
14.3 k D a
l
c
S A
l
I I D
HumanlGF-ll
IB" 32-
PreprolGF-II~
C-A-
D
8-
5
22-
67 a a 7.47kDa
I B- C- A- D []
24 -
180 a a
20.1 kDa
- 89
Fig. 1. Schematic structures of IGF-1, IGF-II and their precursors. The letters A to E represent the 5 regions of the prohormones; "Pre" indicates the amino-terminal signal peptide. The number below each region denotes its length in amino acid residues; the total length in amino acids (aa) and molecular mass in kilodaltons are indicated on the right. The diagram on the left shows the relationship of regions A D within the mature polypeptides, with disulphide bonds indicated.
Roberts et al., 1986). IGF-II m R N A species have been identified in 3.4-4.0, 2.2, 1.6-1.75 and 1.1-1.2 kb forms, as well as some other minor species (Brown et al., 1986; Lund et al., 1986). How these multiple mRNAs arise is not yet understood. Sequence analysis of human IGF-I and I G F - I I genes has revealed that both have a discontinuous structure. The IGF-I gene contains five exons spanning a region of at least 45 kb, and can generate at least two different IGF-I mRNAs by alternative processing of the primary transcript. The I G F - I I gene consists of at least seven exons spanning more than 16kb (de Pagter-Holthuizen et al., 1987; Rotwein et al., 1986); its primary product also appears able to give rise to at least two different mRNAs (Jansen et al., 1985). Recent gene localisation studies indicate that the human I G F - I I gene is on the short arm of chromosome 11, contiguous with the insulin gene, while the gene for IGF-I is on chromosome 12, which is evolutionarily related to chromosome 11 (Brissenden et al., 1984; Tricoli et al., 1984). The proximity of the insulin and I G F - I I genes on chromosome 11, and the similarities between the chromosomes bearing the IGF-I and I G F - I I genes, all suggest a close ancestral relationship between the genes for the three peptides, consistent with the close structural relationship seen between the peptides themselves. CONTROL OF INSULIN-LIKE GROWTH FACTOR PRODUCTION
Although IGFs may be produced by many tissues of the body (D'Ercole et al., 1984), in the adult rat 7.5 kb IGF-I m R N A is up to 50 times more abundant in the liver than in other tissues (Lund et al., 1986), and hepatic IGF-I production has been shown to account for most or all of the circulating levels of the peptide (Scott et al., 1985a). In contrast, I G F - I I m R N A in the adult rat is expressed predominantly in the brain (Lund et al., 1986), and circulating IGF-II levels are virtually undetectable. In foetal and neonatal rats, a different picture is seen, with IGF-II m R N A abundant in liver and many other tissues (though low in brain), consistent with a role for this
peptide in foetal growth (Brown et al., 1986; Lund et al., 1986). IGF-I m R N A is also widespread in foetal tissues, but at very low levels compared to that seen in adult liver. It is not, however, clear whether these patterns of tissue and age distribution of I G F mRNAs are always reflected in the secretory patterns of the peptides. It is also important to note that the patterns seen in the rat may be quite different in other species; for example, circulating I G F - I I levels, while low to undetectable in the adult rat, are very high (600/~g/L) in the adult human (Baxter, 1986). One of the definitive characteristics of IGF-I is its regulation by growth hormone (GH) (Clemmons and Van Wyk, 1984). The close relationship between the secretion of GH, which has a pulsatile secretory pattern in most species, and circulating IGF-I levels (Baxter et al., 1983; Bercu et al., 1986), forms the basis of the use of I G F - I measurement as an indicator of GH secretory status. The regulation of IGFs by GH has been recognised since the demonstration 30 years ago that "sulphation factor" (the factor in serum which stimulated proteoglycan synthesis in cartilage, now known to be IGFs) was absent in the serum of hypophysectomised rats (Salmon and Daughaday, 1957). More recent studies have shown the direct stimulation of I G F production, measured by radioimmunoassay, in cultured hepatocytes treated with GH in vitro, or isolated from rats treated with GH in vivo (Scott et al., 1985b). The other major factors which regulate IGF-I levels are age and nutritional status (Clemmons and Van Wyk, 1984). IGF-I is low in the neonate, rises through the prepubertal years to peak values during puberty, then slowly declines with increasing age (Hall and Sara, 1983). The peripubertal rise, although apparently associated with increased sex steroid production, is in fact seen in rats even if they are castrated prepubertally, indicating that some more basic regulatory mechanism, perhaps at the hypothalamie level, is responsible (Handelsman et al., 1987). The role of nutritional status in the regulation of IGF-I production has also been studied extensively, since malnutrition is often associated with growth retardation. Fasting has been shown to cause
Insulin-like growth factors
231
although the function of this process is not yet understood (Oka et al., 1984). It is impossible in a short review to present more than a brief outline of the actions of IGFs on their target tissues. IGF research developed in the 1970s from a synthesis of three separate areas of investigation: studies of factors which stimulated proteoglycan synthesis in cartilage ("sulphation factor"), factors which possessed insulin-like activity which could not be neutralised by anti-insulin antibodies ("non-suppressible insulin-like activity"), and factors which stimulated cell division ("multiplicationstimulating activity"). All of these activities, and many more, are in fact possessed by both IGFs. Expressed more generally, IGF actions fall into three classes: metabolic, mitogenic, and differentiative. The metabolic activities (principally anabolic) include insulin-like actions such as stimulation of glucose oxidation, glycogen synthesis and amino acid transport. In some cell types (notably adipocytes) these functions may be mediated by cross-reaction of the IGFs at insulin receptors, but in other cells, the INSULIN-LIKE GROWTH FACTOR RECEPTORS AND ACTIONS type I IGF receptor appears to be the mediator (Froesch and Zapf, 1985). Insulin-like activity can Like other peptide hormones and growth factors, also be seen in vivo: injection of IGF-I into rats or the IGFs interact with their target cells by binding to humans elicits a hypoglycaemic response similar to specific cell-surface receptors (Rechler and Nissley, that caused by insulin, and with about 6% of the 1985). Despite the structural similarity between IGF- potency of insulin (Zapf et al., 1985; Guler et al., I and IGF-II, their receptors show quite different 1987). The mitogenic activity of the IGFs has been structures (Kasuga et al., 1981; Massagu6 and Czech, demonstrated in many cell types, with DNA, RNA 1982). The receptor with primary specificity for IGF- and protein synthesis, as well as cell proliferation, I resembles the insulin receptor in having a hetero- stimulated by both IGF-I and IGF-II (Baxter, 1986). tetrameric structure consisting of two extracellular ~t In contrast to other cells, the proliferation of lymphochains of approximately 130 kD, linked by disulphide cytes appears to be inhibited by IGF-I (Hunt and bonds to two transmembrane 90-95 kD fl chains in a Eardley, 1986). In BABL/c 3T3 fibroblasts the IGFs fl-ct.ct-fl formation. This receptor, t~rmed the type I have been shown to exert their mitogenic activity in IGF receptor (or IGF-I receptor) to distinguish it from synergism with other peptides such as platelet-derived the structurally different IGF-II receptor, typically growth factor or fibroblast growth factor, which must shows up to 50% cross-reactivity by IGF-II, and first render the cells "competent" before the IGFs can about i% cross-reactivity by insulin. Its complete stimulate DNA synthesis (Stiles et al., 1979). In primary structure has recently been deduced from the general, mitogenic effects have been shown to be due sequence of its cloned cDNA (Ullrich et aL, 1986), to interaction of IGF-I or IGF-II at the IGF-I revealing a precursor polypeptide of 152 kD which receptor, but the IGF-II receptor may have a similar can be cleaved to yield ~ and fl subunits of 80.4 and role in some tissues. Gross somatic growth effects can 70.9 kD respectively. Glycosylation would increase also be seen in vivo, with IGF infusion over several these subunits to 130 and 90 kD. The ~ chains have days increasing tibial length, body weight and other been shown to contain the IGF binding sites, while growth indices (Zapf et al., 1985). the intracellular portions of the fl chains contain a The important role of the IGFs in stimulating cell tyrosine-specific protein kinase which is activated by differentiation and the expression of differentiated IGF-! binding and is capable of autophosphorylating functions has only recently been recognised. As exthe receptor (Le Bon et al., 1986). amples of effects on differentiation, rat IGF-II stimuIn contrast to the type I IGF receptor, the receptor lates neurite formation in chick sensory neurons with primary specificity for IGF-II (the type II IGF (Bothwell, 1983), while both IGF-I and IGF-II proreceptor, or IGF-II receptor) consists of a single mote myoblast differentiation to myotubes (Schmidt polypeptide chain of approximately 220 kD. Al- et al., 1983). A variety of differentiated functions in though early studies showed quite high IGF-I cross- the reproductive system are influenced by IGFs. In reactivity at the IGF-II receptor, the use of peptides granulosa cells, IGF-I acts synergistically with of high purity, or synthesised by recombinant DNA follicle-stimulating hormone to induce receptors for techniques, reveals that IGF-I has 1% or lower luteinising hormone, increase cyclic AMP procross-reactivity, while insulin shows no reactivity at duction, and stimulate steroidogenesis (Adashi et al., all (Baxter et al., 1987a). The type II IGF receptor 1985); comparable effects have also been described has not been demonstrated to possess intrinsic pro- recently in the testis (Lin et al., 1986) and adrenal tein kinase activity, but can itself be phosphorylated cortex (Morera et al., 1986). In the thyroid gland, (Corvera et al., 1986). In fat cells, this receptor is IGF-I synergises with thyroid stimulating hormone in rapidly translocated from an intracellular pool to the stimulating cell proliferation (Tramontano et al., plasma membrane in response to insulin binding, 1986). Thus it appears that full expression of the a rapid fall in circulating IGF-I, which correlates well with nitrogen balance and is reversed on refeeding (Clemmons and Van Wyk, 1984). Similarly to the regulation of IGF-I by GH (Roberts et al., 1986), regulation by nutritional status occurs at least in part at the level of IGF-I gene transcription (Emler and Schalch, 1987). IGF-II is also said to show partial GH dependence, with reduced circulating levels in GH-deficiency. However, in contrast to IGF-I production by various cell types, IGF-II production by foetal fibroblasts is not stimulated by GH, but by placental lactogen (Adams et al., 1983). Since the circulating half-lives of the IGFs are greatly extended by their association with binding proteins (see below), it is possible that the apparent GH-dependence of IGF-II is simply a reflection of the GH-dependence of the major binding protein.
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ROBERT C. BAXTER
activity of some pituitary hormones on their target endocrine tissues (gonads, adrenal, thyroid) may require the concomitant action of IGF-I. °g~ INSULIN-LIKE GROWTH FACTOR BINDING PROTEINS
Unlike most peptide hormones, the insulin-like growth factors are found in the circulation and in cell culture medium associated with one or more binding proteins (Smith, 1984). Two classes of binding proteins have been described, distinct in structure and regulation (Baxter et al., 1986). The GH-dependent binding protein is found predominantly in a 150 kD complex with most or all of the circulating IGFs (Moses et al., 1976; Daughaday et al., 1982), although it has also been described in a variety of other molecular forms (Wilkins and D'Ercole, 1985; Hossenlopp et al., 1986). On acidification this complex yields an acid-stable GH-dependent subunit of approximately 50 kD with one high-affinity binding site for IGF-I or IGF-II (Martin and Baxter, 1986). How this protein forms a 150 kD complex with the IGFs is not known, but it has been suggested that it combines with another subunit which is irreversibly inactivated upon acidification (Furlanetto, 1980). The concentration of the GH-dependent binding subunit in human plasma, determined by a specific radioimmunoassay, is sufficient to account for the sum of IGF-I and IGF-II concentrations (Baxter and Martin, 1986). As shown in Fig. 2 for 64 samples from normal children, the molar concentration of binding protein closely parallels the total IGF concentration. Similarly, in cultured rat hepatocytes, the production rate of IGF-I (the only IGF produced by adult rat liver) is equal to the production rate of binding protein (Scott et al., 1985a). These observations suggest that there might be co-ordination between the production of binding protein and the IGFs. Alternatively, the IGFs might simply have very short half-lives in the circulation or in culture medium unless they are stabilised by binding to the GH-dependent binding protein. This explanation is supported by the observation that IGF-I or IGF-II infused into hypophysectomised rats (which have low or absent GH-dependent binding protein) disappear with a half-life of 10-20 min, whereas the half-life in normal rats is about 4 hr (Zapf et al., 1985). In addition to the GH-dependent IGF binding protein, another protein of about 30 kD, which does not show GH-dependence, has been described. Although it is reported to be inversely dependent on GH (P6voa et al., 1984a), this protein has recently been shown to have a marked diurnal rhythm, which is apparently unrelated to GH secretion (Baxter and Cowell, 1987). A similar or identical protein is present in very high concentrations in amniotic fluid (Drop et al., 1984), where its concentration appears to decrease with advancing foetal maturity (Baxter et al., 1987b). Comparison of the amino-terminal sequence of this protein (P6voa et al., 1984b) with that of a protein isolated from culture medium conditioned by the rat liver-derived cell line BRL-3A (Lyons and Smith, 1986; Mottola et al., 1986) suggests that they are the human and rat homologues of the same protein (Baxter and Martin, 1987). In contrast, adult rat serum, which does not contain
150
~°°
O
E
OD R
~'~ 100
~~'
/ =
o2
=
5O
50
100
150
IGF-I+IGFql(nmol/L)
200
Fig. 2. The relationship between the molar concentration of circulating IGF-I plus IGF-II, and the molar concentration of the acid-stable 53 kD GH-dependent IGF binding protein BP-53, in 64 normal children.
detectable concentrations of the BRL-3A protein, has a GH-dependent protein with a 50 kD acid-stable subunit highly homologous to that isolated from human plasma (Baxter and Martin, 1987), ROLE OF THE BINDING PROTEINS
Despite increasing interest in the IGF binding proteins in recent years, their functions are still poorly understood. Since IGFs do not appear to be stored within any tissue, the circulating pool is the only storage form of the peptides. In man, the plasma IGF-I plus IGF-II concentration totals about 800 ~g/L or 100 nmol/L, approximately 1000 times higher than the insulin concentration (Baxter, 1986). With an insulin-like potency more than 5% that of insulin itself (Guler et al., 1987), the IGF pool thus has the potential to contribute 50 times more insulinlike activity than insulin. Clearly this activity is not expressed in vivo, presumably as a result of the binding of the IGFs to binding proteins, preventing the association of the growth factors with their cell-surface receptors. Many studies have demonstrated the inhibitory effects of IGF binding proteins on IGF actions. Preparations from human plasma, human amniotic fluid and rat BRL-3A cells have been shown to be inhibitory, and all types of IGF action may be inhibited, e.g. insulin-like activity in fat cells (Zapf et al., 1979), DNA synthesis in chick embryo fibroblasts (Knauer and Smith, 1980) and proteoglycan synthesis in rabbit chondrocytes (Drop et al., 1979). A small plasma protein isolated on the basis of its IGF inhibitory activity has also been shown to bind IGFs (Kuffer and Herington, 1984), and is immunologically-related to the GH-dependent binding protein (Baxter and Martin, 1986). There is, however, some evidence that association with binding proteins may not always inhibit the activity of the IGFs. Cornell et al. (1987) have demonstrated that some high molecular weight IGF
Insulin-like growth factors complexes from human plasma retain biological activity in rat adipocyte assays for insulin-like activity. Cultured h u m a n fibroblasts also secrete a binding protein of about 35 kD which, rather than inhibiting the interaction of the IGFs with their cell-surface receptors, actually increases cell I G F binding (Clemmons et al., 1986). On the assumption that the fibroblast protein is the same as that isolated from amniotic fluid, Elgin et al. (1987) have studied the effect of purified amniotic fluid binding protein on I G F activity. Surprisingly, whereas impure preparations of this protein are inhibitory, a pure preparation significantly potentiated the effect of I G F - I in stimulating D N A synthesis in porcine smooth muscle cells and chicken, mouse and h u m a n fibroblasts. This study suggests that cell types which produce this protein might be able to enhance their I G F responsiveness in an autocrine manner, and raises many new questions about the function of the binding proteins. CONCLUDING COMMENTS The scope of this short review may give some indication of the pace at which I G F research is progressing. Since the preparation of a previous review by the author (Baxter, 1986), many important advances have been made. For example, much of the information on I G F gene organisation, various m R N A species, and precursor and variant forms of the peptides themselves, has appeared within the past two years. The same period has seen significant progress in the purification and structural analysis of the I G F binding proteins, and in studies of I G F receptors at the molecular level. While advances have also been made in I G F physiology, it is in this area that many of the most important questions remain unanswered. These include the following: What is the role of I G F - I I in the adult (particularly in the brain where I G F - I I gene expression is very high), and what functions are mediated by type II I G F receptors? W h a t are the functional relationships between the two classes of binding protein, and how do they deliver I G F s to their sites of action? The prospect of answering these and many other important questions should make the next few years of I G F research an exciting and challenging period. Acknowledgements--Research grants to the author from the
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