Insulin-like growth factors and binding proteins in the fetal rat: Alterations during maternal starvation and effects in fetal brain cell culture

Maternal malnutrition adversely affects fetal body and brain growth during late gestation. We utilized a fetal brain cell culture model to examine whether alternations in circulating factors may contribute to reduce brain growth during maternal starvation; we then used specific immunoassay and western blotting techniques, and purified peptides to investigate the potential role that altered levels of insulin-like growth factors (IGFs) and IGF binding proteins (IGFBPs) may play in impaired growth during maternal nutritional restriction.Fetal, body, liver, and brain weight were reduced after 72 hr maternal starvation, and plasma from starved fetuses were less potent than fed fetal plasma in stimulating brain cell growth. Circulating levels of IGF-I were reduced in starved compared to fed fetuses, while levels of IGF-II were similar in both groups. In contrast, [¹²⁵I]-IGF-I binding assay demonstrated an increase in the availability of plasma IGFBPs following starvation. Western ligand blotting and densitometry indicated that levels of 32 Kd IGFBPs were 2-fold higher in starved compared to fed fetal plasma. Immunoblotting and immunoprecipitation with antiserum against rat IGFBP-1 confirmed that heightened levels of immunoreactive IGFBP-1 accounted for the increase in 32 Kd IGFBPs in starved plasma. Levels of 34 Kd BPs, representing IGFBP-2, were unaffected by starvation. Reconstitution experiments in cell culture showed that IGF-I promoted fetal brain cell growth, and that when they were supplemented with IGF-I, the growth promoting activity of starved fetal plasma was restored to fed levels. These changes were measured using MTT to assess mitochondrial reductase activity. Conversely, addition of physiological amounts of rat IGFBP-1 inhibited the effects of fed fetal plasma on brain cell growth, and bioactivity was reduced even further with higher concentrations of IGFBP-1. Based on these results, we conclude that reciprocal changes in circulating levels of IGFBP-1 (increased) and IGF-I (decreased) may combine to reduce the availability of IGF-I to this tissue and limit fetal brain cell growth when maternal nutrition is impaired.     

Roles of insulin-like growth factors and their binding proteins in the differentiation of mouse tongue myoblasts

To study the roles of insulin-like growth factors (IGFs) and their binding proteins (IGFBPs) in the differentiation of tongue myoblasts, we established a mouse tongue organ culture system and examined the effects of exogenous IGF-I, exogenous IGFBP4, 5, 6, and des(1-3)IGF-I, an IGF-I analogue with reduced affinity for IGFBPs, on the differentiation of tongue myoblasts. The exogenous IGF-I stimulated differentiation of tongue myoblasts and induced the expressions of endogenous IGFBP4, 5, and 6, suggesting that these IGFBPs were involved in the regulation of tongue myoblast differentiation by the IGF-I. Exogenous IGFBP4 and 5 slightly stimulated early tongue myoblast differentiation in which myogenin was involved. The stimulation seems to be due to the protection of endogenous IGFs from proteolytic degradation by the binding of these IGFBPs to endogenous IGFs. A low concentration of des(1-3)IGF-I stimulated tongue myoblast differentiation, whereas high concentrations of des(1-3)IGF-I inhibited it. The abnormal shape of the tongue, low cell density and low staining intensity with hematoxylin and eosin in tongues treated with high concentrations of des(1-3)IGF-I, suggest that the inhibition is due to abnormal reactions of tongue tissues to the toxicity caused by high concentrations of des(1-3)IGF-I. From these results, we suggest that IGFBPs may function to regulate the differentiation of mouse tongue myoblasts by controlling the concentration of free IGFs within a range suitable for the progress of tongue myoblast differentiation.     

Nutritional insult and recovery in the neonatal rat cerebellum: Insulin-like growth factors (IGFs) and their binding proteins (IGFBPs)

Alterations in growth caused by neonatal malnutrition may be mediated in part by changes in insulin-like growth factor (IGF) and IGF binding protein (IGFBP) expression. Since the neonatal rat cerebellum undergoes a transient, proliferative growth phase in the first two weeks of life, this structure was used to determine whether alterations in circulating and tissue IGFs and IGFBPs may mediate effects of impaired nutrition on the developing central nervous system. Gravid rats were placed on a 4% (protein-calorie deprived, D) or 20% (control, C) protein diets one day prior to delivery and allowed to nurse their pups postpartum. Pups nursing from D mothers received a limited volume of milk and were calorically deprived. Some litters of D pups were foster fed by C mothers from day 8 to day 13 to constitute a recovery group (R). Cerebellar weight, protein, and DNA content in D pups were less than C, p<0.001. In R pups, DNA and protein returned to C levels by day 13. Between days 6 and 13, serum IGF-I levels rose from 158±18 to 210±18 ng/ml in C but remained low in D (47±6 ng/ml and 25±3 ng/ml), respectively. In R pups, serum IGF-I partially recovered during this time, and increased from 49±5 to 110±7 ng/ml. In cerebellar extracts, IGF-I levels in both C and D were lower at 13 days than at 6 days, p<0.05 and p<0.005, respectively. IGF-I levels in C were similar at day 9 and day 11 and were consistently higher than D (11.84±0.83 vs 8.56±0.92 ng/g, p<0.02 C vs D). In R, IGF-I was reduced on day 11, but was similar to C on day 13. Serum IGF-II in D was lower than C, p<0.01, and did not increase in the R group. Cerebellar IGF-II was virtually undetectable in either group. Immunoprecipitation and ligand blotting studies of serum demonstrated that circulating levels of 32–34 K IGFBPs were increased 3–4 fold in D vs C, reflecting high levels of IGFBP-1 and/or-2, while levels of 24 K IGFBP-4 were lower in D vs C. By contrast, immunoprecipitation and ligand blotting of cerebellar extracts detected IGFBP-2 and-4, but did not detect IGFBP-1. Further, tissue levels of IGFBP-2 were not increased in D vs C, and levels of IGFBP-4 also were not markedly affected by nutritional deprivation. These results suggest that alterations in tissue content and the availability of IGF-I only modestly contributed to the effects of impaired nutrition in the developing central nervous system.     

The insulin-like growth factors and their binding proteins

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.     

Insulin-like growth factors and their binding proteins in children born small for gestational age: implication for growth hormone therapy

The insulin-like growth factors (IGFs) and their binding proteins (IGFBPs) are important regulators of growth and metabolism and are the key mediators of the actions of growth hormone (GH). Children born small for gestational age (SGA) have a host of medical problems including an increased risk of poor growth later in life, a tendency to develop metabolic abnormalities and a high incidence of learning disabilities. IGFs and related molecules may be linked to all of these concerns. Mouse models of IGF-I and IGF-II deficiencies have phenotypes reminiscent of human SGA, including slow growth, insulin resistance, and mental dysfunction. Humans with IGF-I mutations are born SGA and exhibit very poor subsequent growth, metabolic syndrome and mental retardation. Current management of children born SGA who present with growth failure during childhood includes treatment with GH. SGA children usually have growth factor levels within the normal range; however, as a group, they display lower IGFBP-3 levels in relation to their IGF-I levels. GH is effective in improving growth in children born SGA, but higher doses of GH are required to achieve optimal outcome, suggesting a component of GH insensitivity in SGA children. As in other indications for GH, a rational monitoring approach (focusing on maintaining IGF levels in the high normal range) is prudent.     

Insulin-like growth factor binding proteins and mammary gland development

Mammary gland development is dependent upon insulin-like growth factors (IGFs) as survival factors. The actions of the IGFs are modulated by a family of IGF-binding proteins (IGFBP1-6). Expression of the IGFBPs is both time-dependent and cell-specific during both the developmental phases and the involution of the mammary gland. Although studied extensively in vitro, understanding the roles of IGFBPs in vivo has been difficult, largely due to the fact that IGFBP knock-out mice have no dramatic phenotypes. This review examines the evidence from in vitro studies and the attempts to examine in vivo actions utilising models with IGFBP deficiency or over-expression. In vitro studies demonstrate that IGFBPs can act by inhibition of the survival effects of IGFs, as well as by enhancing the effects of IGFs. Because the IGFBPs are found associated with the extracellular matrix, a role for IGFBPs as a reservoir of IGFs or, alternatively as a potential barrier to IGFs, thereby restricting their entry into particular tissues or cellular compartments was postulated. We also provide evidence with respect to the IGF-independent actions of the IGFBPs which include receptors, nuclear localization, and interaction with the extracellular matrix and cell surface proteins including integrins. We believe that recent findings place some of the IGFBPs in a larger family of extracellular proteins, the secreted cysteine-rich protein (CCN) family, which have similar structural domains (involved in binding to IGFs, extracellular matrix and integrins) and are heavily implicated in tissue re-modeling and morphogenesis.     

Insulin-like growth factors and binding proteins in early milk from mothers of preterm and term infants

Breast-fed preterm infants often show a better outcome, partly ascribed to the benefit of insulin-like growth factors (IGFs) and their binding proteins (IGFBP). We compared IGF-I, IGF-II, IGFBP-2 and IGFBP-3 levels, measured by radioimmunoassays in milk samples from 30 mothers of preterm (<31 weeks) and from 19 mothers of term (>37 weeks) infants at days 7 and 21 postpartum. Proteolysis of IGFBP-2 within mother's milk and digestion of (125)I-IGF-II and (125)I-IGFBP-2 by gastric juice from neonates were assessed by electrophoretic techniques. Mean concentrations did not differ between preterm and term milk: IGF-I (2.8 +/- 0.2 vs. 2.3 +/- 0.1 ng/ml), IGF-II (12.0 +/- 0.4 vs. 12.2 +/- 0.5 ng/ml), IGFBP-3 (100.0 +/- 5.1 vs. 80.0 +/- 5.8 ng/ml), but did so for IGFBP-2 (3,144 +/- 172 vs. 2,428 +/- 188 ng/ml, p < 0.02). Immunoblots revealed 42% (p < 0.05) more IGFBP-2 fragments of 14 and 25 kDa in preterm milk. Incubation with gastric juice caused cleavage of (125)I-IGFBP-2 and partial cleavage of (125)I-IGF-II. Mutual complexation protected IGF-II and IGFBP-2 from cleavage, suggesting that both are likely to arrive in the bowel in an intact form to exert promotive effects. The results provide further evidence that IGFBP-2 and IGF-II in breast milk are relevant factors for the early development of preterm infants.     

Role of insulin-like growth factors and their binding proteins in growth control and carcinogenesis

Interest in the role of the insulin-like growth factor (IGF) axis in growth control and carcinogenesis has recently been increased by the finding of elevated serum insulin-like growth factor I (IGF-I) levels in association with three of the most prevalent cancers in the United States: prostate cancer, colorectal cancer, and lung cancer. IGFs serve as endocrine, autocrine, and paracrine stimulators of mitogenesis, survival, and cellular transformation. These actions are mediated through the type 1 IGF-receptor (IGF-1R), a tyrosine kinase that resembles the insulin receptor. The availability of free IGF for interaction with the IGF-1R is modulated by the insulin-like growth factor-binding proteins (IGFBPs). IGFBPs, especially IGFBP-3, also have IGF-independent effects on cell growth. IGF-independent growth inhibition by IGFBP-3 is believed to occur through IGFBP-3-specific cell surface association proteins or receptors and involves nuclear translocation. IGFBP-3-mediated apoptosis is controlled by numerous cell cycle regulators in both normal and disease processes. IGFBP activity is also regulated by IGFBP proteases, which affect the relative affinities of IGFBPs, IGFs and IGF-1R. Perturbations in each level of the IGF axis have been implicated in cancer formation and progression in various cell types.     

Insulin-like growth factor binding proteins of equine serum.

Ligand blotting analysis of serum from the horse using radiolabelled IGF-I revealed a protein at 96 kDa which was not present in serum from goat, cow, sheep, deer or donkey. These latter species all displayed five labelled bands in the range 24 to 41 kDa. Conversely, these were only weakly labelled in serum from the horse. Size exclusion chromatography of horse serum pre-incubated with radiolabelled IGF-I revealed reduced binding in the 130-kDa peak compared with goat plasma, and ligand blotting analysis indicated the 96-kDa protein was present in this peak. The 96-kDa protein from horse serum binds IGF-I and IGF-II specifically and appears to be unique to this species. The nature of this protein is at present unknown.     

Somatomedins/insulin-like growth factors, their receptors and binding proteins are present during mouse embryogenesis

Somatomedins/insulin-like growth factors (Sm/IGFs) are considered to have important roles in regulating fetal growth; however, because of limited quantities of tissue, few studies have been performed on their effects on embryonic growth. To assess a potential role for these factors, we evaluated mouse embryonic tissues for the presence of Sm/IGF and insulin receptors and Sm/IGF-binding proteins by chemical affinity labelling. In addition, we measured extractable Sm-C/IGF-I radioimmunoactivity in mouse embryonic tissues. Finally, we compared these data with those from the embryonal carcinoma cell line, PC13. All embryos from day 9 (3-4 somites) to day 12 (45 somites) possessed both Sm-C/IGF-I and IGF-II receptors in apparent greater abundance than insulin receptors. The visceral yolk sac appeared to have proportionally more insulin receptors than the corresponding embryonic tissue. Extracts from the embryos contained immunoreactive Sm-C/IGF-I and binding proteins of 30-45 X 10(3) Mr. PC13 cells possessed all three receptors and the apparent abundance of the insulin and IGF-II receptors was reduced after differentiation was induced with retinoic acid. PC13 cells released both immunoreactive Sm-C/IGF-I- and Sm-C/IGF-I-binding proteins into their medium. When differentiated, the binding proteins resembled the native ones extracted from the intact embryos. The presence of Sm/IGF activity, receptors and binding proteins in early embryogenesis suggests a role for these factors in embryonic growth. The PC13 cell line appears to only partially reflect normal development.     

Circulating levels of insulin-like growth factors increase and molecular forms of their serum binding proteins change with human pregnancy.

Insulin-like growth factors (IGF) and binding proteins were measured in serum from pregnant and nonpregnant women. IGF-I determined by immunoassay after acid-ethanol extraction was increased by pregnancy (p less than 0.005) and was highest in the third trimester (p less than 0.01). Size exclusion chromatography of serum in acid before assay (i) gave a very similar IGF-I pattern, (ii) showed that IGF-II was much higher than IGF-I and (iii) revealed less serum IGF-binding protein activity in pregnancy and lactation. All IGF-binding proteins except binding protein-1 were markedly reduced by pregnancy. This indicates a major change in the main carrier protein for IGFs in the circulation and suggests that tissue targetting of IGFs may be altered during pregnancy.     

Insulin-like growth factor binding proteins: roles in regulating IGF physiology.

Insulin-like growth factor binding proteins (IGFBPs) are soluble proteins present in in extracellular fluids. They have high affinity for IGF-I and -II. Blood concentrations are controlled by nutrition and by hormones in a manner that in most, but not all, instances correlates with plasma concentrations of IGF-I or -II. IGF binding proteins are secreted by a range of cell types in a manner that may serve to modulate the functions of the growth factors in a pericellular environment. IGF binding proteins cxan modify IGF interaction with the type I receptor and may thereby alter IGF signal transduction through this transmembrane signalling unit. Binding proteins may also act as inhibitors or potentiators of biological responsiveness and thereby directly cell type specific responses.     

Insulin-like growth factors (IGF) in muscle development. Expression of IGF-I, the IGF-I receptor, and an IGF binding protein during myoblast differentiation

The insulin-like growth factors (IGFs) I and II exert pleiotropic effects on diverse cell types through interaction with specific high affinity cell surface receptors and with locally produced binding proteins. In skeletal muscle and in myoblast cell lines, the functions of IGF-I and -II are complex. Both growth factors appear capable of stimulating cellular proliferation and differentiation, as well as exerting insulin-like effects on intermediary metabolism. We have demonstrated recently that the expression of IGF-II and its receptor is induced during the terminal differentiation of the myoblast cell line, C2, and have suggested that IGF-II may be an autocrine growth factor in these cells (Tollefsen, S.E., Sadow, J.L., and Rotwein, P. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 1543-1547). We now have examined this cell line for expression of other components involved in IGF signaling. The synthesis of IGF-I is low during myoblast proliferation; IGF-I mRNA can be detected only through use of a sensitive solution hybridization assay. Typical IGF-I receptors can be measured in myoblasts, whereas IGF binding proteins cannot be detected in proliferating cells or in conditioned culture medium. During myogenic differentiation, IGF-I mRNA levels increase transiently by 6-10-fold within 48-72 h. The expression of IGF-I mRNA is accompanied by a 2.5-fold accumulation of IGF-I in the culture medium. IGF-I receptors also increase transiently, doubling by 48 h after the onset of differentiation. By contrast, secretion of a Mr 29,000 IGF binding protein is induced 30-fold to 100 ng/ml within 16 h and continues to increase throughout differentiation. These studies demonstrate that several components critical to IGF action are produced in a fusing skeletal muscle cell line in a differentiation-dependent manner and suggest that both IGF-I and IGF-II may be autocrine factors for muscle.     

Insulin-like growth factors and insulin-like growth factor binding proteins in adult patients with severe liver disease before and after orthotopic liver transplantation

INTRODUCTION: The liver is the main source of serum insulin-like growth factors (IGFs) and IGF-binding proteins (IGFBPs) and the concentration of these proteins might reflect liver function. METHODS: In a retrospective longitudinal study we examined serum levels of total and free IGF-I, IGF-II, IGFBP-1, IGFBP-2, IGFBP-3 and IGFBP-6 in 21 adult patients with end-stage liver disease before and after orthotopic liver transplantation (LTX) by sensitive and specific RIAs. In each patient, the mean value of at least three measurements before and after LTX was calculated. RESULTS: Before LTX, serum levels of total and free IGF-I, IGF-II, IGFBP-3 were low and showed a rapid and significant increase in almost all patients after successful LTX (total IGF-I: 30 +/- 7 vs. 256 +/- 30 ng/ml, p < 0.001; free IGF-I: 1.3 +/- 0.3 vs. 3.5 +/- 0.6 ng/ml, p < 0.01; IGF-II: 177 +/- 28 vs. 618 +/- 30 ng/ml, p < 0.001; IGFBP-3: 1,230 +/- 136 vs. 3,665 +/- 264 ng/ml, p < 0.001). In contrast, IGFBP-1 was found to be high immediately before LTX, and declined to normal levels after LTX (210 +/- 40 vs. 90 +/- 15 ng/ml, p < 0.01), while IGFBP-2 did not show any significant changes (1,154 +/- 296 vs. 1,303 +/- 192 ng/ ml). Positive correlations were found between IGF-I, IGF-II or IGFBP-3, and serum pseudocholinesterase (R = 0.50, 0.72 and 0.61 respectively, p < 0.001). Negative correlations were found between IGF-I, IGF-II or IGFBP-3, and prothrombin time (R = 0.50, 0.59 and 0.51 respectively, p < 0.001). CONCLUSION: Patients with severe liver disease show decreased levels of total and free IGF-I, IGF-II and IGFBP-3, and increased levels of IGFBP-1. These abnormalities are promptly normalized after successful LTX. Thus, serum levels of IGF-I, IGF-II and IGFBP-3 might be useful parameters for the assessment of liver function.     

Binding proteins for insulin-like growth factors in adult rat serum. Comparison with other human and rat binding proteins

Insulin-like growth factor (IGF) binding protein has been purified from adult rat serum by affinity chromatography on agarose-IGF-II and high performance reverse-phase chromatography. The final preparation contains two components, of apparent molecular mass 50 and 56 kDa nonreduced, or 44 and 48 kDa reduced, both of which specifically bind IGF-I and IGF-II. Competitive binding data indicate association constants of 5-10 X 10(10) l/mol for both IGFs, with a slightly higher affinity for IGF-II than IGF-I. Amino-terminal sequence analysis yields a unique sequence, identical in 11 of the first 15 amino acids with that of a human plasma IGF binding protein (Martin, J. L., and Baxter, R. C. (1986) J. Biol Chem. 261, 8754-8760), and with slight homology to other human and rat IGF binding proteins characterized to date. By analogy with the binding protein from human plasma, it is likely that the rat protein is part of the growth-hormone dependent complex which appears to carry most or all of the circulating IGFs.     

Biosensor measurement of the interaction kinetics between insulin-like growth factors and their binding proteins.

The binding kinetics of human insulin-like growth factor binding protein (IGFBP) 1-6 for recombinant human insulin-like growth factor (IGF) I and II were measured and compared in the present study using surface plasmon resonance biosensor technique. Different concentrations of IGFBPs (5-100 nM) were allowed to interact with the immobilized IGF-I or IGF-II on sensor chip surface. Both des(1-3)IGF-I and insulin are known to bind weakly to the IGFBPs and therefore are used as negative controls for the binding experiments. The resultant sensorgrams were analyzed by using simple 1:1 binding model to derive both the association rate (k(a)) and dissociation rate (k(d)) constants for IGFBP-IGF interactions. The k(a) values of IGFBPs are in the range of 1x10(4) to 9x10(5) M(-1) s(-1) for IGF-I and 7x10(3) to 1.7x10(6) M(-1) s(-1) for IGF-II, respectively. The orders of k(a) for both IGF-I and IGF-II are IGFBP-3>IGFBP-5>IGFBP-6>IGFBP-4>IGFBP-2>++ +IGFBP-1. The k(d) values of IGFBPs are in the range of 1.5x10(-5) to 2x10(-4) s(-1) for IGF-I and 3.6x10(-5) to 3.7x10(-4) s(-1) for IGF-II, respectively. The order of k(d) for IGF-I is IGFBP-6>IGFBP-5>IGFBP-4>IGFBP-3>IGFBP-2>++ +IGFBP-1 and that for IGF-II is IGFBP-5>IGFBP-6>IGFBP-2>IGFBP-4>IGFBP-3>++ +IGFBP-1, respectively. The equilibrium affinity constants (K(A)) were calculated based on the ratio of k(a)/k(d) and were more precise than the published literature values based on competitive radioligand binding assays. The systematic study enables a direct comparison on the IGF-binding properties among the various IGFBPs, and the kinetic data provide additional information to delineate the physiological role of different IGFBPs in vivo.     

Mechanisms of discrimination between cobalamins and their natural analogues during their binding to the specific B12-transporting proteins

Three proteins, intrinsic factor (IF), transcobalamin (TC), and haptocorrin (HC), all have an extremely high affinity for the cobalamins (Cbls, Kd approximately 5 fM) but discriminate these physiological ligands from Cbl analogues with different efficiencies decreasing in the following order: IF > TC > HC. We investigated interactions of these proteins with a number of ligands: Cbl, fluorescent conjugate CBC, two base-off analogues [pseudo-coenzyme B12 (pB) and adenosyl factor A (fA)], and a baseless corrinoid cobinamide. Protein-ligand encounter and the following internal rearrangements in both molecules were registered as a change in the fluorescence of CBC (alone or mixed with other ligands), a transition in absorbance of pB and fA (base-off --> on-base conversion), and alterations in the molecular mass of two split IF domains. The greater complexity of the binding kinetics followed better Cbl specificity (HC < TC < IF). On the basis of the experimental results, we propose a general binding model with three major steps: (1) initial attachment of the ligand to the high-affinity C-domain, (2) primary assembly of N- and C-domains, and (3) slow adjustments and fixation of the ligand at the domain-domain interface. Since step 3 was characteristic of highly specific TC and especially IF, we suggest its particular importance for ligand recognition. The designed models revealed the absolute Kd values for a group of analogues. Calculations show that most of them could potentially bind to the specific transporters IF and TC under physiological conditions. Implications of this finding and the protective role of HC are discussed.     

Insulin-like growth factor I and its binding proteins: a study of the binding interface using B-domain analogues

The biological activity of the insulin-like growth factors (IGF-I and IGF-II) is regulated by six IGF binding proteins (IGFBPs 1-6). To examine the surface of IGF-I that associates with the IGFBPs, we created a series of six IGF-I analogues, [His(4)]-, [Gln(9)]-, [Lys(9)]-, [Ser(16)]-, [Gln(9),Ser(16)]-, and [Lys(9),Ser(16)]IGF-I, that contained substitutions for residues Thr(4), Glu(9), or Phe(16). Substitution of Ser for Phe(16) did not affect secondary structure but significantly decreased the affinity for all IGFBPs by between 14-fold and >330-fold, indicating that Phe(16) is functionally important for IGFBP association. While His(4) or Gln(9) substitutions had little effect on IGFBP affinity, changing the negative charge of Glu(9) to a positive Lys(9) selectively decreased the affinities of IGFBP-2 and -6 by 140- and 30-fold, respectively. Furthermore, the effects of mutations to both residues 9 and 16 appear to be additive. The analogues are biologically active in rat L6 myoblasts and they retain native structure as assessed by their far-UV circular dichroism (CD) profiles. We propose that Phe(16) and adjacent hydrophobic residues (Leu(5) and Leu(54)) form a functional binding pocket for IGFBP association.