Insulinlike Growth Factor Axis Components

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2.1 Structure of IGF-I and IGF-II

Mature IGF-I and IGF-II are single-chain polypeptides of 70 and 67 amino acids, respectively, with 62% overall sequence identity (4-5). Due to structural identity with insulin, the IGF polypeptide chain has been divided into four domains arranged as B-C-A-D. IGF A- and B-domains have 45% sequence identity with insulin A- and B-chain; however, the connecting peptide C is shorter than the proinsulin C-chain, and the carboxyl-terminal D-domain extension is exclusive to IGF. Another parallel between IGF and insulin structure is the presence of three intrachain disulphide bonds arranged in the same disposition as in insulin, i.e., two connecting B- and A-domains and one intra-A-domain (6). Moreover, the IGF are synthesized as preproproteins with signal peptides of about 25 amino acids at the N-terminus of the B-domain, and further extensions of 35-85 residues at the C-terminus of the D-domain, termed the E-peptide (7). Although the signal peptide and E-domain are deleted sequentially by post-translational processing before secretion, the presence of different IGF-II proforms has been reported in serum (8). Interestingly, these "large" IGF-II forms have the same mitogenic activity as the processed species (8-10).

In Northern hybridization studies, cDNA probes detect several IGF-I mRNA 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. Similarly, IGF-II mRNA species of 3.4-4.0, 2.2, 1.61.75 and 1.1-1.2 kb have been described (11-12). These multiple mRNA arise from alternative splicing, since IGF genes have a discontinuous structure; IGF-I gene contains five exons that span at least 45 kb (13), whereas IGF-II consists of seven exons spanning more than 16 kb (14). The physiological significance of these different splicing forms nonetheless remains unknown.

As mentioned above, IGF interact with four different molecular species: the IGF-1R, the IGF-2R, the IR and the IGFBP. The IGF domains involved in these interactions have been defined mostly by homologous scanning mutagenesis, replacing IGF-I domains with those corresponding to homologous areas of insulin (15). With this approach, it was found that IGF-I residues 1-3 and 49-51 are important for IGFBP and IGF-2R binding, whereas amino acids 21, 23, 24, 44 and tyrosines 31 and 60 are required for binding to the IGF-1R (1522). Extrapolation of these amino acids on a partially-resolved three-dimensional structure of IGF-I (23) indicates that the IGFBP and the IGF-1R binding surfaces are on opposite sides of the IGF-I molecule, suggesting that IGFBP-IGF complexes may bind to the IGF-1R on the cell surface. However, these ternary complexes have not been demonstrated to date. Most studies indicate that IGFBP abrogates IGF-I biological activity; and this inhibition is not observed for IGF-I mutants with low affinity for IGFBP (24). Structure-function analysis with a panel of 28 monoclonal antibodies covering the entire exposed IGF-I surface indicated an overlap of the IGF-I domains involved in IGF-1R and IGFBP binding (25). These results are compatible, since scanning mutagenesis provides direct information about the residues involved in ligand-receptor interaction, but not about the steric hindrance produced by each receptor. Even though IGF amino acids interacting with IGFBP and IGF-1R are on distinct sides of the molecule, the IGFBP and IGF-1R footprints thus overlap on the IGF-I surface.

2.2. IGF binding proteins (IGFBP) and IGFBP proteases

Circulating IGF are tightly bound in serum and in the extracellular milieu to soluble receptors termed IGFBP. Seven members of the IGFBP family have been cloned so far (26). IGFBP-1 to -6 are structurally related proteins of 216-289 amino acids, with highly conserved cysteine-rich N- and C-terminal domains involved in ligand binding; the domains are linked by a central portion that is very dissimilar among each IGFBP. These six IGFBP bind with high affinity to both IGF-I and -II, but do not bind insulin (27). Conversely, IGFBP-7/mac25 binds to insulin with high affinity and with very low affinity to IGF-I or IGF-I (28).

Most IGF-I and IGF-II is found in circulation as 50 and 150 kDa complexes with IGFBP (29-30). These complexes prolong IGF half-life in circulation; indeed, injection of radioactively-labeled IGF-I in rats showed that the 150 kDa form remained in the bloodstream for 3-4 h, compared with 10 minutes recorded for unbound IGF (31). The 150 kDa form, which is the main IGF reservoir in serum (32), is a ternary complex formed by IGF, IGFBP-3 and a glycoprotein termed acid-labile subunit (ALS) (33-34). The presence of ALS in the 150 kDa complex impedes its crossing the endothelial barrier, increasing IGF half-life in the bloodstream (35). Other IGFBP in serum, such as IGFBP-1, -2 and -4, are able to traverse the vasculature, and hence transport IGF from the circulation to peripheral tissues (27, 36). The endocrine effects of IGF on somatic growth or tumourigenesis may therefore be regulated by controlling its availability through the formation of IGF complexes with specific IGFBP.

In addition to their role as IGF reservoirs and carrier proteins in the circulation, IGFBP act as inhibitors (3739) or enhancers (40-41) of IGF biological activity at cell level. The inhibitory effects of IGFBP have been attributed to competitive scavenging of IGF peptides away from the IGF-1R (4243). The enhancer mechanism is poorly understood, and usually involves binding of the proteolytic cleavage of membrane-bound IGF/IGFBP complex, which in turn results in a decrease in IGFBP binding affinity (44-45). Several authors have suggested that the IGFBP enhancing effect is mediated by the sequestering of IGF from the extracellular milieu, which prevents the negative IGF-1R feedback that occurs at high IGF concentrations (41, 46-47). Alternatively, IGFBP may facilitate IGF binding to IGF-1R by anchoring the ligands in close proximity to their cell receptors. In fact, binding of the IGF/IGFBP complex to the cell surface is critical in the IGFBP enhancing effect. IGFBP-3 blocks cell growth if added simultaneously with IGF-I, but increases IGF-I-induced mitogenesis if added prior to IGF-I stimulation (48).

In addition to IGFBP binding to the cell membrane, other processes such as proteolytic cleavage of IGFBP, should account to enhance IGF activity. Several proteases, including serine proteases, cathepsins and matrix metalloproteinases (MMP), are reported to use IGFBP as substrates. In all cases, this proteolytic cleavage results in a drastic decrease in IGFBP affinity for IGF and in the controlled release of the growth factors (49-52). Furthermore, is reported that IGF-I upregulates MMP-2 synthesis in carcinoma cells (53), establishing a positive feedback loop between IGF activity and the IGFBP proteolytic activities. Strikingly, some of the proteases acting on IGFBP are also associated to the cell surface (54-56); this explains the observation that factors that increase IGFBP association to the plasma membrane also contribute to IGFBP-enhanced IGF activity (57).

Several recent lines of evidence in various cell systems have suggested that the IGFBP, especially IGFBP-3, may have more active, IGF-independent roles in cell growth regulation. In support of this hypothesis, high affinity IGFBP-3 binding to the surface of various cell types and IGF-independent direct inhibition of monolayer growth have been shown; both are presumably induced by specific interaction with cell membrane proteins that function as an IGFBP-3 receptor (5859). Interest in IGFBP has increased since it was found that IGFBP-3 could act as an antineoplastic agent. IGFBP-3 inhibits cell proliferation in fibroblasts that express or lack the IGF-1R (60-61), indicating that IGFBP-3-mediated growth suppression is independent of IGF-1R action, IGFBP-3 has also been identified as a p53-regulated target gene (62). Taken together, these data suggest that the IGFBP-3 gene may be a tumour suppressor. The newest member of the family, IGFBP-7, is also suggested to be a tumour suppressor binding protein. The IGFBP-7/mac25 cDNA was originally cloned from leptomeningial cells and subsequently reisolated by differential display as a sequence expressed preferentially in senescent human mammary epithelial cells (63). mac25 mRNA is detectable in a wide range of normal human tissues, with decreased expression in breast, prostate, colon, and lung cancer cell lines, suggesting that IGFBP-7 may function as a growth-suppressing factor (26, 63).

2.3. The IGF receptors

There are at least three receptors in the IGF axis, including the insulin receptor (IR), the type-1 IGF receptor (IGF-1R) and the type-2 IGF receptor (IGF-2R). The IGF-1R is the most active in terms of cellular proliferation, and crossreacts with all three ligands; it shares several functional and structural characteristics with the IR, but the IGF-1R p subunit is ten-fold more mitogenic than the IR (64). Supraphysiological insulin concentrations also activate the IGF-1R, and the mitogenic effects of insulin at microgram concentrations are probably exerted through binding to the IGF-1R (65).

The IGF-1R belongs to the tyrosine kinase receptor family (66) and its amino acid sequence is 70% identical to that of the IR (67). It is a heterotetrameric glycoprotein composed of two ligand-binding subunits, entirely extracellular, and two transmembrane subunits linked by disulfide bonds, which have the enzymatic activity (68-69). The receptor is synthesized as a single preprotein of 1,367 amino acids that is cleaved to generate two half receptors, which are joined by disulfide bonds between the a subunits to form the mature receptor. The IGF-1R binds to all three ligands with distinct affinities; IGF-I and IGF-II bind to the receptor at nanomolar concentrations, whereas insulin binds with 100-fold lower affinity. Ligand binding to the subunits triggers autophosphoryla-

tion of f> subunits (70-71) by an intramolecular trans mechanism similar to that used by other receptors (72-73).

The IGF-2R is a single chain, membrane-spanning glycoprotein that is identical to the cation-independent mannose-6-phosphate receptor (74). The mature human receptor consists of a large extracellular domain and a short intracytoplasmic domain (75). The extracellular domain contains the ligand-binding domain (mannose-6-phosphate and IGF-II) and comprises 15 repeated domains. The intracellular portion has no tyrosine kinase activity and has been implicated mainly in trafficking among different subcellular compartments. The function of IGF-2R remains puzzling. It does not stimulate cell proliferation; in fact, IGF-2R gene deletion causes body weight increase in mice, and blocking of IGF-II binding to the receptor does not alter its mitogenicity (76-77). This supports the argument that the IGF-2R is a specific downregulator of IGF-II (the receptor binds neither IGF-I nor insulin). IGF availability may thus be regulated in two ways, through IGF-2R, which controls IGF-II levels, and through IGFBP, which may serve as storage sites for both IGF-I and IGF-II ligands (insulin does not bind to IGFBP).

An additional receptor in the IGF axis must be considered: the insulin-receptor related receptor (IRRR), which has substantial identity with the IGF-1R and the IR (78). In chimeric constructs with IR or IGF-1R subunits, the IRRR tyrosine kinase domain was shown to be mitogenic, but the ligand involved in the activation of this receptor has not yet been identified. In fact, serum does not activate the IRRR (79), suggesting that the IRRR may be activated by an unknown ligand through a strictly autocrine mechanism.

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