Modulation of IGF Actions

IGFBPs are capable to modulate biological actions of the IGFs. The different cellular effects of IGF-I and IGF-II in highly differentiated cell types are mainly inhibited by IGFBPs. In this case, IGFBP molecules, freely circulating in the extracellular space, block IGF action simply by sequestering free IGFs and preventing the binding of the IGFs to IGF receptors (Jones and Clemmons, 1995; Baxter, 2000). However, some IGFBPs may also modulate action of the IGFs both in positive and in negative manner.

The same IGFBP can have an IGF-inhibiting or potentiating role that is determined by an interaction between IGFBP and the IGFs, which in turn can be controlled by three different mechanisms: (i) posttranslational structural modifications of IGFBP; (ii) proteolytic cleavage of IGFBP; (iii) binding of IGFBP to cell surface and extracellular matrix (ECM).

1.4.2.3.1. Modulation of IGF Actions by Posttranslational Structural Modifications of IGFBPs

Posttranslational structural modifications of IGFBPs include phosphorylation and glycosylation of their molecules. IGFBP-1, IGFBP-3, and IGFBP-5 are all secreted as phosphoproteins(Coverley and Baxter, 1997). Phosphorylation and dephosphorylation status of human IGFBP-1 determines higher or lower binding affinity for IGF, thus

leading to inhibition or potentiation of IGF effects, respectively. Phosphorylation of IGFBP-1 is catalyzed by casein kinase and occurs only on serine residues of IGFBP-1 located in acidic regions of the molecule. In human, in contrast to the highly phosphorylated IGFBP-1 that inhibits IGF actions, the nonphosphorylated form of IGFBP-1 has 4- to 6-fold lower affinity for IGF-I. This might contribute to sequestration of IGF-I by phosphorylated IGFBP-1 and more complete release of IGF-I in the vicinity of the IGF-IR by the nonphosphorylated form, thereby potentiating IGF-I effects.

Although,as described earlier, IGFBP-1 can also interact with cells viathe α5β1-integrin, it is not clear how this interactionis modulated by phosphorylation of IGFBP-1.

IGF IGF--IRIR

IGFBP IGFBP IGF

IGF

Figure 7. Modulation of IGF actions by IGFBPs. In most cases IGFBPs inhibit action of the IGFs by sequestering free IGFs and thereby preventing the binding of the IGFs to IGF receptors.

There is also evidence that phosphorylation inhibits IGFBP-3cell binding. Human skin fibroblasts secrete IGFBP-3into the culture medium as a phosphoprotein, but release of surface-bound IGFBP-3 from fibroblasts using an IGFIR-inactive IGF-I analogue was found to increase total IGFBP-3 but not phospho-IGFBP-3 in the culture medium, implying that surface-bound IGFBP-3 was nonphosphorylated (Coverley and Baxter, 1995). More recently, phosphorylation of IGFBP-3 in vitro by casein kinase CK2 has been shown by direct binding studies to be inhibitory to cell surface association (Coverley et al., 2000).

Some IGFBPs (IGFBP-3, -4, -5, -6) can also be glycosylated. In IGFBP-3,carbohydrate increases the size of core protein from 29 kDa to 40–43 kDa. Of the three potential

an estimated 4 kDa and 4.5 kDa of carbohydrate, respectively, whereas the third site alternatively containseither undetectable or about 5 kDa of carbohydrate, accountingfor the characteristic doublet form of the protein (Firth and Baxter, 1999). Glycosylation of IGFBP-3 has no significant effect on the bindingof IGF-I (Sommer et al., 1993) or ALS (Firth and Baxter, 1999). However, IGFBP-3 formsin which various glycosylation sites have been altered by mutagenesis reveal that decreasing glycosylation tends to increase cell surface association (Firth and Baxter, 1999). This suggests that the carbohydrate present in natural IGFBP-3 might mask potential cell-association sites.

Likewise, association of IGFBP-6 with cell surface also appears to be inhibited by carbohydrate. Binding to glycosaminoglycansis greatly inhibited by glycosylation, and the non-glycosylatedprotein, which is not known to occur in nature, has been shownto bind to cell membranes, whereas the natural, glycosylated form shows no binding (Marinaro et al., 2000). It implies that, as observed in the case of IGFBP-3, cell binding sites of IGFBP-6 arepermanently masked by carbohydrate. Several lines of evidence also suggest that non-glycosylated IGFBP is more susceptible than glycosylated IGFBP to proteolysis, thus glycosylated sites of IGFBP molecules may inhibit access to potential cleavage sites of proteases (Bach, 1999).

PP

Figure 8. Role of posttranslational structural modifications of IGFBPs in modulation of IGF actions. In human, the highly phosphorylated IGFBP-1 (P) has much higher affinity for IGF-I than its nonphosphorylated form. Glycosylated IGFBPs (G) poorly associate with cell surface and are less susceptible to proteolysis than non-glycosylated IGFBP.

1.4.2.3.2. Modulation of IGF Actions by IGFBP Proteolysis

Limited proteolysis of IGFBPs is believed to be the major mechanism for the release of IGF molecules from IGFBP-IGF complexes generating IGFBP fragments with reduced affinity for the IGFs (Bunn and Fowlkes, 2003). Several IGFBP-specific proteases such as kallikrein-like serine proteases, cathepsins and metaloproteinases, active both within circulation and in extravascular fluids, have been described and characterized. Whereas conditioned media from primary cultures of rat liver cells lacked neutral IGFBP protease activities, the presence of acid-activated IGFBP proteases – most likely lysosomal aspartyl and cystein proteases (cathepsins) – was observed. However, when hepatocytes and KC were cocultured at neutral pH in the presence of iodinated IGFBP-3, a time-dependent disappearance of intact IGFBP-3 and the generation of IGFBP-3 fragments of different sizes were observed. These data suggest that either endocytosed IGFBP-3 is degraded in cathepsin-containing organelles accompanied by partial recycling and release of IGFBP-3 fragments into the extracellular medium, or that IGFBP-3 is cleaved by protease(s) localized at the cell surface (Scharf and Braulke, 2003).

Early endosome IGFBP

proteolytic fragments

IGFBP protease IGFBP

receptor?

IGFBP IGF

1

2

3

Figure 9. IGFBP proteolysis. It is believed that major sites for IGFBP proteolysis are: (1) extracellular space, (2) cell surface, (3) intracellular organelles. Cathepsin-mediated IGFBP proteolysis in the endosomal recycling compartment is considered to be accompanied by partial recycling and release of IGFBP fragments into the extracellular compartment.

Proteolytical degradation has been shown for IGFBP-2 to -5. In case of IGFBP-3 and -5, the proteolytic fragments may retain significant affinity for the IGFs, whereas the affinity of IGFBP-2 and -4 fragments is drastically reduced. Furthermore, in case of IGFBP-3 and IGFBP-5, the fragments themselves may potentiate IGF actions even when IGF affinity is substantially reduced (Jones and Clemmons, 1995).

1.4.2.3.3. Modulation of IGF Actions by Binding of IGFBPs to Cell Surface and Extracellular Matrix

Increased adherence of some IGFBPs to cell surface or ECM has been shown to be associated with a decrease in affinity for the IGFs and with an increased mitogenic response. It is speculated that a reservoir of local IGFs bound to the IGFBP localized on the cell surface and in ECM can lead to release the IGFs under the different conditions, thus providing a high local concentration of IGF to stimulate the IGF-IR, thus potentiating its local effects (Firth and Baxter, 2002).

ECM ECM

α β

IGFBP IGFBP

IGF IGF

IGF

IGF--IRIR IGFBP IGFBP Receptor Receptor ?? Integrin

Integrin

Figure 10. Role of tissue- and cell-localized IGFBP in potentiation of IGF actions. Association of IGFBPs with proteins on the cell surface or in the ECM results in an increase in the local concentration of the IGF in the vicinity of the IGF-IR. Association with the cell surface or with the ECM lowers the affinity of IGFBPs for the IGFs. This decreased affinity allows the release of IGF to the receptor, thereby stimulating biological response.