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Endocrinology Vol. 147, No. 11 5268-5274
Copyright © 2006 by The Endocrine Society

The Aminoterminal Insulin-Like Growth Factor (IGF) Binding Domain of IGF Binding Protein-3 Cannot Be Functionally Substituted by the Structurally Homologous Domain of CCN3

Xiaolang Yan, Robert C. Baxter, Bernard Perbal and Sue M. Firth

Kolling Institute of Medical Research (X.Y., R.C.B., S.M.F.), University of Sydney, Royal North Shore Hospital, St. Leonards, New South Wales 2065, Australia; and Laboratoire d’Oncologie Virale et Moléculaire (B.P.), Unités de Formation et de Recherche de Biochimie, Université Paris 75251 cedex 05, France

Address all correspondence and requests for reprints to: Sue M. Firth, Kolling Institute of Medical Research, Royal North Shore Hospital, St. Leonards, New South Wales 2065, Australia. E-mail: sfirth{at}med.usyd.edu.au.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
IGF binding proteins (IGFBPs) are a family of structurally homologous proteins that bind IGFs with high affinities and can modulate IGF activity. The IGF binding site has been shown to comprise residues in both the aminoterminal and carboxyterminal domains. In recent years several proteins including members of the CCN (connective tissue growth factor, Cyr61, and nephroblastoma overexpressed) family were recognized as having structural homology in their aminoterminal domains to the IGFBPs. Despite their low or undetectable IGF binding ability, a proposal was made to rename them as IGFBP-related proteins. To test whether the aminoterminal domain of a CCN protein can fulfill the high-affinity IGF binding function of an IGFBP, we created a chimera in which the aminoterminal domain of IGFBP-3 was substituted with the aminoterminal domain of CCN3 (previously known as Nov). The CCN3-IGFBP-3 chimera bound IGFs and inhibited IGF activity very weakly, similar to CCN3 itself. Although structurally similar, the aminoterminal domain of CCN3 is unable to replace the aminoterminal domain of IGFBP-3 in forming a high-affinity IGF-binding site. These results argue against a direct role of CCN3 in the regulation of IGF bioavailability and indicate that the nomenclature of IGFBP-related proteins (which implies functional relationship to the classical IGFBPs) is inappropriate for CCN proteins.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IGF BINDING PROTEINS (IGFBPs) are a family of six proteins with a conserved primary sequence. All bind the IGFs, IGF-I and IGF-II, with high affinity, although the relative specificities for the two ligands vary among the IGFBPs (1). They show considerable sequence similarity in their cysteine-rich amino- and carboxyterminal domains, whereas their central regions are highly variable. No complete structure has yet been reported for any member of the IGFBP family; therefore, it has been necessary to obtain structural information from a few partial structural determinations, plus a variety of structure-function studies involving point mutations or partial sequence deletions. These studies point to a hydrophobic IGF binding site in the aminoterminal domain (2, 3, 4, 5), plus a second binding site in the carboxyterminal domain (6, 7, 8, 9) involving neutral amino acids in the vicinity of a cluster of basic residues, which, in the case of IGFBP-3 and IGFBP-5, have been shown to include heparin-binding and nuclear localization motifs (1). A recent study of IGFBP-3 has shown that there is cooperativity of IGF binding to two 11-kDa polypeptides representing the amino- and carboxyterminal domains (10), implying that ligand binding by the intact binding proteins may involve cooperativity between the two IGF binding sites.

It has been recognized for several years that some structural features of the aminoterminal domain of the IGFBPs are shared by a wider family of proteins. Specifically the highly conserved organization of disulfide bonds in the aminoterminal part of the protein, characteristic of IGFBPs, is also seen in other groups of proteins, whose genes share structure with exon 1 of the IGFBP genes, which encodes this region of the proteins (11). The term IGFBP-related proteins was proposed for non-IGFBP members of this putative protein superfamily (12), but this was disputed on the grounds that it was confusing in terms of defining both the structure and function of these proteins (13). Subsequently the literature has mainly reverted to alternative nomenclature for these proteins, the best-defined of which are Mac25 (14), a protein related to follistatin, and the family of proteins known as CCN proteins, which include connective tissue growth factor (CTGF), Cyr61, and nephroblastoma overexpressed (Nov) (13, 15, 16, 17). CCN3, originally designated Nov (18) and transiently known as IGFBP-rP3, is a 48-kDa secreted protein that was first identified in avian nephroblastoma. Like other members of the CCN family, CCN3 is described as containing four structural motifs resembling IGFBP, Von Willebrand factor, thrombospondin, and cysteine-knot domains (18). The aminoterminal IGFBP domain of the CCN proteins typically contains 11 of the 12 conserved aminoterminal cysteine residues found in the IGFBP-1 to -5 aminoterminus (11). However, the alignment of these cysteine residues between CCN and IGFBP that was proposed as evidence for functional conservation (11, 19) does not result in alignment of the other aminoterminal residues that are conserved within the CCN family of proteins (20).

Affinity-labeling and ligand-blotting studies suggest weak IGF-binding affinity for at least two members of the CCN family, CTGF (21) and Nov (19), as well as Mac25 (22). Because these proteins share some aminoterminal structure with the IGFBPs, it has been assumed that the structural similarities might account for their IGF-binding activity. The relatively low IGF-binding affinity would be consistent with the low affinity seen in isolated amino-terminal IGFBP fragments (23). However, key hydrophobic residues implicated in the IGFBP aminoterminal binding site are not found in the related proteins (1), raising the question of the structural basis of the weak IGF-binding activity in these proteins. We now report the creation of a chimeric protein in which the aminoterminal domain of IGFBP-3 is substituted by the aminoterminal domain of human CCN3. This protein was created to answer the specific question: can the aminoterminal domain of a CCN-family protein fulfill the IGF-binding function of an IGFBP aminoterminal domain? Our results indicate that the chimeric protein does bind IGFs weakly, similar to but no better than CCN3 itself. This indicates that the aminoterminal domain of a CCN family protein cannot fulfill the IGF-binding function of an IGFBP aminoterminal domain.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
Recombinant human IGF-I was a gift from Genentech Inc. (South San Francisco, CA). Recombinant human IGF-II was purchased from GroPep Pty. Ltd. (Adelaide, South Australia, Australia). The rabbit polyclonal antibody (R-100) was raised in-house against the full-length natural human IGFBP-3, and the goat polyclonal IgG (C-19), raised against a synthetic carboxyl-terminal peptide of human IGFBP-3, was purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Anti-CCN3 polyclonal antibody (K19M) specific for the carboxyl-terminal region of CCN3 has been described previously (24). PCR primers were synthesized by Invitrogen Australia Pty. Ltd. (Mulgrave, Victoria, Australia). All restriction enzymes were purchased from New England Biolabs Inc. (Beverly, MA). 911 retinoblastoma cells and the adenoviral expression system have been described previously (25, 26). Heparin Hi-Trap columns were purchased from Amersham Pharmacia Biotech (Uppsala, Sweden). The HPLC column (5 µm C18 300 Å) was purchased from Phenomenex (Torrance, CA). An IGF-I affinity column was made by covalently linking recombinant human IGF-I to Affi-Gel-10 activated agarose (Bio-Rad Laboratories, Hercules, CA). The type I IGF receptor (IGFRI) overexpressing cells, NIH-3T3-IGFRI, were a generous gift from Dr. Colin Ward (Commonwealth Scientific and Industrial Research Organisation Molecular and Health Technologies, Parkville, Australia). Antiphosphotyrosine monoclonal antibody (PY20) was purchased from BD Bioscience (Palo Alto, CA). Anti-IGFRI ß-subunit polyclonal antibody (C-20) was obtained from Santa Cruz Biotechnology. Silver-staining kits were obtained from Bio-Rad Laboratories. All radiolabeled proteins used were prepared as described previously (27).

Cloning of CCN3 and CCN3/IGFBP-3 chimera into the adenoviral gene expression system
CCN3 cDNA was initially amplified by PCR from the pCB6+/novH plasmid and subcloned into an adenoviral shuttle plasmid, pAdTrack-CMV. To construct CCN3-IGFBP-3 chimera, DNA sequence coding for the N terminus of CCN3 (residues 1–73), and sequence coding for the C terminus of IGFBP-3 (residues 90–264) were amplified separately from plasmids that carried the respective full-length cDNAs. The two amplified fragments were ligated to construct a chimeric sequence that resulted in four amino acids (AAMA) between A73 of CCN3 and A90 of IGFBP-3.

Production of adenoviruses and expression of recombinant CCN3 and CCN3-IGFBP-3 chimeric proteins
After homologous recombination between the recombinant shuttle plasmids and pAdEasy-1 in Escherichia coli strain BJ5183, recombinant plasmid DNA was used to transfect 911 retinoblastoma cells to generate CCN3 and CCN3-IGFBP-3 adenoviruses. High-titer viruses were obtained after three to four rounds of amplification and were used to infect 911 cells in 420-cm2 culture flasks. Cells were grown to confluence in DMEM supplemented with 10% (vol/vol) fetal bovine serum and 20 mM glutamine and then changed to serum-free DMEM supplemented with 1 mg/ml BSA, 20 mM glutamine, and protease inhibitors (0.5 µg/ml leupeptin and 5 µg/ml {alpha}2-macroglobulin) before infection with virus. After 48 h, the conditioned medium was collected and clarified by centrifugation and Na2EDTA added (0.5 mg/ml final concentration) before storage at –20 C (25).

Purification of adenoviral recombinant proteins
Purification of recombinant IGFBP-3 has been described previously (8). Briefly, the conditioned media were loaded onto an IGF-I-AffiGel-10 column at 4 C, and after extensive washes with 50 mM sodium phosphate (pH 6.5), recombinant IGFBP-3 was eluted with 0.5 M acetic acid (pH 3.0). CCN3 and CCN3-IGFBP-3 chimera proteins were purified from conditioned media by heparin-affinity chromatography. The conditioned media were applied to 1-ml heparin-Sepharose (Hi-trap) columns at 0.4 ml/min at 4 C. The columns were washed extensively with 50 mM sodium phosphate (pH 6.5) before elution of bound proteins by applying a gradient of 0–1 M sodium chloride. Samples of the elution fractions were analyzed for the presence of recombinant protein by SDS-PAGE and immunoblots.

Further purification of proteins was achieved by performing reverse-phase HPLC. The proteins were loaded onto a C18 column and a linear gradient of 15–60% acetonitrile in 0.1% trifluoroacetic acid was immediately applied at 1.5 ml/min over 30 min. Purity of the proteins was assessed by silver staining after SDS-PAGE. N-terminal sequencing and quantitative amino acid analysis were performed by Australian Proteome Analysis Facility (North Ryde, New South Wales, Australia).

Immuno- and ligand blotting
SDS-PAGE, silver staining of gels, and immuno- and ligand blotting were performed essentially as described previously (8).

Plasmin digestion
One microgram of IGFBP-3 or CCN3-IGFBP-3 was incubated with 100 ng plasmin (Sigma Aldrich, St. Louis, MO) in a total volume of 12 µl containing 0.02 M Tris-HCl (pH 7.5), 0.15 M NaCl for 4 h at 37 C (28). After separation on SDS-PAGE and Sypro Ruby staining, the IGFBP-3 products were excised from the gel and subjected to N-terminal sequencing. For CCN3-IGFBP-3, the products were transferred to polyvinyl difluoride membrane after SDS-PAGE, stained with Ponceau S, excised from the membrane, and subjected to N-terminal sequencing.

Functional assays
IGF solution binding assays were performed as described (8), with the exception that complexes were precipitated in the presence of 0.025% final concentration of human {gamma}-globulin and 12.5% final concentration of polyethylene glycol. The inhibition of IGF-induced IGFRI phosphorylation was assessed as described previously (8). NIH-3T3-IGFRI cells were treated for 5 min with either 2.5 ng/ml IGF-I or 10 ng/ml IGF-II that had been preincubated with or without IGFBP-3, CCN3, or CCN3-IGFBP-3 in the specified molar ratios for 2 h at room temperature. Cell lysates were separated by SDS-PAGE and immunoblotted with antiphosphotyrosine monoclonal antibody PY20 to detect phosphorylated IGFRI.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression and purification of CCN3, CCN3-IGFBP-3, and IGFBP-3
The amino acid sequences of IGFBP-3, CCN3, and CCN3-IGFBP-3 are shown in Fig. 1Go, with homologies between IGFBP-3 and CCN3 indicated. There is perfect homology between each of the proteins and the consensus IGFBP family signature [GP]C-[GSET][CE][CA]x(2)-C-[ALP]x(6)-C (29). All three proteins were expressed via an adenoviral-mediated expression system, essentially as previously described for IGFBP-3 (8). The cloning strategy to create a cDNA construct coding for the chimeric CCN3-IGFBP-3 is described in Materials and Methods. The CCN3-IGFBP-3 is predicted to consist of 252 amino acids, compared with 264 and 326 amino acids for IGFBP-3 and CCN3, respectively (Fig. 1Go). Sequences corresponding to the glycosaminoglycan binding motifs, BBXB and BBBXXB [B represents basic residue; X represents hydropathic residue (30)], are present in IGFBP-3 (K149KGH and K220KKQCR) and CCN3 (K228KGK) (Fig. 1Go). Both IGFBP-3 and CCN3 are known to bind heparin (31), and in the case of IGFBP-3, the heparin binding domains reside within the C terminus that is present in the chimeric CCN3-IGFBP-3. To compare the relative heparin-binding affinities of the proteins, infection media containing each protein were purified by heparin-affinity chromatography, and immunoblots of the elution fractions are shown in Fig. 2AGo. IGFBP-3 and CCN3-IGFBP-3 appear to have similar affinities for heparin because both proteins were eluted from the column by approximately 0.7 M NaCl (Fig. 2BGo). In contrast, CCN3 eluted with approximately 0.6 M NaCl, confirming that CCN3 has weaker affinity for heparin (24). It has been shown previously that the major heparin binding site on IGFBP-3 resides in its carboxyl region (32). Because the affinity of CCN3-IGFBP-3 for heparin was not increased, compared with that of IGFBP-3, we assume that the amino terminus of CCN3 does not have any significant binding to heparin.


Figure 1
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  		FIG. 1. Sequence alignment of IGFBP-3, CCN3, and CCN3-IGFBP-3. Homologous and similar residues between IGFBP-3 and CCN3 are denoted by asterisks and dots, respectively. Sequences corresponding to the consensus IGFBP family signature [GP]C-[GSET][CE][CA]x(2)-C-[ALP]x(6)-C are shown in the gray box. N-glycosylation sites are shown in open boxes, and glycosaminoglycan binding motifs are indicated by italics. The arrows indicate the cleavage site of plasmin in IGFBP-3 and CCN3-IGFBP-3. Swiss-Prot accession number for IGFBP-3 and CCN3 is P17936 and P48745, respectively.

 

Figure 2
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  		FIG. 2. Heparin binding by recombinant IGFBP-3, CCN3, and CCN3-IGFBP-3. Conditioned media containing recombinant proteins were loaded onto heparin-affinity columns and eluted by a 0–1 M NaCl gradient, as described in Materials and Methods. A, Immunoblots of elution fractions for recombinant IGFBP-3, CCN3, and CCN3-IGFBP-3 by their respective specific antibodies. B, The elution profiles of IGFBP-3 (squares), CCN3 (circles) and CCN3-IGFBP-3 (triangles) were generated by plotting the eluting NaCl concentration vs. the densitometric values obtained from the immunoblots in Fig. 2AGo for each fraction.

 
CCN3 and CCN3-IGFBP-3 were further purified by reverse-phase HPLC after heparin-affinity chromatography, whereas IGFBP-3 was purified by reverse-phase HPLC after IGF-affinity chromatography, as described previously (8). The purity of the proteins was analyzed by SDS-PAGE and silver staining (Fig. 3AGo). IGFBP-3 resolved as the characteristic 40- to 45-kDa doublet with a proteolytic fragment of 30 kDa. Both CCN3 and CCN3-IGFBP-3 resolved as 44-kDa bands, the mass of CCN3 being consistent with previous publications (24). The apparent molecular weights of all three proteins were larger than that predicted from their amino acid sequences (molecular mass of 28,717 for IGFBP-3, 35,702 for CCN3, 27,625 for CCN3-IGFBP-3). Both IGFBP-3 and CCN3 are known to be glycosylated (24, 33). There are three potential N-glycosylation sites at N89ASA, N109ASE, and N172FSS in IGFBP-3; and two potential sites at N66QTG and N249CTS in CCN3; and three potential sites at N66QTG, N97ASE, and N160FSS in CCN3-IGFBP-3 (Fig. 1Go). IGFBP-3 and CCN3-IGFBP-3 were both detectable by polyclonal antibodies raised against either whole IGFBP-3 or a synthetic C-terminal sequence peptide of IGFBP-3 (Fig. 3BGo). The K19M antibody raised against C-terminal CCN3 could detect only CCN3. The 30-kDa proteolyzed fragment of IGFBP-3 appears to be lacking the C-terminal region because it was not detectable by the C-19 antibody raised against that region. On the other hand, the 30-kDa proteolyzed fragment of CCN3 contains its C-terminal region because it was detected by the K19M antibody.


Figure 3
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  		FIG. 3. Assessment of the purity of recombinant proteins by silver staining. A, Approximately 100 ng IGFBP-3 (lane 1) and 200 ng CCN3 (lane 2) or CCN3-IGFBP-3 (lane 3) were separated by SDS-PAGE, and the gel was silver stained. Arrow indicates intact recombinant proteins. B, Detection of recombinant proteins by specific antibodies. Recombinant IGFBP-3 (lane 1), CCN3 (lane 2), and CCN3-IGFBP-3 (lane 3) were immunoblotted with IGFBP-3 polyclonal antibody ({alpha}-BP-3), antibody against the C terminus of CCN3 ({alpha}-CCN3), or antibody against the C terminus of IGFBP-3 (C-19). C, Plasmin digestion of IGFBP-3 and CCN3-IGFBP-3. After plasmin digestion, the products were separated by SDS-PAGE and visualized by Sypro Ruby staining (IGFBP-3) or transferred to polyvinyl difluoride membrane and stained with Ponceau S. Indicated intact and proteolysed fragments were subjected to N-terminal sequencing.

 
To verify the presence of the N terminus of CCN3 in CCN3-IGFBP-3, the chimeric protein was subjected to N-terminal sequencing. The sequence obtained was TQRXP (X is most likely a cysteine residue that was not derivatized), which matched the published mature sequence of CCN3 (34). The identical amino-terminal sequence was also obtained for the recombinant CCN3 purified in this study. Quantitative amino acid analysis of CCN3-IGFBP-3 correlated with its expected composition (data not shown), thus confirming the sequence of the chimeric protein.

For the CCN3-IGFBP-3 chimeric protein to adopt a similar conformation to IGFBP-3, one would expect the cysteine residues in the amino-terminal domain of CCN3 and the carboxyl-terminal domain of IGFBP-3 to form covalent disuphide linkages within their respective domains. To test this, the CCN3-IGFBP-3 and IGFBP-3 proteins were subjected to proteolysis by plasmin, a protease known to cleave in the midregion of IGFBP-3 between residues 160 and 161 (35). Both proteins yielded a similar fragmentation pattern in a nonreducing sodium dodecyl sulfate-polyacrylamide gel, resulting in 30-, 20-, and 17-kDa fragments (Fig. 3CGo). For IGFBP-3, N-terminal sequencing of the 30-kDa fragment (1GASSG) indicated that it began at the amino terminus. The 20- and 17-kDa fragments shared the same amino terminus (161VDYE), thus confirming the previous study (35). For CCN3-IGFBP-3, the 30-kDa fragment also began at the N terminus (1TQRXPP), whereas the 20- and 17-kDa fragments shared the same amino terminus (126FHPLH). This suggests that although the cleavage by plasmin is in the midregion of both proteins, the site of cleavage is different (Fig. 1Go). Nevertheless, N- and C-terminal sequences unique to distinct fragments were found for each protein, indicating that there were no disuphide linkages between N- and C-terminal domains.

IGF binding
The ability of the proteins to bind IGFs was tested by ligand blotting and solution binding assays using radiolabeled IGFs. Figure 4AGo shows that there was a strong signal of radiolabeled IGF-I and IGF-II binding to 50 ng of IGFBP-3. In contrast, there was little or no detectable binding of radiolabeled IGF-I to 500 ng of either CCN3 or CCN3-IGFBP-3. Some binding to radiolabeled IGF-II was detectable in CCN3 and CCN3-IGFBP-3; however, this was extremely weak, compared with IGFBP-3. In a previous study, it was also reported that binding of CCN3 to radiolabeled IGF-I or IGF-II was detected by ligand blotting and affinity cross-linking (19) with affinities that were several orders of magnitude lower than that of IGFBP-3. Using the solution binding assay, we were unable to detect any binding of IGF-I (Fig. 4BGo) or IGF-II (Fig. 4CGo) to CCN3, even when CCN3 was present in microgram amounts. In accordance with the ligand blots, there was no detectable binding of CCN3-IGFBP-3 to IGF-I and minimal binding to IGF-II.


Figure 4
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  		FIG. 4. IGF binding of IGFBP-3, CCN3, and CCN3-IGFBP-3. A, IGF ligand blots: 50 ng or 500 ng recombinant IGFBP-3, CCN3, and CCN3-IGFBP-3 were blotted with 125I-IGF-I or 125I-IGF-II. B and C, Solution binding assays: 0.1–10 ng of recombinant IGFBP-3 (squares) and up to 3 µg of recombinant CCN3 (circles) or CCN3-IGFBP-3 (triangles) were used in IGF solution binding assays with 125I-IGF-I (B) or 125I-IGF-II (C).

 
Functional inhibition of IGFRI activation by IGF
To assess the ability of CCN3 and CCN3-IGFBP-3 to modulate IGF function, we tested their ability to inhibit IGF-induced IGFRI autophosphorylation in NIH-3T3 cells that are overexpressing IGFRI (Fig. 5Go). IGFRI autophosphorylation was induced 8-fold by 2.5 ng/ml IGF-I and 4-fold by 10 ng/ml IGF-II. When IGFBP-3 was preincubated with IGF-I or IGF-II before treatment of the cells, the IGF-induced activation of IGFRI is inhibited dose dependently, presumably due to the sequestration of IGFs by IGFBP-3. Preincubation of either IGF with 5- to 10-molar excess of CCN3 appeared to inhibit IGF-induced IGFRI autophosphorylation by 15–20%, but this was not statistically significant. CCN3-IGFBP-3 appeared slightly more inhibitory to IGF-induced IGFRI autophosphorylation. When present at 5- to 10-fold molar excess to IGF, CCN3-IGFBP-3 inhibited both IGF-I and IGF-II induction of IGFRI activation by approximately 40–45% (Table 1Go). This was not significantly different from the inhibitory effect of CCN3 itself. By comparison, equimolar IGFBP-3 inhibited IGF-I and IGF-II induction by 62 and 85%, respectively, and higher concentrations showed further dose-dependent inhibition.


Figure 5
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  		FIG. 5. Inhibition of IGF-induced IGFRI phosphorylation by IGFBP-3, CCN3, and CCN3-IGFBP-3. NIH-3T3-IGFRI cells were treated for 5 min with either 2.5 ng/ml IGF-I (A) or 10 ng/ml IGF-II (B) preincubated with IGFBP-3, CCN3, or CCN3-IGFBP-3 at the indicated molar ratios. As controls, cells were either untreated (–) or treated (+) with the IGF alone. Cell lysates were separated by SDS-PAGE and immunoblotted for phosphorylated IGFRI ß-subunit (indicated by arrowhead) with antiphosphotyrosine antibody. Representative blots are shown.

 

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  		TABLE 1. Inhibition of IGF-stimulated IGFRI phosphorylation by IGFBP-3, CCN3, and CCN3-IGFBP-3

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The IGFBP family was initially identified as circulating carrier proteins for the IGFs and modulators of endocrine IGF action. All six members of the family have high affinities (affinity constant > 109 liters/mol) for both IGF-I and IGF-II. It was then recognized that IGFBPs are also present in the cellular environment and can modulate IGF activity at the tissue level. More recently it was determined that some IGFBPs, especially IGFBP-3 and IGFBP-5, have intrinsic biological activity that is independent of IGF binding or IGFRI activation (36).

Apart from the functional similarity among the members of the IGFBP family, these proteins also share primary structural similarity. The most striking similarity is the 12 and six cysteine residues in the amino- and carboxyl-domains, respectively, that are conserved in five of the six IGFBPs. IGFBP-6 has 10 of the 12 cysteine residues in the amino-terminal domain. Among the IGFBPs, there is 31–40% similarity in the amino-terminal domains and 27–35% similarity in the carboxyl-terminal domains. The central region of each IGFBP is unique.

It was proposed a few years ago that the primary structure of the IGFBP amino terminus is shared by several other proteins including Mac25 and members of the CCN family of growth factors (CTGF, Cyr61, and Nov), and it was suggested that these IGFBP-related proteins and the IGFBPs constitute an IGFBP superfamily (11). It was subsequently proposed that along with the Twisted gastrulation family of proteins, the IGFBP and CCN proteins form three separate families that together constitute a superfamily of cysteine-rich secreted factors based on a similar overall structure of a conserved amino-terminal cysteine-rich domain, a nonconserved spacer domain, and a conserved carboxyl-terminal cysteine-rich domain (37). Based on the modularity of the protein domain structures present in IGFBP and CCN proteins and the observation that the conserved amino-terminal domain is encoded by a single exon in their gene structures, it was suggested that the IGFBP and CCN proteins may share a common ancestral gene for the amino-terminal domain (11, 37, 38). The evolutionary sequence of events that led to the divergence of the remaining DNA sequence coding for the central and carboxyl-terminal domains of these two families of proteins remains to be elucidated.

Amino acid sequence conservation may indicate structural and/or functional significance. The so-called IGFBP-related proteins demonstrate low-affinity binding for IGFs, and it was proposed that this may be due to the absence of the conserved carboxyl-terminal cysteine-rich domain that is present in the high-affinity IGF binders, IGFBPs. Our previous studies with IGFBP-3 have shown that both domains are required for high-affinity IGF binding and indeed that isolated N- and C-domains functionally cooperate to form a high-affinity binding site (10). We tested this hypothesis by constructing a chimera of the amino-terminal domain of a low-affinity IGF binder (CCN3) with the linker and carboxyl-terminal domains of a high-affinity IGF binder (IGFBP-3). We showed that although the chimera was not different from CCN3 itself in its IGF binding ability, it appeared slightly more effective than CCN3 (although not significantly) in inhibiting IGFRI phosphorylation induced by IGF-I and IGF-II. However, in dose-response experiments, both were much less than one tenth as effective than IGFBP-3. These results suggest that either the conserved cysteine residues in the amino-terminal domain of CCN3 are not sufficient to confer the conformation required for high-affinity IGF binding or that the functionally cooperative IGF binding site is absent from the amino-terminal domain of CCN3.

When added at 5- to 10-fold molar excess, the CCN3-IGFBP-3 chimera was weakly inhibitory to IGF activity, although not significantly more so than CCN3 itself. This is intriguing because the chimera showed no evidence of binding to IGF in the equilibrium binding assays. We previously reported that there are independent low-affinity IGF binding sites within the amino- and carboxylterminal domains of IGFBP-3 that can together cooperatively form the high-affinity IGF binding site (10). It is likely that the significant IGF inhibitory activity shown by the chimera in the IGF-induced IGFRI phosphorylation assay can be attributed to the presence of the low-affinity IGF binding site on the carboxyl-terminal domain of the chimera, which is absent in CCN3.

Chimeras of domains from various high-affinity IGFBPs have been reported. Chimeras of IGFBP-3 and IGFBP-2 (39) and chimeras of IGFBP-5 and IGFBP-6 (40) display IGF binding affinities that are 2- to 6-fold lower than that of their parent IGFBP, indicating that domain swapping between IGFBPs did not have a major effect on IGF binding. This is in contrast to the CCN3-IGFBP-3 chimera in which IGF binding was barely detectable. We and others have suggested that the IGF binding site on IGFBP-3 includes hydrophobic residues (77Leu, 80Leu, 81Leu) in the amino-terminal domain (3, 4, 5, 8) and neutral residues (217Gly, 223Gln) in the carboxyl-terminal domain (8), in accordance with similar studies on IGFBP-5 (2, 4, 7). These residues are not conserved in CCN3, thus suggesting that the potentially similar conformation conferred by the conserved cysteine residues in the CCN3-IGFBP-3 chimera is not sufficient for high-affinity IGF binding.

The conserved cysteine residues in IGFBPs are known to be involved in disulfide bonds and dictate the tertiary structure of these proteins (41). It is also known that the disulfide bonds in IGFBPs are intradomain, and therefore, it is likely that the homologous cysteine residues in the amino-terminal domain of CCN3 would adopt a similar tertiary structure to the amino-terminal domain of IGFBPs. It may well be that this tertiary structure acts as a scaffold for diverse IGFBP and CCN sequences that result in diverse functionality.

In conclusion, this study has shown that although the IGFBP and CCN families are structurally related, the amino-terminal domain of an IGFBP family protein cannot be substituted functionally, in terms of IGF binding, by the amino-terminal domain of a CCN family protein. This supports previous commentaries that the nomenclature of IGFBP-related proteins (which implies functional relationship to the classical IGFBPs) should not be applied to the CCN proteins (37, 38) and argues against a direct role of CCN3 in the modulation of IGF activity.


   Footnotes
 
This work was supported by Project Grant DP 0345171 (to R.C.B. and S.M.F.) from the Australian Research Council. S.M.F. is a Cancer Institute NSW Fellow. B.P. was supported by the Ministére de la Recherche et de l’Education Nationale and a grant from Ligue Nationale le Cancer (Comité du Cher).

Disclosure statement: X.Y., R.C.B., B.P., and S.M.F have nothing to declare.

First Published Online August 24, 2006

Abbreviations: CCN, CTGF, Cyr61, and Nov; CTGF, connective tissue growth factor; IGFBP, IGF binding protein; IGFRI, type I IGF receptor; Nov, nephroblastoma overexpressed.

Received December 9, 2005.

Accepted for publication August 11, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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