These findings show that IGFBP-5 binds to pSMC/ECM and that specific basic amino acids within the region between amino acid positions 201 and 218 mediate ECM binding. This region of IGFBP-5 contains 10 basic amino acids, and several of these have been shown to be important for its binding to fibroblast ECM and to heparan sulfate–containing proteoglycans (Arai et al., 1996b
; Parker et al., 1996
). Our previous studies had shown that residues 206, 207, 214, 217, and 218 appeared to be the most important for binding to fibroblast ECM. Therefore, these studies focused on the importance of those residues. The results show that the basic amino acids R207 and R214 appear to be very important for binding to pSMC ECM. Two mutants that each contained substitutions at position 207 and a mutant that contained a single substitution at 214 had a significant reduction in ECM binding. In contrast, mutants containing neutral substitutions at positions 201, 202, 206, 208, and 211 had no decrease in ECM binding, and a mutant with substitution for positions K217 and R218 showed a minimal reduction. Taken together, the findings suggest that R207 and R214 are the most important determinants of ECM binding and that the other basic residues are not important or contribute minimally.
Analysis of IGFBP-5 abundance in ECM prepared from transfected pSMCs that constitutively expressed the IGFBP-5 mutants confirmed the importance of the basic amino acids at positions 207 and 214. The two mutants containing alterations in these residues showed major reductions in IGFBP-5 content in the ECM, whereas a mutant containing four substitutions at positions 201, 202, 206, and 208 had only a 14% reduction. This suggests that the presence of these basic residues is important for IGFBP-5 incorporation into the ECM as it is synthesized de novo.
Helical wheel analysis of the 201–218 region of IGFBP-5 shows that the amino acids in positions 207, 211, 214, and 218 align asymmetrically on one side of the helical wheel (Cardin and Weintraub, 1989
; Pratt et al., 1992
) (Figure ). Of note is the observation that the amino acids in positions 207 and 214 are present in this cluster. In contrast, none of the basic amino acids in the four-point mutant with substitutions at positions 201, 202, 206, and 208 are within this cluster, and this mutant had a minimal alteration in ECM binding. This suggests that the helical wheel analysis of the spatial alignment of the basic residues that are required for binding may be predictive of the optimum charged amino acid alignment that mediates the binding of IGFBP-5 to ECM. Our results do not definitively prove that some residues outside this motif do not contribute to binding, although the contribution of residues such as K217 must be minimal.
Helical wheel alignment of the region of IGFBP-5 between amino acids 201 and 218. The basic residues are shown in black.
Previously we showed that when IGFBP-5 is layered onto fibroblast ECM, its affinity for IGF-I is markedly reduced, and when fibroblasts are plated on a substratum that has been enriched in IGFBP-5, their cellular growth response to IGF-I is markedly enhanced (Jones et al., 1993a
). The results of this study extend that observation in two important ways. First, using transfected cultures that are constituitively synthesizing IGFBP-5, we show that cultures expressing the mutant forms that have reduced ECM affinity have less IGFBP-5 deposited in the ECM. Second, we show that these cultures have significantly reduced DNA synthesis responses to IGF-I. In contrast, the cultures expressing the R201A/K202N/K206N/R208N mutant have a minimal reduction in the amount of IGFBP-5 in their ECM and respond to IGF-I in a manner similar to the mock-transfected cells. The cultures that were expressing native IGFBP-5 constituitively had increased IGFBP-5 in their ECM and an enhanced DNA synthesis response to IGF-I. Therefore, there appears to be a relationship between the amount of IGFBP-5 within the ECM and the cellular DNA synthesis response to IGF-I. Because mutagenesis does not result in a change in the affinity in these mutants for IGF-I (Arai et al., 1996b
), we conclude that the reduction in IGFBP-5 binding to ECM results in a reduction in ECM-associated IGF-I (Parker et al., 1996
) and that this reduction in IGF-I and IGFBP-5 content leads to an attenuation of IGF-I actions.
Resistance or sensitivity of IGFBP-5 to proteolysis is also an important parameter of pSMC responsiveness (Imai et al., 1997
). IGFBP-5 in the ECM is resistant to proteolysis and has a major reduction in its affinity for IGF-I (Jones et al., 1993a
). In contrast, intact IGFBP-5 in extracellular fluids has a high affinity for IGF-I, and, if a 4:1 molar excess of intact IGFBP-5 to IGF-I is present, it can inhibit IGF-I interaction with its receptor and cellular responsiveness to IGF-I (Imai et al., 1997
). However, IGFBP-5 is cleaved in both pSMC and fibroblast culture media (Nam et al., 1994
; Duan et al., 1996
), and the fragments that are generated bind IGF-I with very low affinity. Therefore, IGFBP-5 may function to enhance IGF-I actions if there is a high concentration of intact, low-affinity IGFBP-5 in the ECM and a minimal amount of intact, high-affinity IGFBP-5 in the extracellular fluid. This suggests that proteolysis high-affinity IGFBP-5 in the interstitial fluid is also a major determinant of pSMC responsiveness.
IGFBP-5 is unique among members of the IGFBP family for its capacity to adhere to ECM. When IGFBP-1, -2, and -4 are added exogenously to fibroblast ECM, no binding can be detected (Jones et al., 1993a
). In the case of IGFBP-2, binding is detectable if an excess of IGF-I is added simultaneously (Arai et al., 1996a
). IGFBP-3 binds to fibroblast ECM but with at least 20-fold lower affinity compared with IGFBP-5 (Jones et al., 1993a
; Imai et al., 1997
). Because IGFBP-3 contains the same amino acid sequence that is present in the 201–218 region of IGFBP-5, the presumed explanation for this difference is that this region of IGFBP-3 is not surface exposed. IGFBP-3 also has a much lower affinity for heparan sulfate–containing glycosaminoglycans compared with IGFBP-5, and this may account for some of its reduced binding to ECM (Arai et al., 1996b
). Because ECM binding of IGFBP-5 appears to be an important component of the cellular response to IGF-I, and connective tissue cells, such as fibroblasts and osteoblasts, have abundant IGFBP-5 within their ECM, this may account for part of their IGF-I responsiveness compared with cell types that do not have this property.
The specific components of pSMC ECM that bind to IGFBP-5 have not been determined. For fibroblast ECM we have shown that tenascin (Imai et al., 1997
), type IV collagen (Jones et al., 1993a
), and plasminogen activator inhibitor-1 all bind with IGFBP-5 high affinity. Undoubtedly, other heparan sulfate–containing proteoglycans that are present in fibroblast ECM will be shown to bind this protein. Two specific components of pSMC ECM (thrombospondin and osteopontin) have been preliminarily reported to bind to IGFBP-5. These proteins are abundant components of the ECM within atherosclerotic lesions (Giachelli et al., 1993
; Borstein and Sage, 1994
). Therefore, they have the potential to focally concentrate IGF-I and IGFBP-5 within the lesion ECM.
Several other growth factors have been shown to associate with ECM either by binding it directly or indirectly through binding to other ECM proteins (Gordon et al., 1987
; Yayon et al., 1991
; Gitay-Goren et al., 1992
; Nam et al., 1997
). In several cases, this association is required for growth factor action or for potentiating growth factor activity (Roberts et al., 1988
; Yayon et al., 1991
; Lopez et al., 1993
). Specific examples that meet these criteria, but are not identical to the IGF–IGFBP-5 system, include FGF association with heparin sulfate proteoglycans in ECM and TGF-β association with β-glycan, a cell surface–associated proteoglycan. In both cases, growth factor association with the proteoglycans facilitates receptor interaction (Gordon et al., 1987
; Yayon et al., 1991
). Growth factors that interact in this way with these types of extracellular molecules have been termed crinopectins (Feige and Baird, 1995
). Our data show that the IGF-I–IGFBP-5 interaction fulfills the criteria to be termed type I crinopectin interaction.
In summary, we have determined the specific amino acids in IGFBP-5 that are necessary for IGFBP-5 binding to pSMC/ECM. Synthesis of IGFBP-5 needs to remain high enough to maintain a critical level of low-affinity IGFBP-5 in the ECM to act as a reservoir for IGF-I, and a loss of ECM-associated IGFBP-5 results in reduced IGF-I response. In contrast, if a high concentration of intact IGFBP-5 is present in the media, it inhibits IGF-I response (Imai et al., 1997
). Therefore, the factors that determine ECM association and proteolysis of IGFBP-5 in interstitial fluids are important determinants of IGF-I actions.