Protease-resistant form of insulin-like growth factor-binding protein 5 is an inhibitor of insulin-like growth factor-I actions on porcine smooth muscle cells in culture.

Imai, Yumi; Busby, Walker H.; Smith, Christine E.; Clarke, Jane; Garmong, Aaron; Horwitz, Gayle D.; Rees, Catherine; Clemmons, David R.

doi:10.1172/jci119803

Cited by 84 publications

(94 citation statements)

References 38 publications

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“…Peptide sequencing of the proteolytic fragments suggested Lys 138 -Lys 139 as the primary cleavage site (see Fig. 1) and this was confirmed by the creation of a protease-resistant mutant of IGFBP-5 by mutating these two residues (Imai et al 1997). Cleavage at this site could potentially produce an N-terminal fragment that retains GAG binding sites in the central domain, including Arg 136 and Arg…”

Section: Discussionmentioning

confidence: 84%

“…This initially raises the interesting possibility that heparin binding to the C-terminal site in IGFBP-5 may itself inhibit subsequent heparin binding to the central site. In support of this, others have shown that heparin binding to the C-terminal domain of IGFBP-5 does in fact reduce the activity of an IGFBP-5-specific serine protease, which cleaves within the central GAG binding consensus sites (Nam et al 1994, Imai et al 1997). However, we have shown with our Hep-mutant that prevention of heparin binding to the 201-218 region of IGFBP-5 does not facilitate heparin binding to the central domain.…”

Section: Discussionmentioning

confidence: 85%

“…It is particularly relevant to the proteolytic cleavage of IGFBP-5, which has been the subject of many studies. Human fibroblasts and porcine smooth muscle cells have been shown to secrete a calcium-dependent serine protease that is specific for cleaving IGFBP-5 (Nam et al 1994, Imai et al 1997. Peptide sequencing of the proteolytic fragments suggested Lys 138 -Lys 139 as the primary cleavage site (see Fig.…”

Section: Discussionmentioning

confidence: 99%

“…(Hodgkinson et al 1994) are indicated by a black rectangle, as is the C-terminal heparin binding site located between residues 201 and 218 (Hodgkinson et al 1994). The primary cleavage site of the IGFBP-5-specific serine protease at Lys 138 -Lys 139 is indicated () (Imai et al 1997) (Nam et al 1994). (B) Schematic representation of the domains present in the various mutant IGFBP proteins used in this study.…”

Section: In Vitro Mutagenesis and Construction Of Igfbp Chimerasmentioning

confidence: 99%

“…Alignment of all six IGFBP protein sequences from different species demonstrates that the cysteine-rich amino (N)-and carboxyl (C)-terminal domains (encoded by exon 1 and exons 3 and 4 respectively) are highly conserved, whereas the central 'hinge' domain (encoded by exon 2) is not conserved (Kiefer et al 1991a,b, Landale et al 1995. Both the N-and C-terminal domains have been shown to be required for high-affinity IGF binding (Hwa et al 1999), whereas the central domain would appear to contain most of the cleavage sites for specific proteases (Chernausek et al 1995, Conover et al 1995, Booth et al 1996, Imai et al 1997.…”

Section: Introductionmentioning

confidence: 99%

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The carboxy-terminal domain of IGF-binding protein-5 inhibits heparin binding to a site in the central domain

Song

Shand²,

Beattie³

et al. 2001

Journal of Molecular Endocrinology

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The IGF-binding protein (IGFBP)-5 protein contains consensus heparin binding motifs in both its carboxy (C)-terminal and central domains, although only the C-terminal site has previously been shown to be functional. We have made two chimeric IGFBP proteins by switching domains between rat IGFBP-5 and -2, named BP552 and BP522 to reflect the domains present, and a truncated rat IGFBP-5 mutant (1-168), named BP550. The ability of these proteins and wild-type (wt) IGFBPs-5 and -2 to bind to either IGFs or heparin was determined using biosensor real-time analysis and heparin ligand blotting respectively. We report that the chimeric molecules have IGF binding affinities comparable to those of the native IGFBPs from which they were derived and, as expected, the binding of BP550 to IGFs was greatly compromised. More surprising was the finding that the ability of BP552 and BP550 to bind to heparin was equivalent to that of wtIGFBP-5, whereas wtIGFBP-2 and BP522 failed to bind. These results demonstrate that the active heparin binding site in BP552 and BP550 is contained within the central domain of IGFBP-5, and that this site is active only in the absence of the C-terminal domain. We subsequently mutated two basic amino acids (R136A:R137A) in the central consensus binding sites between residues 132-140. This resulted in the loss of heparin binding for BP550, confirming the importance of these two basic amino acids in the central domain heparin binding activity. In light of these findings, we suggest that C-terminally truncated fragments of IGFBP-5 generated in vivo by proteolysis could retain heparin/extracellular matrix binding properties.

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Section: Discussionmentioning

confidence: 84%

Section: Discussionmentioning

confidence: 85%

Section: Discussionmentioning

confidence: 99%

Section: In Vitro Mutagenesis and Construction Of Igfbp Chimerasmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

The carboxy-terminal domain of IGF-binding protein-5 inhibits heparin binding to a site in the central domain

Song

Shand²,

Beattie³

et al. 2001

Journal of Molecular Endocrinology

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Inhibition of IGFBP-5 binding to extracellular matrix and IGF-I–stimulated DNA synthesis by a peptide fragment of IGFBP-5

Rees

Clemmons

1998

J. Cell. Biochem.

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Insulin-like growth factor binding protein-5 (IGFBP-5) is synthesized and secreted by smooth muscle cells (SMC). IGFBP-5 synthesis is stimulated five- to sixfold by IGF-I, and IGFBP-5 has been shown to augment IGF-I-stimulated DNA synthesis in this cell type. The ability of IGFBP-5 to augment the SMC response to IGF-I is dependent upon its binding to extracellular matrix. A highly charged region of IGFBP-5 that contains amino acids in positions 201-218 has been shown to mediate binding of IGFBP-5 to human fibroblast extracellular matrix (ECM), and a synthetic peptide containing this sequence inhibits IGFBP-5 binding to fibroblast ECM. In this study we show that exposure of SMC cultures that are constitutively synthesizing IGFBP-5 to a synthetic peptide (termed peptide A) containing this sequence has no effect on its synthesis but reduces its abundance within the ECM. The addition of increasing concentrations of the peptide to SMC cultures resulted in a concentration-dependent reduction in ECM-associated IGFBP-5. In contrast, a control peptide (peptide B), which contained the region of amino acids in positions 131-141 and had a similar charge-to-mass ratio, caused a minimal decrease in ECM binding. This effect was functionally significant since the addition of 10 microg/ml of peptide A inhibited the cellular replication response to 10 ng/ml IGF-I by 51%, and peptide B had no effect. The effects of peptide A were not due to nonspecific cytotoxicity since it had no inhibitory effect on the response of these cells to human serum and was associated with only minimal inhibition of the cellular response to platelet-derived growth factor. The findings suggest that inhibiting IGFBP-5 binding to porcine SMC ECM results in reduced cellular responses to IGF-I.

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Inhibition of IGFBP‐5 binding to extracellular matrix and IGF‐I–stimulated DNA synthesis by a peptide fragment of IGFBP‐5

Rees¹,

Clemmons²

1998

J. Cell. Biochem.

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Protease-resistant form of insulin-like growth factor-binding protein 5 is an inhibitor of insulin-like growth factor-I actions on porcine smooth muscle cells in culture.

Cited by 84 publications

References 38 publications

The carboxy-terminal domain of IGF-binding protein-5 inhibits heparin binding to a site in the central domain

The carboxy-terminal domain of IGF-binding protein-5 inhibits heparin binding to a site in the central domain

Inhibition of IGFBP-5 binding to extracellular matrix and IGF-I–stimulated DNA synthesis by a peptide fragment of IGFBP-5

Inhibition of IGFBP‐5 binding to extracellular matrix and IGF‐I–stimulated DNA synthesis by a peptide fragment of IGFBP‐5

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