Insulin-like growth factor binding protein-3 : structure and function

Insulin-like growth factor (IGF) -I and -II share structural homology with insulin. Their receptors and intracellular signaling are similar. Consequently, the biological actions of IGFs and insulin are overlapping. Insulin is secreted on demand depending on e.g. blood glucose while IGFs are continuously produced. IGFs are bound to specific IGF binding proteins (IGFBPs) with affinities higher than to the signaling receptor.

The focus of this thesis is IGFBP-3, the major carrier of IGFs in the circulation. We have studied posttranslational modification of IGFBP-3, such as proteolysis and glycosylation, and their impact on IGF binding and actions. The IGFBPs have IGF binding sites in the N- and C-terminal domains. Prior to this thesis the binding domains in IGFBP-3 had not been fully characterized. We studied amino acids in the N-terminal hydrophobic binding pocket of IGFBP-3 and their importance for IGF binding. Directed mutations of residues I56, L80 and L81 with substitution for glycine resulted in single mutations with markedly decreased IGF binding. Double L80 and L81 mutations yielded an IGFBP-3 mutant with almost complete loss of affinity for IGF-I and -II and the triple mutant did not have detectable IGF binding. The ability of the IGFBP-3 mutants to inhibit IGF-Istimulated phosphorylation of its receptor reflected the IGF binding affinity.

In states of insulin resistance, including pregnancy and diabetes, IGFBP-3 is proteolyzed. However, the identity of the proteolytic fragment was unknown prior to this work. We isolated a 30 kDa IGFBP-3 fragment from human pregnancy serum and identified, by N- and C-terminal amino acid sequence analysis and mass spectrometry, a fragment corresponding to residues 1-212 of the intact protein. The same fragment was also isolated from non-pregnancy serum in which it coexists with intact IGFBP-3. Using biosensor technology, we determined IGF binding kinetics. Compared to intact IGFBP-3, (1-212)IGFBP-3 had 11-fold lower affinity for IGF-I and 4-fold lower affinity for IGF-II entirely due to faster dissociations. Glycosylation of IGFBP-3 does not affect IGF binding.

We demonstrated that glycosylation of IGFBP-3 protects against proteolysis in pregnancy and diabetes serum in vitro. Thus, glycosylation may indirectly affect IGF binding. In vitro cleavage of non-glycosylated IGFBP-3 occurs at four different major sites, none of which is identical to the cleavage site generating the naturally occurring (1-212)IGFBP-3 fragment in pregnancy. These data suggest that proteolysis of endogenous glycosylated IGFBP-3 takes place outside the circulation.

Finally, we demonstrated that intact IGFBP-3 inhibited insulin stimulated glucose uptake in isolated skeletal muscles from mouse and rat. This effect, previously reported in vivo and in fat cells, was specific to fast twitch EDL and independent of Akt (protein kinase B) phosphorylation. This thesis has contributed to the current understanding of IGF interactions with IGFBP-3. Glycosylation of IGFBP-3 protects from serum proteolysis, suggesting that the dominant (1-212)IGFBP-3 species in pregnancy is produced by tissue proteases. The inhibitory effects of IGFBP-3 on insulin stimulated glucose uptake indicate that, in addition to reducing IGF binding, IGFBP-3 proteolysis may have other effects on insulin and IGF signaling. These changes may be essential to the pregnant woman in supporting optimal fetal growth.

List of scientific papers

I. Buckway CK, Wilson EM, Ahlsén M, Bang P, Oh Y, Rosenfeld RG (2001). "Mutation of three critical amino acids of the N-terminal domain of IGF-binding protein-3 essential for high affinity IGF binding." J Clin Endocrinol Metab 86(10): 4943-50
https://pubmed.ncbi.nlm.nih.gov/11600567

II. Ahlsén M, Carlsson-Skwirut C, Jonsson AP, Cederlund E, Bergman T, Bang P (2007). "A 30-kDa fragment of insulin-like growth factor (IGF) binding protein-3 in human pregnancy serum with strongly reduced IGF-I binding." Cell Mol Life Sci 64(14): 1870-80
https://pubmed.ncbi.nlm.nih.gov/17593323

III. Ahlsén M, Carlsson-Skwirut C, Cederlund E, Bergman T, Bang P (2007). "Glycosylation protects insulin-like growth factor binding protein-3 against proteolysis in pregnancy and diabetes serum." (Submitted)

IV. Ahlsén M, Zhang SJ, Katz A, Carlsson-Skwirut C, Bang P, Westerblad H (2007). "Insulin like growth factor binding protein-3 inhibits insulin stimulated glucose uptake in isolated fast-twitch but not slow-twitch muscle." (Submitted)