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Use of Mutagenesis to Probe IGF-Binding Protein Structure/Function Relationships

Department of Medicine, University of North Carolina, Chapel Hill, North Carolina 27599-7170

Correspondence: Address all correspondence and requests for reprints to: David R. Clemmons, M.D., CB 7170, 6111 Thurston-Bowles, Division of Endocrinology, University of North Carolina, Chapel Hill, North Carolina 27599-7170. E-mail: endo{at}med.unc.edu

The IGF-binding proteins (IGFBPs) are multifunctional proteins that modulate IGF actions. To determine whether specific domains within these proteins account for specific functions, we and other laboratories have used in vitro mutagenesis. Prior experiments that used a variety of techniques had identified discrete regions within each protein that were proposed to account for specific functions. Alterations of these regions by substituting charged residues with neutral residues or hydrophobic residues with nonhydrophobic residues as well as domain swapping, i.e., substituting a domain from one specific form of IGFBP for the homologous domain in another form, has resulted in the elucidation of the functions of many of these specific sequences. Because the areas of protein sequence that are altered involve a limited number of amino acids, they generally do not alter the conformation of the entire protein; therefore, these specific substitutions can often be correlated with the functional changes that occur after mutagenesis. Mutants have been particularly useful for performing functional analyses in which the purified mutant protein is added to a biological test system. In some cases it has been possible to overexpress the mutagenized protein and determine whether the constitutively synthesized, mutant form of IGFBP has altered functional activity. These results have revealed that discrete regions of IGFBP sequence can mediate important and specific functional properties of these proteins.

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