ENGINEERING THE C-REGION OF HUMAN INSULIN-LIKE GROWTH-FACTOR-I - IMPLICATIONS FOR RECEPTOR-BINDING

Recombinant wild-type human IGF-1 and a C-region mutant in which resid ues 28-37 have been replaced by a 4-glycine bridge (4-Gly IGF-1) were secreted and purified from yeast. An IGF-1 analogue in which residues 29-41 of the C-region have been deleted (mini IGF-1) was created by si te-directed mutagenesis and also expressed. All three proteins adopted the insulin-fold as determined by circular dichroism. The significant ly raised expression levels of mini IGF-1 allowed the recording of two -dimensional NMR spectra. The affinity of 4-Gly IGF-1 for the IGF-1 re ceptor was similar to 100-fold lower than that of wild-type IGF-1 and the affinity for the insulin receptor was similar to 10-fold lower Min i IGF-1 showed no affinity for either receptor. Not only does the C-re gion of IGF-1 contribute directly to the free energy of binding to the IGF-1 receptor, but also the absence of flexibility in this region el iminates binding altogether. As postulated for the binding of insulin to its own receptor, it is proposed that binding of IGF-1 to the IGF-1 receptor also involves a conformational change in which the C-termina l B-region residues detach from the body of the molecule to expose the underlying A-region residues.