Alanine Scanning Mutagenesis of a Type 1 Insulin-like Growth Factor Receptor Ligand Binding Site *

The high resolution crystal structure of an N-terminal fragment of the IGF-I receptor, has been reported. While this fragment is itself devoid of ligand binding activity, mutational analysis has indicated that its N terminus (L1, amino acids 1–150) and the C terminus of its cysteine-rich domain (amino acids 190–300) contain ligand binding determinants. Mutational analysis also suggests that amino acids 692–702 from the C terminus of the α subunit are critical for ligand binding. A fusion protein, formed from these fragments, binds IGF-I with an affinity similar to that of the whole extracellular domain, suggesting that these are the minimal structural elements of the IGF-I binding site. To further characterize the binding site, we have performed structure directed and alanine-scanning mutagenesis of L1, the cysteine-rich domain and amino acids 692–702. Alanine mutants of residues in these regions were transiently expressed as secreted recombinant receptors and their affinity was determined. In L1 alanine mutants of Asp 8, Asn 11, Tyr 28, His 30, Leu 33, Leu 56, Phe 58, Arg 59, and Trp 79 produced a 2- to 10-fold decrease in affinity and alanine mutation of Phe 90 resulted in a 23-fold decrease in affinity. In the cysteine-rich domain, mutation of Arg 240, Phe 241, Glu 242, and Phe 251 produced a 2- to 10-fold decrease in affinity. In the region between amino acids 692 and 702, alanine mutation of Phe 701 produced a receptor devoid of binding activity and alanine mutations of Phe 693, Glu 693, Asn 694, Leu 696, His 697, Asn 698, and Ile 700 exhibited decreases in affinity ranging from 10- to 30-fold. With the exception of Trp 79, the disruptive mutants in L1 form a discrete epitope on the surface of the receptor. Those in the cysteine-rich domain essential for intact affinity also form a discrete epitope together with Trp 79.