J. Whittaker et al., Alanine scanning mutagenesis of a type 1 insulin-like growth factor receptor ligand binding site, J BIOL CHEM, 276(47), 2001, pp. 43980-43986
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 liga
nd binding activity, mutational analysis has indicated that its N terminus
(L1 amino acids 1-150) and the C terminus of its cysteine-rich domain (amin
o acids 190-300) contain ligand binding determinants. Mutational analysis a
lso suggests that amino acids 692-702 from the C terminus of the a subunit
are critical for ligand binding. A fusion protein, formed from these fragme
nts, binds IGF-I with an affinity similar to that of the whole extracellula
r domain, suggesting that these are the minimal structural elements of the
IGF-I binding site. To further characterize the binding site, we have perfo
rmed structure directed and alanine-scanning mutagenesis of L1, the cystein
e-rich domain and amino acids 692-702. Alanine mutants of residues in these
regions were transiently expressed as secreted recombinant receptors and t
heir affinity was determined. In L1 alanine mutants of Asp(8), Asn(11), Tyr
(28), Hi(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 i
n a 23-fold decrease in affinity. In the cysteine-rich domain, mutation of
Are, 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 o
f Phe(701) produced a receptor devoid of binding activity and alanine mutat
ions 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 essentia
l for intact affinity also form a discrete epitope together with Trp(79).