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 alpha 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).
Whittaker
J
J
Receptor Biology Laboratory, Hagedorn Research Institute, Gentofte 2820, Denmark. jow@novonordisk.com
Groth
A V
AV
Mynarcik
D C
DC
Pluzek
L
L
Gadsbøll
V L
VL
Whittaker
L J
LJ
D000409:Alanine; D001665:Binding Sites; D007334:Insulin-Like Growth Factor I / Q000378:metabolism*; D008024:Ligands; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017526:Receptor, IGF Type 1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D008024:Ligands; D007334:Insulin-Like Growth Factor I; D017526:Receptor, IGF Type 1; D000409:Alanine