Alanine scanning mutagenesis of a type 1 insulin-like growth factor receptor ligand binding site

Citation
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
Citations number
52
Categorie Soggetti
Biochemistry & Biophysics
Journal title
JOURNAL OF BIOLOGICAL CHEMISTRY
ISSN journal
00219258 → ACNP
Volume
276
Issue
47
Year of publication
2001
Pages
43980 - 43986
Database
ISI
SICI code
0021-9258(20011123)276:47<43980:ASMOAT>2.0.ZU;2-C
Abstract
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).