Asparagine-344 is a key residue for ligand binding in keratinocyte growth factor receptor.
The membrane proximal, immunoglobulin- (Ig-) like domain 3 of KGFR shows significant sequence similarity to the Ig light chain variable (V) domain. According to our model, based on this similarity, the F-G loop in KGFR corresponds to the complementarity determining region (CDR) 3 of the Ig V domain. The F-G loop in the membrane proximal domain of the keratinocyte growth factor receptor has previously been shown to participate in determining the FGF ligand binding specificity of KGFR [Gray, T. E., Eisenstein, M., Shimon, T., Givol, D., & Yayon, A. (1995) Biochemistry 34, 10325-10333]. Here, we report the effects of additional mutations in this F-G loop. Both a single mutant KGFR Q348-->I and a double mutant KGFR Q348-->I, Q351-->H are found to have relatively mild effects on ligand binding, as was previously found for three other F-G loop mutant receptors. In contrast, a single mutation N344-->A in the F-G loop of KGFR is sufficient to abolish essentially all affinity of this receptor for its primary ligand KGF, while some affinity for aFGF is retained. Asparagine-344 is, therefore, essential for ligand binding by KGFR. We discuss the likelihood of this effect being due to global or local structural changes or to the removal of a specific interaction with the ligand, in relation to various known and model structures. Taking into account the mild effects of other mutations in the region and various other considerations, we tend to favor the idea that asparagine-344 is a key residue in determining the local conformation of the F-G loop.
Gray
T E
TE
Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel.
Eisenstein
M
M
Yayon
A
A
Givol
D
D
D000595:Amino Acid Sequence; D001216:Asparagine / Q000235:genetics* / Q000378:metabolism; D001665:Binding Sites; D015153:Blotting, Western; D002846:Chromatography, Affinity; D003432:Cross-Linking Reagents / Q000378:metabolism; D017931:DNA Primers; D005346:Fibroblast Growth Factors / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D009154:Mutation / Q000235:genetics; D011485:Protein Binding / Q000235:genetics; D011487:Protein Conformation; D051497:Receptor, Fibroblast Growth Factor, Type 2; D017468:Receptors, Fibroblast Growth Factor; D017978:Receptors, Growth Factor / Q000737:chemistry* / Q000235:genetics; D016415:Sequence Alignment; D013388:Succinimides / Q000378:metabolism
D003432:Cross-Linking Reagents; D017931:DNA Primers; D017468:Receptors, Fibroblast Growth Factor; D017978:Receptors, Growth Factor; D013388:Succinimides; D005346:Fibroblast Growth Factors; D001216:Asparagine; D051497:Receptor, Fibroblast Growth Factor, Type 2; C065104:keratinocyte growth factor receptor; C019358:disuccinimidyl suberate