Complete mapping of divergent amino acids responsible for differential ligand binding of folate receptors alpha and beta.
The folate receptor (FR) type alpha may be distinguished from FR-beta by its higher affinity for the circulating folate coenzyme, (6S)-5-methyltetrahydrofolate (5-CH3H4folate), and its opposite stereospecificity for reduced folate coenzymes. Previous studies showed that a single leucine to alanine substitution at position 49 of the mature protein sequence is responsible for the functional divergence of FR-beta (Shen, F., Zheng, X., Wang, H., and Ratnam, M. (1997) Biochemistry 36, 6157-6163); however, the results also indicated that the minimum requirement for conversion of FR-beta to the functional equivalent of FR-alpha should include amino acid substitution(s) downstream of residue 92 in addition to mutation of L49A. To pinpoint those residues, chimeric FR-betaL49A/FR-alpha constructs including progressively shorter segments of FR-alpha downstream of position 92 as well as selected point mutants were studied. Simultaneous substitution of Leu-49, Phe-104, and Gly-166 in FR-beta with the corresponding FR-alpha residues Ala, Val, and Glu, respectively, reconstituted the ligand binding characteristics of FR-alpha. The results also exclude a role for other residues in FR-alpha in determining its functional divergence. A homology model of FR-alpha based on the three-dimensional structure of the chicken riboflavin-binding protein is used to show the position of residues 49, 104, and 166 in relation to the hydrophobic cleft corresponding to the riboflavin-binding pocket.
Maziarz
K M
KM
Department of Biochemistry and Molecular Biology, Medical College of Ohio, Toledo, Ohio 43614-5804, USA.
Monaco
H L
HL
Shen
F
F
Ratnam
M
M
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism*; D001483:Base Sequence; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D017931:DNA Primers; D058973:Folate Receptors, GPI-Anchored; D006801:Humans; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010449:Peptide Mapping; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
D000596:Amino Acids; D002352:Carrier Proteins; D017931:DNA Primers; D058973:Folate Receptors, GPI-Anchored; D008024:Ligands; D020033:Protein Isoforms; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins