title: Identification of amino acid residues that determine the differential ligand specificities of folate receptors alpha and beta.
abstract: The homologous folate receptor (FR) types alpha and beta from both human and murine sources have opposite stereospecificities for reduced folate coenzymes and different affinities for a variety of (anti)folate compounds. The present study identifies the critical amino acid sequence divergence underlying functional differences between FR-alpha and FR-beta. Chimeric constructs of the cDNAs encoding human FR-alpha and FR-beta were expressed in human 293 fibroblasts. The resulting membrane associated proteins were characterized in terms of their ability to bind [3H]folic acid and their relative affinities for the (6S) and (6R) diastereoisomers of N5-methyltetrahydrofolate. Substitution of the amino-terminal portion (residues 1-92) in the mature FR-alpha polypeptide with the corresponding segment of FR-beta resulted in folate binding characteristics similar to FR-beta. Next, a series of chimeric constructs were generated, involving substitution of progressively shorter segments within residues 1-92 in FR-alpha with the corresponding peptides of FR-beta. In this fashion, it was determined that the alanine residue at position 49 in FR-alpha was critical for its functional divergence from FR-beta, since substitution at this position with Leu (the corresponding residue in FR-beta) resulted in the folate binding characteristics of FR-beta. Reciprocal substitution in FR-beta with peptide 1-92 of FR-alpha resulted in poor expression of a [3H]folic acid binding protein. By analysis of chimeric constructs, the poor [3H]folic acid binding of the FR-alpha(1-92)/beta(93-237) chimera could be attributed to interference of a short segment from FR-alpha in the vicinity of Ala 49 (peptide 39-59) with proper folding of the chimera. Conversion of the ligand binding properties of FR-beta to those of FR-alpha required the reciprocal mutation of Leu 49 to Ala, but in addition, substitution of one or more residues downstream of amino acid 92 of FR-beta with the corresponding residues in FR-alpha was essential. The homologous murine FR types alpha and beta, which are functionally analogous to the human receptor isoforms, also contain a similar Ala vs Leu substitution. These results indicate that steric/hydrophobic effects of the side chains of Leu vs Ala at position 49 will critically modulate the affinities and stereospecificities of FR isoforms for folate compounds. Furthermore, additional amino acid sequence divergence at one or more positions downstream of residue 92 in FR-alpha is also an essential determinant of the unique functional characteristics of this receptor isoform.
authors.affiliation: Department of Biochemistry & Molecular Biology, Medical College of Ohio, Toledo 43699-0008, USA.
authors.forename: F
authors.initials: F
authors.lastname: Shen
authors.forename: X
authors.initials: X
authors.lastname: Zheng
authors.forename: J
authors.initials: J
authors.lastname: Wang
authors.forename: M
authors.initials: M
authors.lastname: Ratnam
mesh_terms: D000818:Animals; D002352:Carrier Proteins / Q000145:classification / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D058973:Folate Receptors, GPI-Anchored; D006801:Humans; D007930:Leucine / Q000235:genetics; D008024:Ligands; D051379:Mice; D016296:Mutagenesis; D011956:Receptors, Cell Surface / Q000145:classification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D013237:Stereoisomerism; D013329:Structure-Activity Relationship; D013763:Tetrahydrofolates / Q000737:chemistry / Q000378:metabolism*
chemical_list: D002352:Carrier Proteins; D058973:Folate Receptors, GPI-Anchored; D008024:Ligands; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; D013763:Tetrahydrofolates; D007930:Leucine; C005984:5-methyltetrahydrofolate