Site-directed mutagenesis of tryptophan residues to conserved hydrophobic residues inhibits the processing of human KB cell folate receptor.
We are interested in identifying the ligand binding site of the human folate receptor (hFR). Previous reports have suggested a role of tryptophan(W) residues in ligand binding. We used site-directed mutagenesis to change the conserved W residues in positions 86, 116, 142, 143, 156, 160, and 193 of the hFR to either leucine(L) or phenylalanine(F) to examine the role of these W residues in hFR function. Although all W to L changes except W86L produced unstable proteins, W to F changes were tolerated. Based on total folate binding and transport studies, Chinese hamster ovary (CHO) cells transfected with W86L, W116F, and W143F expressed high levels of functional hFR, equivalent to cells transfected with wt hFR. CHO cells transfected with W142F, W156F, W160F, and W193F expressed low or undetectable levels of functional hFR although mRNA was present. Of these four mutants, only W142F expressed easily detectable immunoprecipitable protein but it was not fully glycosylated. Since glycosylation may affect the ability of hFR to bind folate, we expressed W142F in Xenopus oocytes which glycosylate the mutant and wild type proteins to the same apparent extent. In oocytes, the stoichiometry of folate binding was identical between the fully processed mutant protein and the wild type hFR. These results indicate that in CHO cells three of the seven W mutations (W86L, W116F, and W143F) function normally, whereas four of the seven W mutations (W142F, W156F, W160F, and W193F) produce unstable or abnormally processed protein.(ABSTRACT TRUNCATED AT 250 WORDS)
Chung
K N
KN
Medicine Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
Paik
T H
TH
Roberts
S
S
Kim
C H
CH
Kirassova
M
M
Weinstein
J N
JN
Trepel
J B
JB
Elwood
P C
PC
D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D002460:Cell Line; D058973:Folate Receptors, GPI-Anchored; D015870:Gene Expression; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D009865:Oocytes; D011499:Protein Processing, Post-Translational; D012333:RNA, Messenger / Q000235:genetics; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D014364:Tryptophan / Q000737:chemistry; D014982:Xenopus laevis
D002352:Carrier Proteins; D058973:Folate Receptors, GPI-Anchored; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; D014364:Tryptophan