Functional effect of point mutations in the alpha-folate receptor gene of CABA I ovarian carcinoma cells.
The alpha-folate receptor (alpha FR) is overexpressed in 90% of nonmucinous ovarian carcinomas. In addition to the known role of alpha FR binding and mediating the internalization of folates, functional interaction of alpha FR with signaling molecules was recently shown. To identify a model to study the role of alpha FR in ovarian carcinoma, we characterized the alpha FR gene in the ovarian carcinoma cell line CABA I in comparison to a reference line, IGROV1. In CABA I cells, Northern blot analysis revealed an alpha FR transcript of the expected length and FACS analysis indicated receptor expression on the cell membrane; however, RNase protection assay revealed no specific signals. Southern blot and genomic PCR analysis suggested the presence of a rearrangement(s) involving the 5' region of the gene in CABA I cells as compared to IGROV1 cells. Cloning and sequencing of CABA I alpha FR cDNA revealed several point mutations. The partitioning of alpha FR in membrane microdomains from CABA I cells and its association with regulatory molecules was comparable to that of IGROV1 cells. By contrast, the alpha FR expressed on the CABA I cell membrane bound folic acid with lower affinity, and ectopic expression of the corresponding cDNA in CHO cells confirmed impaired folic acid binding. Thus, CABA I cells may provide a tool to delineate functional domains of the alpha FR.
Istituto Nazionale Tumori, Department of Experimental Oncology, Unit of Molecular Therapies, via Venezian 1, 20133 Milan, Italy.
F
F
Mangiarotti
S
S
Miotti
E
E
Galmozzi
M
M
Mazzi
S
S
Sforzini
S
S
Canevari
A
A
Tomassetti
D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002352:Carrier Proteins / Q000235:genetics*; D003001:Cloning, Molecular; D006224:Cricetinae; D018076:DNA, Complementary; D005260:Female; D058973:Folate Receptors, GPI-Anchored; D005492:Folic Acid / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D017354:Point Mutation; D011485:Protein Binding; D011956:Receptors, Cell Surface; D014407:Tumor Cells, Cultured
D002352:Carrier Proteins; D018076:DNA, Complementary; D058973:Folate Receptors, GPI-Anchored; D011956:Receptors, Cell Surface; D005492:Folic Acid