Identification of a Novel Variant Form of Fibroblast Growth Factor Receptor 3 (FGFR3 IIIb) in Human Colonic Epithelium

Abstract

Although several tyrosine kinase-type growth factor receptors have been demonstrated in human colonic epithelial cells, the full spectrum of growth factor receptors has not been identified. Low stringency screening of a complementary DNA library prepared from the human colon cancerderived cell line HT-29 with a probe containing the tyrosine kinase domain of human c-src kinase led to the identification and isolation of a clone containing a receptor class tyrosine kinase. This putative receptor was found to be identical to the human fibroblast growth factor receptor 3 (FGFR3) except for a region of 150 nucleotides (50 amino acids) encoding the presumptive ligand-binding domain, where it exhibited only 32% homology with the previously described FGFR3. The variant domain corresponded precisely to the splicing junctions of the exon encoding the carboxyl terminal half of the third immunoglobulin-like domain, suggesting that two forms of FGFR3 result from splicing of alternate exons in a manner similar to that previously found for FGFR1 and FGFR2. By prior convention, the previously reported form of FGFR3 was designated IIIc due to its high degree of homology with the IIIc domain of FGFR1 (83% homology) and the IIIc domain of FGFR2 (81% homology). However, the ligand-binding domain of FGFR3 found in the HT-29 cell line was more highly divergent from all previously reported FGFR immunoglobulin-like domain IIIs than any other two members of this receptor family. Therefore, we propose to designate the newly reported form as the FGFR3 IIIb variant. Genomic polymerase chain reaction confirmed that the IIIb-containing exon occupies a position 5′ relative to the IIIc-containing exon within the FGFR3 gene. Northern blot analysis using a probe encompassing sequences unique to the FGFR3 IIIb mRNA confirmed the expression of a 4.4-kilobase transcript in two colon cancer-derived cell lines as well as normal human colonic mucosa. Using a technique combining reverse transcriptase polymerase chain reaction with restriction endonuclease digestion, cell lines, primary cells, and tissues were assessed for IIIb and IIIc transcripts; expression of the IIIb variant was associated with an epithelial lineage, while the IIIc variant was expressed predominantly in nonepithelial cells and tissues.