cDNA cloning and analysis of a flg variant fibroblast growth factor receptor from Xenopus laevis

Abstract

A cDNA library was constructed with mRNA isolated from stage 8 Xenopus embryos. -- 2. A full length cDNA clone encoding a FGFR-1/flg gene was isolated by screening this cDNA library. It is designated XFGFR-A3. -- 3. The XFGFR-A3 clone was sequenced by dideoxynucleotide chain termination method on both strands with synthetic oligonucleotides. It is 3863 bp in length and is predicted to encode an 810 aa protein. -- 4. The XFGFR-A3 clone contains two dipeptide deletions, Val⁴²³⁻ Thr⁴²⁴ and Pro⁴⁴¹Ser⁴⁴², which are different from the published XFGFR-1 sequence ( Figure 8 ) . The Pro⁴⁴¹Ser⁴⁴² deletion has been described previously, however, this is the first report of the Val⁴²³⁻Thr⁴²⁴ deletion in Xenopus. Both dipeptide deletions result in the removal of consensus phosphorylation sites for Protein Kinase C ( PKC ) which may have consequences on intracellular signal transduction and the regulation of embryonic development. -- 5. RT-PCR results showed that XFGFR-A3 was expressed at all stages of Xenopus embryonic development. -- 6. The RNase protection experiment showed that XFGFR-A3 is a minor form of the XFGFR-1 in all stages of Xenopus embryonic development. Like the wild type XFGFR-1, XFGFR-A3 is also uniformly expressed throughout Xenopus development. -- 7. The XFGFR-A3 genomic DNA sequence covering the VT deletion region was sequenced. The VT deletion is located at an exon/intron boundary and comparison with the cDNA sequence suggests that the XFGFR-A3 variant arises from the use of an alternate 5'splice donor site. -- 8. Expression vectors containing an insert covering the VT deletion region fused to the cDNA encoding GST were constructed by using XFGFR-A2 and XFGFR-A3. A PKC assay using the purified fusion proteins products showed that Thr⁴²⁴ of XFGFR-A2 can be phosphorylated by PKC in vitro.