Murgue B, Tsunekawa S, Rosenberg I, deBeaumont M, Podolsky D K
Gastrointestinal Unit, Massachusetts General Hospital, Boston 02114.
Cancer Res. 1994 Oct 1;54(19):5206-11.
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 cancer-derived 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 from 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.
尽管在人结肠上皮细胞中已证实存在几种酪氨酸激酶型生长因子受体，但尚未确定生长因子受体的完整谱系。用人c-src激酶酪氨酸激酶结构域的探针，对从人结肠癌来源的细胞系HT-29制备的互补DNA文库进行低严格度筛选，从而鉴定并分离出一个含有受体类酪氨酸激酶的克隆。发现这个推定的受体与人成纤维细胞生长因子受体3（FGFR3）相同，只是在编码推定配体结合结构域的150个核苷酸（50个氨基酸）区域，它与先前描述的FGFR3仅表现出32%的同源性。该变异结构域恰好对应于编码第三个免疫球蛋白样结构域羧基末端一半的外显子的剪接连接处，这表明FGFR3的两种形式是由交替外显子的剪接产生的，其方式类似于先前在FGFR1和FGFR2中发现的情况。按照先前的惯例，先前报道的FGFR3形式由于与FGFR1的IIIc结构域具有高度同源性（83%同源性）以及与FGFR2的IIIc结构域具有高度同源性（81%同源性）而被指定为IIIc。然而，在HT-29细胞系中发现的FGFR3的配体结合结构域与所有先前报道的FGFR免疫球蛋白样结构域III相比，其差异程度比该受体家族的任何其他两个成员都要大。因此，我们建议将新报道的形式指定为FGFR3 IIIb变体。基因组聚合酶链反应证实，含IIIb的外显子在FGFR3基因中位于含IIIc的外显子的5'端位置。使用包含FGFR3 IIIb mRNA特有序列的探针进行Northern印迹分析，证实了在两种结肠癌来源的细胞系以及正常人结肠黏膜中存在4.4千碱基转录本的表达。使用逆转录聚合酶链反应与限制性内切酶消化相结合的技术，对细胞系、原代细胞和组织进行了IIIb和IIIc转录本的评估；IIIb变体的表达与上皮谱系相关，而IIIc变体主要在非上皮细胞和组织中表达。
