Identification of a novel splice variant of C3G which shows tissue-specific expression

Abstract

C3G is a guanine nucleotide-releasing protein that binds to the Src homology 3 (SH3) domain of the adapter protein Crk. In this study, we isolated cDNAs coding for rat C3G. Northern blot analysis of RNA from various rat tissues and cell lines showed a major transcript of about 7 kb which was present at the highest level in testis. A comparison of the amino acid sequence (derived from the cDNA sequence) of rat C3G with the human form showed 87.3% sequence identity. The principal difference was the presence of an additional 51 amino acids in the rat C3G sequence after the fifth PXXP motif. This difference may be attributable to alternative splicing of the primary transcript. This interpretation was supported by reverse transcription-polymerase chain reaction (RT-PCR) assays, which resulted in two products differing by 153 bp. The RT-PCR analysis of RNA from various rat tissues showed that the relative expression levels of the two splice forms were variable. The form of C3G with the insertion of 51 amino acids (named C3G-2) was present in rat testis at a high level and, to a lesser extent, in brain, but it was not seen (or was present at a very low level) in other rat tissues and certain rat and mouse cell lines. This expression pattern of the C3G-2 form was confirmed by Northern blotting using the insert region as a probe. The C3G-1 form, without the insertion of 51 amino acids, was present in almost all rat tissues except testis and in cell lines of rat, mouse, or human origin. Thus, in rat cells, we have identified a novel splice variant of C3G. The expression pattern indicates that the form of C3G described here is likely to serve a tissue-or cell-specific physiological function.