Characterization of two splice variants of EGFR and their effects on the growth of the razor clam

Abstract

The epidermal growth factor receptor (EGFR) plays a vital role in cell growth, proliferation, and body growth. In this study, two EGFR isoforms (Sc-EGFR-1a and Sc-EGFR-1b) were obtained from Sinonovacula constricta. Sequence analysis of the coding region of Sc-EGFRs, revealed the 2 isoforms were generated from a common gene by alternative splicing and that one form possessed an extra 81 bp that corresponded to an insertion of 27 amino acids in the C-terminus. Expression of the Sc-EGFR isoforms were detectable in the early embryo stages and increased in the middle-late stages. Both isoforms were widely expressed in tissues, but the highest levels were detected in the siphon. Although both isoforms had a typical extracellular region, transmembrane region, and intracellular region, the insertion/deletion of 27 aa in the C-terminus changed the phosphorylation sites and may have influenced downstream kinase activities. In vitro, the proliferating cell nuclear antigen indicator and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay showed that HEK293T cells transfected with Sc-EGFR-1a and Sc-EGFR-1b exhibited different proliferation patterns. Sc-EGFR-1a caused more rapid proliferation than Sc-EGFR-1b and the EGFP-N1 control. In vivo, Sc-EGFRs were successfully knocked-down for at least 15 days by injecting the clams with the dsRNA and was associated with a significant reduction in shell length. Our results reveal for the first time in mollusks the existence of alternative splicing of EGFR and provide the basis for further studies to establish the role of EGFR in growth and development of S. constricta. Keywords: Sinonovacula constricta, EGFR, Alternative splicing, Cell proliferation