Molecular cloning, identification and characterization of four distinct receptor subtypes for insulin and IGF-I in Japanese flounder, Paralichthys olivaceus

Abstract

Insulin receptor (IR) and IGF-I receptor (IGF-IR) are structurally and functionally related and belong to the tyrosine kinase receptor family. In teleosti such as salmonids and turbot, occurrence of multiple IR and IGF-IR members has been reported, but the structures of a complete set of both IR and IGF-IR members in a single teleost species have not yet been characterized. In this study, we cloned and analysed four distinct cDNA clones for IR and IGF-IR members from the liver and kidney of the Japanese flounder (Paralichthys olivaceus). Deduced amino acid sequence analyses and phylogenetic analysis have revealed that two of them (fIR-1 and fIR-2) belong to IR members and the other two (fIGF-IR-1 and fIGF-IR-2) are IGF-IRs. fIR-1 and fIR-2 comprised 1369 and 1368 amino acid residues respectively, and fIGF-IR-1 and fIGF-IR-2 comprised 1412 and 1418 residues respectively. All the receptor proteins contained cysteine-rich domains in their α-subunits, and conserved each transmembrane and tyrosine kinase domains in their β-subunits. The amino acid sequences of fIRs and fIGF-IRs showed more than 90% sequence identity with turbot IR and IGF-IR respectively. When compared with their mammalian homologues, fIGF-IR-1 and fIGF-IR-2 proteins contained large insertions at their C-termini, as was observed in the corresponding region of turbot IGF-IR. Occurrence of multiple species of mRNA for each IR and IGF-IR was suggested by Northern blot analyses. A ribonuclease protection assay revealed diverse expressions of four receptor mRNAs in a wide range of tissues including heart, liver, ovary, testis, brain, gill arch, kidney, skeletal muscle, intestine, stomach, spleen and eye of the flounder.