A NOVEL INSULIN-RECEPTOR SUBSTRATE PROTEIN, XIRS-U, POTENTIATES INSULIN SIGNALING - FUNCTIONAL IMPORTANCE OF ITS PLECKSTRIN HOMOLOGY DOMAIN

A novel Xenopus insulin receptor substrate cDNA was isolated by hybrid ization screening using the rat insulin receptor substrate-1 (IRS-1) c DNA as a probe. The xIRS-u cDNA encodes an open reading frame of 1003 amino acids including a putative amino-terminal pleckstrin homology (P H) domain and phosphotyrosine-binding (PTB) domain. The carboxy termin us of xIRS-u contains several potential Src homology 2 (SH2)-binding s ites, five of which are in the context of YM/LXM (presumptive binding sites for phosphatidylinositol 3-kinase). It also contains a putative binding site for Grb2 (YINID). Pair-wise amino acid sequence compariso ns with the previously identified xIRS-1 and the four members of the m ammalian IRS family (1 through 4) indicated that xIRS-u has similar ov erall sequence homology (33-45% identity) to all mammalian IRS protein s. In contrast, the previously Isolated xIRS-1 is particularly similar (67% identical) to IRS-1 and considerably less similar (31-46%) to th e other IRS family members (2 through 4). xIRS-u is also distinct from xIRS-1, having an overall sequence identity of 47%. These sequence an alyses suggest that xIRS-u is a novel member of the IRS family rather than a Xenopus homolog of an existing member. Microinjection of mRNA e ncoding a Myc-tagged xIRS-u into Xenopus oocytes resulted in the expre ssion of a 120-kDa protein (including 5 copies of the 13-amino acid My c tag). The injection of xIRS-u mRNA accelerated insulin-induced MAP k inase activation with a concomitant acceleration of insulin-induced oo cyte maturation. An amino-terminal deletion of the PH domain (xIRS-u D elta PH) significantly reduced the ability of xIRS-u to potentiate ins ulin signaling. In contrast to the bull-length protein, injection of x IRS-u (1-299), which encoded the PH and PTB domain, or xIRS-u (1-170), which encoded only the PH domain, blocked insulin signaling in Xenopu s oocytes. Finally, xIRS-u (119-299), which had a truncated PH domain and an intact PTB domain, had no effect on insulin signaling. This is the first report that the PH domain of an IRS protein can function in a dominant negative manner to inhibit insulin signaling.