Interactions of signaling proteins with the insulin and insulin-like growth factor-I receptors

Abstract

The insulin and insulin-like growth factor I (IGFI) receptors are members of the receptor tyrosine kinase family, and have been implicated in regulation of cellular growth, differentiation, and metabolism. In the present studies we have examined the interactions between the signaling proteins IRS-1 and SHC and the IGFIR using the yeast two-hybrid assay of protein:protein interaction. We have shown that SHC and IRS-1 interact with tyrosine 950 in the IGFIR juxtamembrane NPEY domain in a phosphotyrosine-dependent manner. We have identified a region of homology between SHC and IRS-1 in the regions capable of mediating this interaction, termed SAIN for SHC and IRS-1 NPXYinteration domain, also called PTB for phosphotyrosine binding domain. In order to characterize the molecular nature of the SHC PTB interaction with the IGFIR, we designed a random mutagenesis protocol to identify residues necessary for mediating this interaction. We identified six SHC PTB mutants which showed a decreased interaction with the IGFIR. Five out of the six mutations were found to lie within or in proximity to the phosphotyrosine binding pocket. When injected into fibroblasts these mutants are unable to block insulin-induced uptake of BrdUrd, indicating a lack of in vivo interaction with the receptor. Finally, we examined the interaction of 14-3-3, a putative signaling protein, with the IGFIR. 14-3-3 does nor interact with the insulin receptor. We showed that 14-3-3 interacts with the activated IGFIR in the two-hybrid assay and in vitro. 14-3-3 also interacts with IRS-1. These interactions appear to be dependent on phosphoserine, since serine phosphopeptides to either the raf 14-3-3 binding site or the IGFIR C-terminus can efficiently block the interaction between 14-3-3 and both the IGFIR and IRS-1. The C-terminal region of the IGFIR which mediates the interaction with 14-3-3 has previously been shown to be necessary for IGFIR-mediated cellular transformation. 14-3-3 may be involved in this transformation pathway.