Insulin receptor substrates-5 and -6 are poor substrates for the insulin receptor

Abstract

Abstract. insulin receptor substrates (irS)-5 and -6 are two recently identified members of the IRS family. We investigated their roles as insulin receptor substrates and compared them with Src-homology-2-containing (Shc) protein, a well-established substrate. Bioluminescence resonance energy transfer (BRET) experiments showed no interaction between the receptor and IRS-5, while interaction with IRS-6 was not enhanced by insulin. By contrast, Shc showed an insulin-induced BRET response, as did a truncated form of IRS-1 (1-262). While Shc was heavily phosphorylated after stimulation of the insulin receptor, IRS-5 and -6 showed very weak phosphorylation levels. These results suggest that, although these two adaptors have previously been proposed as substrates for the insulin receptor, they are poor substrates for the insulin receptor. This calls into question their relevance to insulin signalling. IntroductionInsulin is crucial for the regulation of metabolism, growth and development. Binding of insulin to the insulin receptor (IR) leads to the activation of receptor tyrosine kinase and receptor phosphorylation, which enables the binding of docking proteins such as insulin receptor substrates (IRS)-1, -2, -3 and -4 and Src-homology-2-containing protein (Shc). This in turn leads to their phosphorylation and, thereby, intracellular signalling (1).Until recently, the IRS family included IRS-1, -2, -3 and -4 (2) and three downstream of kinases, Dok-1, -2 and -3. These seven proteins have similar amino-terminal pleckstrin homology (PH) and similar phosphotyrosine binding (PTB) and carboxyl-terminal phosphorylation domains which, when tyrosine-phosphorylated, dock Src-homology-2 (SH2) domain proteins. Despite their similar domain architecture, the Dok proteins can be distinguished from the IRS family based on sequence homology and functional interactions. Recently, two new members of the family were identified: IRS-5 and -6 (also called Dok-4 and -5, respectively). Based on PH and PTB domain sequence identity, IRS-5 and -6 have been shown to be more closely related to each other than to either the IRS or the Dok family (3,4). Dok-6, defined as a novel member of the Dok-4/5 'subclass' of the Dok family (5), and Dok-7 have also recently been identified (6).The physiological processes regulated by the Dok family are poorly understood. IRS family members and other receptor tyrosine kinase-associated adaptor molecules are generally involved in signal amplification. Dok-1, -2 and -3 function primarily as inhibitors of tyrosine kinase signalling (7-9). Dok-4 and -5, however, have been shown to be positively involved in tyrosine kinase signalling (3,10-12), although Dok-4 was suggested to be an inhibitor of tyrosine kinase signalling in epithelial cells (13,14). It has been suggested that Dok-4 and -5 are involved in insulin and related insulin-like growth factor (IGF)-I signalling, and play a role as substrates for the insulin and the IGF-I receptor (3). However, contradictory results have been obtained in other studies (13...

Differential Effects of Camel Milk on Insulin Receptor Signaling – Toward Understanding the Insulin-Like Properties of Camel Milk

“…First, we wanted to validate the applicability of BRET approach to investigate the activation of hIR as previously reported for this receptor ( 23 , 24 ) as well as EGFR ( 25 ) and various GPCRs ( 26 – 28 ). This is mostly based on agonist-promoted recruitment of cytosolic signaling proteins to the membrane receptor reflecting its activation.…”

“…To test this hypothesis, we examined the effect of camel milk on the activation of human insulin receptor (hIR) by investigating its physical association with two key signaling proteins, insulin receptor signaling proteins (IRS1) and growth factor receptor-bound protein 2 (Grb2), known to bind directly or indirectly to the receptor upon its activation/phosphorylation by insulin binding. For this, we used bioluminescence resonance energy transfer (BRET) technology as previously reported for insulin receptor ( 23 , 24 ), epidermal growth factor receptor (EGFR) ( 25 ), and various G protein-coupled receptors (GPCRs) ( 26 – 28 ). This approach allowed us to assess the activation of hIR through the monitoring of IRS1 and Grb2 binding to the protein complex involving the receptor, in real-time and live cells, before and upon activation with insulin.…”

From signal transduction to signal interpretation: An alternative model for the molecular function of insulin receptor substrates

“…Relatively little is known about the function of IRS5 and IRS6. They are only distantly related to IRS1-4 and appear to be poor substrates for the IR (Versteyhe et al, 2010). While humans only express IRS1, IRS2 and IRS4, rodents also express IRS3.…”

Insulin Receptor Signaling in Normal and Insulin-Resistant States

“…IRS-4 mRNA is present in skeletal muscle, liver, heart, brain, and kidney (Fantin et al 1999), and IRS-4 KO mice show only very minimal growth retardation and glucose intolerance (Fantin et al 2000). IRS-5 (also called DOK4) and IRS-6 (DOK5) have limited tissue expression (Cai et al 2003) and are relatively poor IR substrates (Versteyhe et al 2010).…”