Abstract
To characterize the structural basis for the interactions between the insulin receptor (IR) and its major substrate, insulin receptor substrate-1 (IRS-1), a segment of the NH2-terminal region of IRS-1 (Pro5-Pro65) was deleted. This region contains the first four conserved boxes of a pleckstrin homology (PH) domain, located at the NH2-terminal part of IRS-1. COS-7 cells were then cotransfected with the genes coding for IR and a wild-type (WT) or a mutated form of IRS-1. IRS-1 delta PH underwent significantly reduced insulin-dependent tyrosine phosphorylation compared with WT IRS-1. The reduced in vivo tyrosine phosphorylation of IRS-1 delta PH was accompanied by reduced association between IRS-1 delta PH and its downstream effector p85 regulatory subunit of phosphatidylinositol-3 kinase. In contrast, both WT IRS-1 and IRS-1 delta PH underwent comparable insulin-dependent tyrosine phosphorylation in vitro when incubated with partially purified insulin receptor kinase. These findings suggest that the overall structure of IRS-1 is not altered by deletion of its PH domain and that the PH domain is not the main site for protein-protein interactions between the insulin receptor and IRS-1, at least in vitro. In conclusion, the PH region might facilitate in vivo binding of IRS-1 to membrane phospholipids or other cellular constituents in close proximity to the IR, whereas the actual interactions with the IR are presumably mediated through other domains of the IRS-1 molecule. This could account for the fact that partial deletion of the PH domain selectively impairs the in vivo interactions between the insulin receptor and IRS-1, whereas their in vitro interactions remain unaffected.
Highlights
To characterize the structural basis for the interactions between the insulin receptor (IR) and its major substrate, insulin receptor substrate-! (IRS-1), a segment of the NH2-terminal region of IRS-1 (Pro5-Pro65) was deleted
The present study provides evidence that deletion of four out of six pleckstrin homology (PH) subdomains of IRS-1 largely impairs its capacity to serve as an in vivo substrate for the insulin receptor kinase
The reduced phosphorylation could not be accounted for by deletion of tyrosine phosphorylation sites, because only 2 (Tyr46 and Tyr47 ) out of 21 such potential sites were deleted in IRS-1.lPH
Summary
Mater_ials-Restriction enzymes were purchased from Boeheringer Mannhmm and Promega. Radiolabeled nucleotides and [35S]methionine were from Amersham Corp. Immunoprecipitation-Anti-IRS-1 antibodies (at a 1:10 dilution) were added to 60 ILl of 50% protein A-Sepharose in 0.1 MTris buffer, pH 8.5, and were incubated for 1 hat 4 °C. The preparation of membranes, solubilization in Triton X-100, and affinity chromatography of insulin receptors on wheat germ agglutinin coupled to agarose were carried out as described previously [36]. Cytosolic extracts derived from CHO cells that overexpress either WT-IRS-1 or IRS-1"PH were added, and phosphorylation in a final volume of 450 ILl was initiated with 80 ILl of a "reaction mix" to yield the following final concentrations: 50 mM Hepes, 1 mM ATP, 10 mM magnesium acetate, 4 mM manganese acetate, and 0.05% Triton X-100.
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