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.

A structural analysis has been carried out to determine which part of the intracellular domain of the insulin receptor (IR) ␤ subunit is involved in direct interaction with the receptor substrates IRS-1 and Shc. Toward this end, the juxtamembrane (JM) domain (amino acids 943-984) and the carboxyl-terminal (CT) region (amino acids 1245-1331) of IR were expressed in bacteria as (His) 6 -fusion peptides, and their interaction with IRS-1 and Shc was studied. We could demonstrate that the CT region of IR was sufficient to bind Shc, although significant, but much lower binding of Shc to the JM region could be detected as well. Furthermore, in vitro Tyr phosphorylation of the CT region potentiated its interactions with Shc 2-fold. In contrast, the JM region, but not the CT domain of the IR, was sufficient to mediate interactions between the IR and IRS-1. These interactions did not involve the pleckstrin homology (PH) region of IRS-1, since an IRS-1 mutant, in which four "blocks" of the PH domain (Pro 5 -Pro 65 ) were deleted, interacted with the JM region of IR with the same efficiency as native IRS-1. These results suggest that the IR interacts with its downstream effectors through distinct receptor regions, and that autophosphorylation of Tyr residues located at the CT domain of the IR can modulate these interactions. The insulin receptor (IR)1 is an heterotetrameric transmembrane glycoprotein composed of two extracellular ␣ subunits and two transmembrane ␤ subunits linked by disulfide bonds. The ␣ subunits contain the insulin-binding domain, while the transmembrane ␤ subunits function as a tyrosine-specific protein kinase that undergoes autophosphorylation following insulin binding (for reviews, see Refs. 1 and 2). Autophosphorylation activates the insulin receptor kinase (3) and enables it to phosphorylate endogenous proteins, including insulin receptor substrate-1 (IRS-1) (4), IRS-2 (5), and Shc (6).Several structural regions have been defined within the intracellular part of the ␤ subunit. These are the juxtamembrane (JM) region, the kinase region, and the carboxyl-terminal (CT) region (7,8 ), which resides in an LX 4 NPXYXSXSD motif (numbering of IR amino acids is according to Ullrich et al. (7)). Replacement of Tyr 960 with Phe or Ala impairs receptor signal transmission and abolishes both the metabolic and growth-promoting effects of insulin, even though autophosphorylation in other regions is normal and the kinase is fully active in vitro (9, 10). This appears to be due to an inability of the mutant receptors to mediate the phosphorylation of endogenous receptor substrates, including IRS-1. Indeed, overexpression of IRS-1 can rescue certain biological effects in cells overexpressing the mutated receptor (1). Similarly, the IGF-1 and the IL-4 receptors, which contain LX 4 NPXYXS motifs, also phosphorylate IRS-1 (11, 12). These findings strongly suggest that the JM region of IR is involved in the interactions with IRS-1.The CT region (Leu 1245 -Ser 1343) possesses only limited (44%) homology with the related IG...

Internalization of the insulin receptor (IR) is a highly regulated multi-step process whose underlying molecular basis is not fully understood. Here we undertook to study the role of extracellular matrix (ECM) proteins in the modulation of IR internalization. Employing Chinese hamster ovary cells that overexpress IR (CHO-T cells), our results indicate that IR internalization proceeds unaffected even when Tyr phosphorylation of IR substrates, such as IRS-1, is impaired (e.g. in CHO-T cells overexpressing IRS-1 whose pleckstrin-homology domain has been deleted or in CHO-T cells that overexpress the PH/PTB domain of IRS-1). In contrast, IR internalization is affected by the context of the ECM proteins to which the cells adhere. Hence, IR internalization was inhibited 40 -60% in CHO-T cells adherent onto galectin-8 (an ECM protein and an integrin ligand of the galectin family) when compared with cells adherent onto fibronectin, collagen, or laminin. Cells adherent to galectin-8 manifested a unique cytoskeletal organization, which involved formation of cortical actin and generation of F-actin microspikes that contrasted with the prominent stress-fibers formed when cells adhered to fibronectin. To better establish a role for actin filament organization in IR endocytosis, this process was assayed in CHO-T cells (adherent onto fibronectin), whose actin filaments were disrupted upon treatment with latrunculin B. Latrunculin B did not affect insulininduced Tyr phosphorylation of IR or its ability to phosphorylate its substrates; still, a 30 -50% reduction in the rate of IR internalization was observed in cells treated with latrunculin B. Treatment of cells with nocodazole, which disrupts formation of microtubules, did not affect IR internalization. These results indicate that proper actin, but not microtubular, organization is a critical requirement for IR internalization and suggest that integrin-mediated signaling pathways emitted upon cell adhesion to different extracellular matrices and the altered cytoskeletal organizations generated thereof affect the itinerary of the insulin receptor.Receptor tyrosine kinases (RTKs) rapidly internalize following ligand binding. Internalized receptors are then sorted to distinct subcellular pathways that lead either to degradation or recycling to the cell surface (1-3). Similarly, internalization of the insulin receptor is a multi-step process (4). Following surface redistribution (5, 6) the receptor-insulin complex progressively concentrates in clathrin-coated pits that represent the internalization gates (cf. Ref. 4, for review). The internalized receptor undergoes sorting, which determines whether it will be subjected to degradation in lysosomes or whether it will recycle back to the plane of the membrane (7). Stimulation of the intrinsic Tyr kinase activity of the insulin receptor (IR) 1 following insulin binding is a prerequisite for surface redistribution of receptor-insulin complexes; accordingly, mutations of IR that abolish its kinase activity or mutations that replace amin...