Extracellular Matrix Proteins Modulate Endocytosis of the Insulin Receptor

Abstract

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...

Importance of a N-terminal aspartate in the internalization of the neuropeptide Y Y2 receptor

“…The receptor internalization can be controlled by specific proteins of the extracellular matrix (e.g. (Boura-Halfon et al, 2003) for the insulin receptor), via multiple electrostatic interactions (Kauf et al, 2001). The stimulation of the neuropeptide Y Y 2 receptor internalization by heparin that we observed in the A 34 A 35 mutant should relate to inhibition of contacts of the residues 36–39 (PEPE), possibly weakened due to the absence of residues P 34 and D 35 .…”

Thromboxane A 2 Receptor Agonists Antagonize the Proangiogenic Effects of Fibroblast Growth Factor-2

“…46 ECM components have been shown to influence the internalization of other growth factor receptors, such as the insulin receptor. 47 The present report additionally describes the integrin-mediated regulation of receptor tyrosine kinase internalization in response to ligand and that perturbation of this mechanism can alter the angiogenic properties of the agent. Indeed, FGFR1 and ␣ v ␤ 3 appear to form a complex on FGF-2 stimulation as assessed by coimmunoprecipitation experiments, which is abrogated by TP␤ costimulation (Figure 6).…”

Selective gene activation by spatial segregation of insulin receptor B signaling

“…Transfected INS1 cells were biotinylated with Sulfo‐NHS‐LC‐biotin (Pierce, Rockford, IL, USA) (10, 11) and IR‐B inter‐nalization was studied using Pronase digestion as described in (10). INS1 cells (5×10 6 cells/10 cm dish) were transfected with FLAG‐tagged insulin receptor constructs by the calcium phosphate method.…”