Site-Directed Mutagenesis of the Fibronectin Domains in Insulin Receptor-Related Receptor

Abstract

The orphan insulin receptor-related receptor (IRR), in contrast to its close homologs, the insulin receptor (IR) and insulin-like growth factor receptor (IGF-IR) can be activated by mildly alkaline extracellular medium. We have previously demonstrated that IRR activation is defined by its extracellular region, involves multiple domains, and shows positive cooperativity with two synergistic sites. By the analyses of point mutants and chimeras of IRR with IR in, we now address the role of the fibronectin type III (FnIII) repeats in the IRR pH-sensing. The first activation site includes the intrinsically disordered subdomain ID (646-716) within the FnIII-2 domain at the C-terminus of IRR alpha subunit together with closely located residues L135, G188, R244, H318, and K319 of L1 and C domains of the second subunit. The second site involves residue T582 of FnIII-1 domain at the top of IRR lambda-shape pyramid together with M406, V407, and D408 from L2 domain within the second subunit. A possible importance of the IRR carbohydrate moiety for its activation was also assessed. IRR is normally less glycosylated than IR and IGF-IR. Swapping both FnIII-2 and FnIII-3 IRR domains with those of IR shifted beta-subunit mass from 68 kDa for IRR to about 100 kDa due to increased glycosylation and abolished the IRR pH response. However, mutations of four asparagine residues, potential glycosylation sites in chimera IRR with swapped FnIII-2/3 domains of IR, decreased the chimera glycosylation and resulted in a partial restoration of IRR pH-sensing activity, suggesting that the extensive glycosylation of FnIII-2/3 provides steric hindrance for the alkali-induced rearrangement of the IRR ectodomain.

Keywords: alkaline medium; insulin receptor-related receptor; pH sensor; protein glycosylation; receptor tyrosine kinase activation; site directed mutagenesis; tyrosine phosphorylation.

Figure 1

The role of T582, L868, R869 (red) and region (646–716) (green) within fibronectin repeats domains. (A) Alignment of insulin receptor (IR), insulin-like growth factor receptor (IGF-IR) and insulin receptor-related receptor (IRR) sequences with indication of T582 and region (646–716). (B) Alignment of IR, IGF-IR and IRR sequences with indication of L868 and R869. (C) Domain models of the 6xHis-tagged T582-TK(N), LR-TK(N), and AED-TK(N) alanine mutants or chimera with partial tyrosine kinase domain swapping TK(N). (D) Quantitative analysis of the activation of indicated constructs by alkali in comparison with IRR-TK(N) as 100%. The Western blots with samples from in vitro autophosphorylation assays were blotted with anti-pIR/IRR antibodies and, after stripping, with anti-IR/IRR antibodies. Quantitative analysis of the activation of the constructs performed as described in Materials and Methods. Asterisks indicate p < 0.05 in comparison with the IRR-TK(N). Values are means ± SE (n ≥ 4).

Figure 2

The pH dependence of IRR chimeric mutants. (A) Domain models of the HA-tagged T582 and AED mutants and chimeras. (B) HEK 293 cells were transfected with indicated plasmid and incubated for 10 min with PBS medium adjusted to the indicated pH by 60 mM Tris-HCl buffer, followed by lysis and blotting with anti-pIR/IRR and anti-IR/IRR antibodies after stripping. Western blot phosphorylation signals were quantified and normalized according to the anti-IR/IRR signals. On each graph normalized signals were plotted versus pH of the tested solutions. On each plot, the Y-axis shows percentage of the maximum average value of activation at pH 9.4. The values of EC₅₀ were calculated by GraphPad software. Values are means ± SE (n = 4). (C) The pH dependence of AED chimera phosphorylation. Pictures represented at least four independent experiments. (D) Cell surface expression of AED chimera as described in Materials and Methods.

Figure 3

Treatment of Fn2&3-IRR with PNGase. Proteins from HEK 293 transfected cells was immunoprecipitated with anti-IR/IRR antibodies. The immunoprecipitates were treated without or with PNGase. After digestion, the reaction mixture was analyzed by Western blotting with indicated antibodies.

Figure 4

The role of glycosylation within fibronectin repeats domains. (A) Alignment of human IR isoform b and human IRR sequences with indication of mutated resides. N2l (left), N2m (middle) and N2r (right) mutations indicated by green, red, or blue colors, correspondently. Sites of proteolysis also indicated by underlying; (B) Domain models of the HA-tagged Fn2&3-IRR, N2l-IRR, N2m-IRR, and N2r-IRR alanine mutants or chimeras; (C) Western blotting of indicated constructs with anti-IR/IRR antibody (against C-end of IRR) and with anti-IRR ectodomain antibody (against alpha-subunit of IRR); (D) Quantitative analysis of the activation of indicated constructs by alkali. For each constructs basal phosphorylation at pH 7.4 indicated as 100%. Transfected cells were incubated with two set of Tris-buffered physiological saline solutions with pH from 7.4 or 9.0. Lysates of transfected cells were directly analyzed by Western blotting with anti-pIR/IRR antibodies and after stripping with anti-IR/IRR antibodies. For the quantitative analysis of Western blots we used Fusion Solo system (Vilber Lourmat, France). Asterisks indicate p < 0.05 in comparison with the basal phosphorylation at pH 7.4 of same constructs. Values are means ± SE (n ≥ 4).

Figure 5

(A) Positions of indicated T582, L868, R869, and region (646–716) within fibronectin repeats domains and their IR substitutes on a putative three-dimensional model of IRR built according to the described structure of IR ectodomain [23] with Cn3D 4.3.1 software (NCBI, Bethesda, MD, USA). Region, which were swapped in AED chimera construct, was indicated by yellow color (presented in IR ectodomain structure) and red color (unstructured part of IR alpha subunit). Blue color showed rest of unstructured part of IR alpha subunit. (B) Two-site activation model of IRR. Important residues are indicated. Domains indicated by different colors. Monomers indicated by trait.