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

Igor Deyev,Natalia Chachina,Egor Zhevlenev,Alexander Petrenko

doi:10.3390/ijms18112461

Igor Deyev, Natalia Chachina + Show 2 more

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https://doi.org/10.3390/ijms18112461

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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.

Highlights

Insulin receptor-related receptor (IRR) belongs to the insulin receptor minifamily of receptor tyrosine kinases that includes the insulin receptor (IR) and insulin-like growth factor receptor (IGF-IR). [1,2,3,4]
To analyze the role of fibronectin type-III repeats regions (FnIII-1, fibronectin type III (FnIII)-2, and FnIII-3) in detail, we have constructed plasmids with alanine mutagenesis of potentially important amino acid residues, which were identified by the multiple alignment of the fibronectin domains of IR, IGF-IR, and IRR sequences from different species (Figure 1A,B)
We searched for the residues that were evolutionarily conserved in IRR but differed significantly from those of IR or IGF-IR by polarity, size, or charge

Summary

Introduction

Insulin receptor-related receptor (IRR) belongs to the insulin receptor minifamily of receptor tyrosine kinases that includes the insulin receptor (IR) and insulin-like growth factor receptor (IGF-IR). [1,2,3,4]. Insulin receptor-related receptor (IRR) belongs to the insulin receptor minifamily of receptor tyrosine kinases that includes the insulin receptor (IR) and insulin-like growth factor receptor (IGF-IR). Due to endogenous proteolytic processing of the precursor protein, mature receptor monomers consist of the disulfide-linked hydrophilic extracellular α and membrane-spanning β-subunits, the latter bearing an intracellular catalytic phosphotyrosine kinase domain [8]. No peptide or protein has been yet found to activate IRR. In contrast with insulin binding to IR, the activation of IRR demonstrates positive cooperativity with Hill coefficient around 2.3 [14]. The triple knockout of all the members of the insulin receptor minifamily suggested the role of IRR in the reproductive system development by an unknown mechanism [16]

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