Determination of Alkali-Sensing Parts of the Insulin Receptor-Related Receptor Using the Bioinformatic Approach

Abstract

IRR (insulin receptor-related receptor) is a receptor tyrosine kinase belonging to the insulin receptor family, which also includes insulin receptor and IGF-IR receptor. We have previously shown that IRR is activated by extracellular fluid with pH > 7.9 and regulates excess alkali excretion in the body. We performed a bioinformatic analysis of the pH-sensitive potential of all three members of the insulin receptor family of various animal species (from frog to man) and their chimeras with swapping of different domains in the extracellular region. An analysis using the AcalPred program showed that insulin receptor family proteins are divided into two classes: one class with the optimal working pH in the acidic medium (virtually all insulin receptor and insulin-like growth factor receptor orthologs, except for the IGF-IR ortholog from Xenopus laevis) and the second class with the optimal working pH in the alkaline medium (all IRR orthologs). The program had predicted that the most noticeable effect on the pH-sensitive property of IRR would be caused by the replacement of the L1 and C domains in its extracellular region, as well as the replacement of the second and third fibronectin repeats. It had also been assumed that replacement of the L2 domain would have the least significant effect on the alkaline sensitivity of IRR. To test the in silico predictions, we obtained three constructs with swapping of the L1C domains, the third L2 domain, and all three domains L1CL2 of IRR with similar domains of the insulin-like growth factor receptor. We found that replacement of the L1C and L1CL2 domains reduces the receptor’s ability to be activated with alkaline pH, thus increasing the half-maximal effective concentration by about 100%. Replacement of the L2 domain increased the half-maximal effective concentration by 40%. Thus, our results indicate the high predictive potential of the AcalPred algorithm, not only for the pH-sensitive enzymes, but also for pH-sensitive receptors.

The dimeric ectodomain of the alkali-sensing insulin receptor–related receptor (ectoIRR) has a droplike shape

“…5a). This structural feature agrees with the mutant analysis of ectoIRR indicating that FnIII-2 and FnIII-3 domains are most critical for IRR activity (5). Swapping FnIII-2 and FnIII-3 domains or all FnIII domains of IRR with the IR led to significant glycosylation of these chimeras and completely abolished the IRR pH-sensing property.…”

“…The activation of IRR by alkaline media has typical features of the ligand-receptor interaction (2,4,5). Because IR and type I insulin-like growth factor receptor (IGF-IR) cannot be activated by either alkali or acid, the IRR activation is quite specific.…”

“…Actin cytoskeleton rearrangement was also observed in beta pancreatic MIN6 cells where IRR is expressed endogenously (4, 6, 7). Finally, analyses of several chimeras of IR and IRR revealed the key importance of the IRR extracellular region in its pH sensitivity (5,6).…”

IGF system targeted therapy: Therapeutic opportunities for ovarian cancer

“…In contrast to IGF-1R and IR, IRR does not bind IGF-I, IGF-II or insulin and to date no IRR activating ligand has been identified. Instead, IRR is proposed to be an external pH sensor as it is activated at an external cellular pH of 7.9 and higher [32]. Importantly, IRR does form heterodimer receptors with IGF-1R and IR, possibly affecting their function.…”

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

“…By the analysis of IRR/IR chimeras and IRR point mutants, it was shown that the alkali-induced activation of IRR is defined by its extracellular region and involves multiple domains with the key role of L1C domains and FnIII repeats [ 14 , 21 , 22 ]. This study was focused on the role of conserved amino acid residues within fibronectin type-III repeats regions of IRR (FnIII-1, FnIII-2, and FnIII-3).…”