Laboratory of Receptor Cell Biology

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Laboratory investigates functions of cell receptors. First area is adhesion G-protein coupled receptor CIRL. CIRLs are natural hybrids of two protein classes – signal receptors and cell adhesion molecules. These heptahelical receptors are of interest because of their potential to couple extracellular adhesion interactions with G protein-mediated intracellular signaling. However, the natural ligand(s) of CIRLs remained completely unknown to date.

Second direction concerns receptor tyrosine kinase – IRR (insulin receptor-related receptor). IRR is a member of the family of three structurally related receptors tyrosine kinases that includes insulin receptor (IR) and insulin-like growth factor receptor (IGF-IR). Natural agonists of the latter two are endogenous peptides: insulin and two insulin-like growth factors, IGF-I and IGF-II. The physiological role of IRR has remained enigmatic primarily because no endogenous ligands for IRR have been identified since its discovery in 1989, despite significant efforts that included the genome analysis.

All publications (show selected)

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Alexander Petrenko

Orphan Receptor Tyrosine Kinase of the Insulin Receptor Family Takes Shape: Structural study by Small-Angle X-Ray Scattering and AFM

In collaboration with Group of Molecular Physiology

Activation of the IRR can be achieved by increasing the extracellular pH value. The activation by alkaline media is specific, dose-dependent and reversible, which resembles typical features of ligand-receptor interaction. It was also revealed that the pH sensitivity of IRR is defined by its extracellular region, that is similar to other members of the IR minifamily. Since the activation of the IRR is determined by its extracellular part (ectodomain), the isolation and study of the structure of the ectodomain IRR (ectoIRR) is of particular interest for understanding the fundamental basis of the mechanism of alkaline sensitivity. This work is devoted to the determination of possible conformational rearrangements in the ectodomain of IRR induced by pH changing using small-angle X-ray scattering (SAXS). SAXS is a particularly useful tool for investigation of the non-crystallizable proteins, the structural organization of multidomain proteins and it allows for the rigid body modeling of the quaternary structure from subunits for which crystal structures or other models of the constituent domains are available.

From the data obtained from SAXS experiments it can be concluded that the protein (soluble IRR ectodomain) in solution exists as a dimer having a molecular mass close to that calculated from an amino acid sequence with the contribution of glycosylation, but we can’t find significant differences in X-ray scattering between conditions of pH 7.0 and pH 9.0.

To obtain detailed structural organization of the ectoIRR in solution, hybrid modeling was performed using CORAL software. Available high-resolution X-ray crystal structure of insulin receptor ectodomain as the closest IRR homologue was split into individual subdomains. In total, two polypeptide chains (ectoIRR dimer) were modeled in CORAL, followed by the search for optimal positions and orientation of the rigid subdomains and feasible conformation of the linkers to achieve the best fit to the experimental data. The modeling yields good fit with χ2 = 1.3 confirming that the chosen modeling scheme with two domains per chain was adequate for the data fitting.

The data obtained by atomic force microscopy also demonstrates good agreement with the results of structural analysis by SAXS. AFM experiments of the IRR ectodomain adsorbed on the surface of atomically flat mica revealed the structure similar to the one observed by SAXS, with no significant differences between conditions of pH 7.0 and pH 9.0.

Profile of Gene Expression in the Kidneys of Mice with the insrr Gene Knockout

In collaboration with Group of Molecular Physiology

The insulin receptor-related receptor (IRR) is a receptor tyrosine kinase and a close homologue of the insulin receptor and the insulin-like growth factor receptor; however, peptide and protein ligands for this receptor have not been identified. IRR is encoded by the insrr gene and is found in some cell populations of kidneys, stomach, pancreas, as well as in some sympathetic and cholinergic neurons. In the present study, a comparative analysis of the transcriptomes of kidneys from wild-type mice and IRR-knockout mice has been performed by the method of deep sequencing and by the analysis of gene expression on microarrays and in real-time PCR. A significant change (a more than 1.5 fold increase in gene expression in knockout animals) was found for the transcripts of two genes: hsd3b2 and igf2. The results suggest that the line of IRR-knockout mice can find application as an animal model in studies of the role of these genes in the kidney.

Publications

  1. Deyev IE, Shayahmetova DM, Zhenilo SV, Radionov NV, Petrenko AG (2018). Profile of Gene Expression in the Kidneys of Mice with the insrr Gene Knockout. Russ. J. Bioorganic Chem. 44 (2), 256–260

Alkaline pH induces IRR-mediated phosphorylation of IRS-1 and actin cytoskeleton remodeling in a pancreatic beta cell line

In collaboration with Group of Molecular Physiology

Secretion of mildly alkaline (pH 8.0-8.5) juice to intestines is one of the key functions of the pancreas. Recent reports indicate that the pancreatic duct system containing the alkaline juice may adjoin the endocrine cells of pancreatic islets. We have previously identified the insulin receptor-related receptor (IRR) that is expressed in islets as a sensor of mildly alkaline extracellular media.  In this study, we show that those islet cells that are in contact with the excretory ducts are also IRR-expressing cells. We further analyzed the effects of alkaline media on pancreatic beta cell line MIN6. Activation of endogenous IRR but not of the insulin receptor was detected that could be inhibited with linsitinib. The IRR autophosphorylation correlated with pH-dependent linsitinib-sensitive activation of insulin receptor substrate 1 (IRS-1), the primary adaptor in the insulin signaling pathway. However, in contrast with insulin stimulation, no protein kinase B (Akt/PKB) phosphorylation was detected as a result of alkali treatment. We observed overexpression of several early response genes (EGR2, IER2, FOSB, EGR1 and NPAS4) upon alkali treatment of MIN6 cells but those were IRR-independent. The alkaline medium but not insulin also triggered actin cytoskeleton remodeling that was blocked by pre-incubation with linsitinib. We propose that the activation of IRR by alkali might be part of a local loop of signaling between the exocrine and endocrine parts of the pancreas where alkalinization of the juice facilitate  insulin release which, in turn, might induce further duct secretion.

Site-directed mutagenesis of the fibronectin domains in insulin receptor-related receptor

In collaboration with Group of Molecular Physiology

We have previously demonstrated that IRR activation is defined by its extracellular region, involves multiple domains and show positive cooperativity with two synergistic sites. By the analyses of point mutants and chimeras of IRR with IR, we now address the role of the FnIII repeats in the IRR pH-sensing. We found that 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, 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, 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.