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.

Selected publications

  1. Deyev I.E., Chachina N.A., Zhevlenev E.S., Petrenko A.G. (2017). Site-Directed Mutagenesis of the Fibronectin Domains in Insulin Receptor-Related Receptor. Int J Mol Sci 18 (11), [+]

    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.

    ID:1951
  2. Deyev I.E., Popova N.V., Serova O.V., Zhenilo S.V., Regoli M., Bertelli E., Petrenko A.G. (2017). Alkaline pH induces IRR-mediated phosphorylation of IRS-1 and actin cytoskeleton remodeling in a pancreatic beta cell line. Biochimie 138, 62–69 [+]

    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 that increases the volume of secreted juice to control its pH and bicabonate content.

    ID:1952
  3. Можаев А.А., Ерохина Т.Н., Серова О.В., Деев И.Е., Петренко А.Г. (2017). Получение и иммунохимическая характеристика моноклонального антитела к эктодомену рецептора, подобного рецептору инсулина (IRR). Биоорг. хим. 43 (6), 631–636 [+]
    Insulin receptor-related receptor (IRR) is the only known metabotropic sensor of extracellular alkaline
    medium involved in the regulation of the acid-base balance in the body. IRR is expressed in separate cell
    populations of the kidney, stomach and pancreas, that can contact with the extracellular fluids with alkaline
    pH. To study IRR structure and function we obtained a stable hybridoma cell line producing antibody to the
    extracellular portion of the receptor. The monoclonal antibody isolated from ascitic fluids showed positive
    reaction with the antigen in the ELISA test. The minimum working concentration of antibodies was 12.5 ng/ml.
    The ability of the antibodies to specifically recognize purified ectodomain IRR and the full-length receptor
    was confirmed by Western-blot, immunoprecipitation and immunocytochemistry.
    ID:1871
  4. Deyev I.E., Mitrofanova A.V., Zhevlenev E.S., Radionov N., Berchatova A.A., Popova N.V., Serova O.V., Petrenko A.G. (2013). Structural determinants of the insulin receptor-related receptor activation by alkali. J. Biol. Chem. , [+]

    IRR is a member of the insulin receptor (IR) family that does not have any known agonist of a peptide nature but can be activated by mildly alkaline media and was thus proposed to function as an extracellular pH sensor. IRR activation by alkali is defined by its N-terminal extracellular region. To reveal key structural elements involved in alkali sensing, we developed an in vitro method to quantify activity of IRR and its mutants. Replacing the IRR L1C domains (residues 1-333) or L2 domain (residues 334-462), or both with the homologous fragments of IR reduced the receptor activity to 35, 64, and 7% percent, respectively. Within L1C domains, five amino acid residues (L135, G188, R244, and vicinal H318 and K319) were identified as IRR-specific by species conservation analysis of the IR family. These residues are exposed and located in junctions between secondary structure folds. The quintuple mutation of these residues to alanine had the same negative effect as the entire L1C domains replacement, whereas none of the single mutations was as effective. Separate mutations of these five residues and of L2 produced partial negative effects that were additive. The pH dependence of cell-expressed mutants (L1C and L2 swap, L2 plus triple LGR mutation, and L2 plus quintuple LGRHK mutation) was shifted towards alkalinity and, in contrast with IRR, did not show significant positive cooperativity. Our data suggest that IRR activation is not based on a single residue deprotonation in the IRR ectodomain but rather involves synergistic conformational changes at multiple points.

    ID:876
  5. Petrenko A.G., Zozulya S.A., Deyev I.E., Eladari D. (2013). Insulin receptor-related receptor as an extracellular pH sensor involved in the regulation of acid-base balance. Biochim. Biophys. Acta 1834 (10), 2170–5 [+]

    Recent studies of insulin receptor-related receptor (IRR) revealed its unusual property to activate upon extracellular application of mildly alkaline media, pH>7.9. The activation of IRR with hydroxyl anion has typical features of ligand-receptor interaction; it is specific, dose-dependent, involves the IRR extracellular domain and is accompanied by a major conformational change. IRR is a member of the insulin receptor minifamily and has been long viewed as an orphan receptor tyrosine kinase since no peptide or protein agonist of IRR was found. In the evolution, IRR is highly conserved since its divergence from the insulin and insulin-like growth factor receptors in amphibia. The latter two cannot be activated by alkali. Another major difference between them is that unlike ubiquitously expressed insulin and insulin-like growth factor receptors, IRR is found in specific sets of cells of only some tissues, most of them being exposed to extracorporeal liquids of extreme pH. In particular, largest concentrations of IRR are in beta-intercalated cells of the kidneys. The primary physiological function of these cells is to excrete excessive alkali as bicarbonate into urine. When IRR is removed genetically, animals loose the property to excrete bicarbonate upon experimentally induced alkalosis. In this review, we will discuss the available in vitro and in vivo data that support the hypothesis of IRR role as a physiological alkali sensor that regulates acid-base balance. This article is part of a Special Issue entitled: Emerging recognition and activation mechanisms of receptor tyrosine kinases.

    ID:878
  6. Deyev I.E., Rzhevsky D.I., Berchatova A.A., Serova O.V., Popova N.V., Murashev A.N., Petrenko A.G. (2011). Deficient Response to Experimentally Induced Alkalosis in Mice with the Inactivated insrr Gene. Acta Naturae 3 (4), 114–7 [+]

    Currently, the molecular mechanisms of the acid-base equilibrium maintenance in the body remain poorly understood. The development of alkalosis under various pathological conditions poses an immediate threat to human life. Understanding the physiological mechanisms of alkalosis compensation may stimulate the development of new therapeutic approaches and new drugs for treatment. It was previously shown that the orphan insulin receptor-related receptor (IRR) is activated by mildly alkaline media. In this study, we analyzed mutant mice with targeted inactivation of theinsrr gene encoding IRR, and revealed their phenotype related to disorders of the acid-base equilibrium. Higher concentrations of bicarbonate and CO(2)were found in the blood ofinsrr knockout mice in response to metabolic alkalosis.

    ID:879
  7. Popova N.V., Deyev I.E., Petrenko A.G. (2009). Analysis of structural determinants of alkali sensor IRR positive cooperativity. Dokl. Biochem. Biophys. 450, 160–3 ID:877

Alexander Petrenko

Alkaline pH induces IRR-mediated phosphorylation of IRS-1 and actin cytoskeleton remodeling in a pancreatic beta cell line (2017-11-27)

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.

Publications

  1. Deyev I.E., Popova N.V., Serova O.V., Zhenilo S.V., Regoli M., Bertelli E., Petrenko A.G. (2017). Alkaline pH induces IRR-mediated phosphorylation of IRS-1 and actin cytoskeleton remodeling in a pancreatic beta cell line. Biochimie 138, 62–69 [+]

    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 that increases the volume of secreted juice to control its pH and bicabonate content.

    ID:1952