Autophosphorylation of Orphan Receptor ERBB2 Can Be Induced by Extracellular Treatment with Mildly Alkaline Media

ErbB2 is an oncogene receptor tyrosine kinase linked to breast cancer. It is a member of the epidermal growth factor receptor (EGFR) minifamily. ErbB2 is currently viewed as an orphan receptor since, by itself, it does not bind EGF-like ligands and can be activated only when overexpressed in malignant cells or complexed with ErbB3, another member of the EGFR minifamily. Here, we report that ErbB2 can be activated by extracellular application of mildly alkaline (pH 8–9) media to ErbB2-transfected cells. We also show that the activation of the ErbB2 receptor by alkali is dose-dependent and buffer-independent. The endogenous ErbB2 receptor of A431 cell line can also undergo alkali-dependent autophosphorylation. Thus, we describe a novel ligand-independent mechanism of ErbB2 receptor activation

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

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

Insulin receptor-related receptor in the pancreas and in a β-cell line

Insulin receptor-related receptor (IRR) belongs to the family of the insulin receptor (IR) along with the IR itself and the insulin-like growth factor receptor. Whereas the ligands of the latter receptors are known, identification of an IRR ligand has eluded investigations so that IRR has been considered an orphan receptor. A recent breakthrough in the understanding of IRR functional role came from the finding that IRR can be activated by mildly alkali media in absence of any protein agonist [1]. IRR shows highly specific tissue distribution, with highest concentration in kidney intercalated cells. However, significant amounts of the receptor are also found in the stomach and in α- and β-cells of the islets of Langerhans. Recent reports indicate that the pancreatic duct system is frequently associated with islet cells. Here, we show that those islet cells that are in contact with the excretory ducts are also IRR-expressing cells. Thus, when the exocrine pancreas is in an active state of secretion duct-associated islet cell behavior is potentially influenced by an IRR-mediated alkaline-induced signalling pathway. To explore this issue, we analyzed the effects of alkaline media on the pancreatic β-cell line MIN6. Activation of endogenous IRR was detected and could be inhibited with linsitinib, a synthetic inhibitor of the IR family of receptors. IRR autophosphorylation correlated with pH-dependent linsitinib-sensitive activation of IR substrate 1 (IRS-1). In contrast to insulin stimulation, no protein kinase B (Akt/PKB) phosphorylation was detected as a result of the alkali treatment. The alkaline medium but not insulin also triggered actin cytoskeleton remodeling in MIN6 cells that was blocked by pre-incubation with linsitinib. We propose that the activation of IRR by alkali is a component of a local loop of signaling between the exocrine and endocrine parts of the pancreas

FLIM-Based Intracellular and Extracellular pH Measurements Using Genetically Encoded pH Sensor

The determination of pH in live cells and tissues is of high importance in physiology and cell biology. In this report, we outline the process of the creation of SypHerExtra, a genetically encoded fluorescent sensor that is capable of measuring extracellular media pH in a mildly alkaline range. SypHerExtra is a protein created by fusing the previously described pH sensor SypHer3s with the neurexin transmembrane domain that targets its expression to the cytoplasmic membrane. We showed that with excitation at 445 nm, the fluorescence lifetime of both SypHer3s and SypHerExtra strongly depend on pH. Using FLIM microscopy in live eukaryotic cells, we demonstrated that SypHerExtra can be successfully used to determine extracellular pH, while SypHer3s can be applied to measure intracellular pH. Thus, these two sensors are suitable for quantitative measurements using the FLIM method, to determine intracellular and extracellular pH in a range from pH 7.5 to 9.5 in different biological systems

Insulin receptor-related receptor as an extracellular alkali sensor

17sireservedThe insulin receptor-related receptor (IRR), an orphan receptor tyrosine kinase of the insulin receptor family, can be activated by alkaline media both in vitro and in vivo at pH >7.9. The alkali-sensing property of IRR is conserved in frog, mouse, and human. IRR activation is specific, dose-dependent and quickly reversible and demonstrates positive cooperativity. It also triggers receptor conformational changes and elicits intracellular signaling. The pH sensitivity of IRR is primarily defined by its L1F extracellular domains. IRR is predominantly expressed in organs that come in contact with mildly alkaline media. In particular, IRR is expressed in the cell subsets of the kidney that secrete bicarbonate into urine. Disruption of IRR in mice impairs the renal response to alkali loading attested by development of metabolic alkalosis and decreased urinary bicarbonate excretion in response to this challenge. We therefore postulate that IRR is an alkali sensor that functions in the kidney to manage metabolic bicarbonate excess.mixedDeyev, Igor E; Sohet, Fabien; Vassilenko, Konstantin P; Serova, Oxana V; Popova, Nadezhda V; Zozulya, Sergey A; Burova, Elena B; Houillier, Pascal; Rzhevsky, Dmitry I; Berchatova, Anastasija A; Murashev, Arkady N; Chugunov, Anton O; Efremov, Roman G; Nikol'Sky, Nikolai N; Bertelli, Eugenio; Eladari, Dominique; Petrenko, Alexander G.Deyev, Igor E; Sohet, Fabien; Vassilenko, Konstantin P; Serova, Oxana V; Popova, Nadezhda V; Zozulya, Sergey A; Burova, Elena B; Houillier, Pascal; Rzhevsky, Dmitry I; Berchatova, Anastasija A; Murashev, Arkady N; Chugunov, Anton O; Efremov, Roman G; Nikol'Sky, Nikolai N; Bertelli, Eugenio; Eladari, Dominique; Petrenko, Alexander G

Structural Determinants of the Insulin Receptor-related Receptor Activation by Alkali

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 medium 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 (Leu-135, Gly-188, Arg-244, and vicinal His-318 and Lys-319) 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 domain 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 toward 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