SUMMARY The 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, 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.

Macromolecules gain access to the cytoplasm of eukaryotic cells using one of several ways of which clathrin-dependent endocytosis is the most researched. Although the mechanism of clathrin-mediated endocytosis is well understood in general, novel adaptor proteins that play various roles in ensuring specific regulation of the mentioned process are being discovered all the time. This review provides a detailed account of the mechanism of clathrin-mediated internalization of activated G protein-coupled receptors, as well as a description of the major proteins involved in this process.

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The last decade has witnessed significant advance in the imaging of living systems using fluorescent markers. This progress has been primarily associated with the discovery of different spectral variants of fluorescent proteins. However, the fluorescent protein technology has its own limitations and, in some cases, the use of low-molecular-weight fluorophores is preferable. In this review, we describe the arsenal of synthetic fluorescent tools that are currently in researchers’ hands and span virtually the entire spectrum, from the UV to visible and, further, to the near-infrared region. An overview of recent advances in site-directed introduction of synthetic fluorophores into target cellular objects is provided. Application of these fluorescent probes to the solution of a wide range of biological problems, in particular, to the determination of local ion concentrations and pH in living systems, is discussed.

Background:The IRR is a member of the insulin receptor family that functions as a sensor of alkaline medium. Results: We have identified key motifs of IRR ectodomain that are involved in alkali sensing. Conclusion: IRR activation by alkali is a complex multipoint process. Significance: Understanding activation of IRR potentially similar to insulin receptor activation.

Kaiso is a member of the BTB/POZ zinc finger family, which is involved in cancer progression, cell cycle control, apoptosis, and WNT signaling. Depending on promoter context, it may function as either a transcriptional repressor or activator. Previous studies found that Kaiso might be SUMOylated due to heat shock, but the biological significance of Kaiso SUMOylation is unclear. Here, we find that K42 is the only amino acid within Kaiso that is modified with SUMO. Kaiso is monoSUMOylated at lysine 42 in cell lines of kidney origin under normal physiological conditions. SUMOylated Kaiso can activate transcription from exogenous methylated promoters, wherein the deSUMOylated form of the protein kept the ability to be a repressor. Rapid Kaiso deSUMOylation occurs in response to hyperosmotic stress and is reversible upon return to an isotonic environment. DeSUMOylation occurs within minutes in HEK293 cells treated with 100 mM NaCl and relaxes in 3 h even in a salt-containing medium. Genomic editing of Kaiso by conversion of K42 into R42 (K42R) in HEK293 cells that resulted in fully deSUMOylated endogenous protein led to misregulation of genes associated with ion transport, blood pressure, and the immune response. TRIM25 was significantly repressed in two K42R HEK293 clones. By a series of rescue experiments with K42R and KO HEK293 cells, we show that TRIM25 is a direct transcriptional target for Kaiso. In the absence of Kaiso, the level of TRIM25 is insensitive to hyperosmotic stress. Extending our observations to animal models, we show that in response to a high salt diet, Kaiso knockout mice are characterized by significantly higher blood pressure increases when compared to wild-type animals. Thus, we propose a novel biological role for Kaiso in the regulation of homeostasis.

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CIRL (the calcium-independent receptor of α-latrotoxin), a neuronal cell surface receptor implicated in the regulation of exocytosis, is a member of the GPS family of chimeric cell adhesion/G protein-coupled receptors. The predominant form of CIRL is a membrane-bound complex of two subunits, p120 and p85. Extracellularly oriented p120 contains hydrophilic cell adhesion domains, whereas p85 is a heptahelical membrane protein. Both subunits are encoded by the same gene and represent products of intracellular proteolytic processing of the CIRL precursor. In this study, we demonstrate that a soluble form of CIRL also exists in vitro and in vivo. It results from the further cleavage of CIRL by a second protease. The site of the second cleavage is located in the short N-terminal extracellular tail of p85, between the GPS domain and the first transmembrane segment of CIRL. Thus, the soluble form of CIRL represents a complex of p120 non-covalently bound to a 15 amino acid residue N-terminal peptide fragment of p85. We have previously shown that mutations of CIRL in the GPS domain inhibit intracellular proteolytic processing and also result in the absence of the receptors from the cell surface. Our current data suggest that although CIRL trafficking to the cell membrane is impaired by mutations in the GPS region, it is not blocked completely. However, at the cell surface, the non-cleaved mutants are preferentially targeted by the second protease that sheds the extracellular subunit. Therefore, the two-step proteolytic processing may represent a regulatory mechanism that controls cell surface expression of membrane-bound and soluble forms of CIRL.