The orphan insulin receptor-related receptor (IRR), in contrast to its homologs from the insulin receptor family, is activated by a mildly alkaline extracellular medium. We have previously demonstrated that IRR activation is defined by two synergistic sites located in the dimeric extracellular domain. Here, we describe artificial mutations in the IRR transmembrane domain that promote receptor activation. First, using molecular modeling based on the NMR-derived structure, we proposed amino acid substitutions that could enhance non-covalent interactions between the transmembrane segments of the IRR dimer. These mutations were subsequently tested for effects on pH sensing by IRR. We showed that double-mutant A938E-A939R was highly phosphorylated at neutral pH and still sensitive to alkaline pH. Remarkably, the double substitution of V929E-G930R resulted in strong basal phosphorylation of the receptor over the pH titration range. Through site-directed mutagenesis, we demonstrated that the transmembrane domain plays a critical role in IRR activation, allowing for targeted control of functioning of the receptor, including its pH sensitivity.

There has been a notable increase in interest in the transcriptional regulator Kaiso, which has been linked to the regulation of clonal hematopoiesis, myelodysplastic syndrome, and tumorigenesis. Nevertheless, there are no consistent data on the binding sites of Kaiso in vivo in the genome. Previous ChIP-seq analyses for Kaiso contradicted the accumulated data of Kaiso binding sites obtained in vitro. Here, we studied this discrepancy by characterizing the distribution profile of Kaiso binding sites in Caki-1 cells using Kaiso-deficient cells as a negative control, and compared its pattern on chromatin with that in lymphoblastoid cell lines. We employed Caki-1 kidney carcinoma cells and their derivative, which lacks the Kaiso gene, as a model system to identify the genomic targets of Kaiso. The principal binding motifs for Kaiso are CGCG and CTGCNAT, with 60

This review discusses the importance of homeostasis with a particular emphasis on the acid-base (AB) balance, a crucial aspect of pH regulation in living systems. Two primary organ systems correct deviations from the standard pH balance: the respiratory system via gas exchange and the kidneys via proton/bicarbonate secretion and reabsorption. Focusing on kidney functions, we describe the complexity of renal architecture and its challenges for experimental research. We address specific roles of different nephron segments (the proximal convoluted tubule, the loop of Henle and the distal convoluted tubule) in pH homeostasis, while explaining the physiological significance of ion exchange processes maintained by the kidneys, particularly the role of bicarbonate ions (HCO3−) as an essential buffer system of the body. The review will be of interest to researchers in the fields of physiology, biochemistry and molecular biology, which builds a strong foundation and critically evaluates existing studies. Our review helps identify the gaps of knowledge by thoroughly understanding the existing literature related to kidney acid-base homeostasis.

The pancreas plays a key role in the endocrine system of animals, as well as in the digestion and absorption of nutrients. The exocrine and endocrine portions of the pancreas are structurally separated from each other, however, multiple studies suggest the anatomical and functional unity between them. While previously, the interactions of these two portions received less attention, nowadays the pancreas is considered as a single organ consisting of functionally interlinked components, which coordinates endocrine and exocrine responses. The review addresses the latest data indicating the functional relationship and reciprocal influence of the endocrine and exocrine pancreatic portions. In addition, we consider the impact of SARS-CoV-2 infection on pancreatic function.

The pancreas plays a key role in the endocrine system of animals and in the digestion and absorption of nutrients. The exocrine and endocrine pancreas are structurally separate from each other, but numerous studies suggest anatomical and functional connections between these parts. Previously, less attention was paid to these interactions, but the pancreas is now viewed as a single organ consisting of functionally related components that coordinates endocrine and exocrine responses. Our review examines the latest data indicating the functional connection and mutual influence of the endocrine and exocrine parts of the pancreas. In addition, we will also look at the impact of SARS-CoV-2 infection on pancreatic function.

GPR55 is a non-canonical cannabinoid receptor, important for cancer proliferation. Depending on the ligand, it induces either cell proliferation or death. The objective of the study was to establish the mechanisms of this multidirectional signaling. Using the CRISPR-Cas9 system, the GPR55, CB1, CB2, and GPR18 receptor knockouts of the MDA-MB-231 line were obtained. After the CB2 receptor knockout, the pro-apoptotic activity of the pro-apoptotic ligand docosahexaenoyl dopamine (DHA-DA) slightly increased, while the pro-proliferative activity of the most active synthetic ligand of the GPR55 receptor (ML-184) completely disappeared. On the original cell line, the stimulatory effect of ML-184 was removed by the CB2 receptor blocker and by GPR55 receptor knockout. Thus, it can be confidently assumed that when proliferation is stimulated with the participation of the GPR55 receptor, a signal is transmitted from the CB2 receptor to the GPR55 receptor due to the formation of a heterodimer. GPR18 was additionally involved in the implementation of the pro-apoptotic effect of DHA-DA, while the CB1 receptor is not involved. In the implementation of the pro-apoptotic action of DHA-DA, the elimination of Gα13 led to a decrease in cytotoxicity. The obtained data provide novel details to the mechanism of the pro-proliferative action of GPR55.

The maintenance of plasma pH is critical for life in all organisms. The kidney plays a critical role in acid–base regulation in vertebrates by controlling the plasma concentration of bicarbonate. The receptor tyrosine kinase IRR (insulin receptor-related receptor) is expressed in renal β-intercalated cells and is involved in alkali sensing due to its ability to autophosphorylate under alkalization of extracellular medium (pH > 7.9). In mice with a knockout of the insrr gene, which encodes for IRR, urinary bicarbonate secretion in response to alkali loading is impaired. The specific regulatory mechanisms in the kidney that are under the control of IRR remain unknown. To address this issue, we analyzed and compared the kidney transcriptomes of wild-type and insrr knockout mice under basal or bicarbonate-loaded conditions. Transcriptomic analyses revealed a differential regulation of a number of genes in the kidney. Using TaqMan real-time PCR, we confirmed different expressions of the slc26a4, rps7, slc5a2, aqp6, plcd1, gapdh, rny3, kcnk5, slc6a6 and atp6v1g3 genes in IRR knockout mice. Also, we found that the expression of the kcnk5 gene is increased in wild-type mice after bicarbonate loading but not in knockout mice. Gene set enrichment analysis between the IRR knockout and wild-type samples identified that insrr knockout causes alterations in expression of genes related mostly to the ATP metabolic and electron transport chain processes.

Molecular engineering is an upstream engineering method for creating functional materials and devices using molecules, and atoms as building blocks. In 2000, Japan became the first country to genetically modify silkworms (Bombyx mori, hereinafter referred to as B. mori). The subsequent research in the development of new materials expanded the possibilities of using sericulture products, characterizing this situation as a "sericulture revolution". In the Russian Federation, molecular engineering in life sciences is aimed at solving the tasks of developing world-class technological platforms to create tools to produce new molecules (biopolymers, proteins, enzymes), bioproducts, cells and organisms. The main "bottom-up" approaches applied in the upstream process (USP) stage of silk production to improve the production and economic performance and quality characteristics of raw materials have been studied. The variety of improvement methods includes: the possibility of using artificial nutrient medium, molecular engineering based on transient expression techniques or stable germline transformation, genetic selection methods, breeding management, etc. The advantages of the mulberry silkworm (B. mori) such as low breeding costs, significantly higher production yield compared to other protein expression systems, favor its use as an effective producer of recombinant proteins, antimicrobial peptides and biologically active substances. USP stage biotechnologies enable the production of new raw materials for downstream processing (DSP) to produce a wide range of products that contribute to improving the quality of human life. A set of biotechnological solutions forms the modern basis of the mulberry silkworm bioindustrial platform.

Human InsR, IGF1R, and IRR receptor tyrosine kinases (RTK) of the insulin receptor subfamily play an important role in signaling pathways for a wide range of physiological processes and are directly associated with many pathologies, including neurodegenerative diseases. The disulfide-linked dimeric structure of these receptors is unique among RTKs. Sharing high sequence and structure homology, the receptors differ dramatically in their localization, expression, and functions. In this work, using high-resolution NMR spectroscopy supported by atomistic computer modeling, conformational variability of the transmembrane domains and their interactions with surrounding lipids were found to differ significantly between representatives of the subfamily. Therefore, we suggest that the heterogeneous and highly dynamic membrane environment should be taken into account in the observed diversity of the structural/dynamic organization and mechanisms of activation of InsR, IGF1R, and IRR receptors. This membrane-mediated control of receptor signaling offers an attractive prospect for the development of new targeted therapies for diseases associated with dysfunction of insulin subfamily receptors.

Using TrkA or TrkB receptor gene knockout HT-22 cells, the selectivity of the interaction of the low-molecular-weight dipeptide BDNF mimetic GSB-106 (hexamethylenediamide bis(N-monosuccinyl-L-seryl-L-lysine)) with TrkB receptors was shown.

The orphan insulin receptor-related receptor (IRR) encoded by insrr gene is the third member of the insulin receptor family, also including the insulin receptor (IR) and the insulin-like growth factor receptor (IGF-1R). IRR is the extracellular alkaline medium sensor. In mice, insrr is expressed only in small populations of cells in specific tissues, which contain extracorporeal liquids of extreme pH. In particular, IRR regulates the metabolic bicarbonate excess in the kidney. In contrast, the role of IRR during Xenopus laevis embryogenesis is unknown, although insrr is highly expressed in frog embryos. Here, we examined the insrr function during the Xenopus laevis early development by the morpholino-induced knockdown. We demonstrated that insrr downregulation leads to development retardation, which can be restored by the incubation of embryos in an alkaline medium. Using bulk RNA-seq of embryos at the middle neurula stage, we showed that insrr downregulation elicited a general shift of expression towards genes specifically expressed before and at the onset of gastrulation. At the same time, alkali treatment partially restored the expression of the neurula-specific genes. Thus, our results demonstrate the critical role of insrr in the regulation of the early development rate in Xenopus laevis.

The most important property of a living organism is the maintenance of optimal acid–base balance and the ionic composition of the internal environment. The kidneys are one of the main pH-regulating organs in the body. Receptor tyrosine kinase IRR (an insulin receptor-related receptor) is an alkaline pH-sensor. In mice (Mus Musculus) with a knockout of the insrr gene encoding the IRR receptor, bicarbonate secretion is impaired under the conditions of alkaline loading, which indicates the role of the receptor tyrosine kinase IRR in the regulation of acid–base balance in the body. In order to search for proteins functionally associated with the receptor tyrosine kinase IRR, we performed a large-scale sequencing of the mouse kidney transcriptome of wild type and insrr knockout mice kept under normal conditions and under alkaline conditions. As a result, we found a decrease in the gapdh gene expression in the kidneys of insrr knockout mice compared to wild type mice. RNA sequencing data were confirmed by TaqMan real-time PCR and Western blotting. Using the TaqMan real-time PCR method, we revealed a decrease in the level of gapdh expression not only in the kidneys, but also in the liver and brain of insrr knockout mice. Thus, the changes in the gapdh gene expression in the kidneys of insrr knockout mice may indicate a functional relationship between genes and a possible role of GAPDH in previously undescribed molecular mechanisms of regulation of acid–base balance in the body.

— Neurexins are a family of synaptic adhesion proteins that play a key role in synapse formation and maintenance. Neurexins undergo extensive alternative splicing at six sites (SS1–SS6) resulting in expression of multiplicity of different isoforms. Alternative splicing regulates the functional activity of neurexins in different types of tissues and cells and presumably plays a key role in determining the specificity of the interaction of various neurons. In this study, we have investigated the pattern of tissue expression of neurexin-1α mRNA isoforms including an insert in the recently discovered splice site SS6 using TaqMan Real-Time PCR in different organs of Wistar rats. The isoform containing the insert in the SS6 site was found only in neural tissues suggesting its potential functional importance. Position of the SS6 insert in the hinge region between the LNS5 and LNS6 domains increases variability of possible conformations of the molecule which may represent an additional mechanism for regulating functional activity of the neurexin-1α in the brain.

Maintaining an optimal acid-base balance of the organism has a pivotal role in the regulation of metabolism. It is provided by the functioning of endogenous pH-sensors, the molecules with abilities to change their activity with the changes in the pH of the medium. Receptor tyrosine kinase IRR (insulin receptor-related receptor) is an alkaline pH sensor that is activated when the pH of the extracellular medium rises above 7.9. The expression of IRR is specific; the receptor is found in some organs, in certain types of cells. It has been established that the IRR receptor is involved in the excretion of bicarbonate by kidneys. The mechanism of action and function of the IRR receptor as an alkaline pH sensor in other organs is not understood yet. To reveal the role of the IRR receptor in the embryogenesis, we carried out experiments to evaluate the development of preimplantation embryos of wild-type and insrr knockout mice using the MEA (Mouse Embryo Assay) test. The development was assessed using the blastocyst yield index, the percentage of blastocyst formation from the total number of obtained zygotes. The yield of blastocysts in knockout animals was lower than in wild-type animals; it was 6.7% of the total number of extracted cells for knockout animals and 43.8% for wild-type animals. The number of obtained zygotes in case of wild-type and insrr knockout mice also differed. The average number of extracted zygotes from one female was 18.9 zygotes for wild-type mice and 11.8 zygotes for insrr knockout mice. Our results reveal a possible role for the receptor tyrosine kinase IRR in the development of preimplantation embryos.

The insulin receptor (IR), insulin-like growth factor 1 receptor (IGF-1R), and insulin receptor-related receptor (IRR) form a mini family of predimerized receptor-like tyrosine kinases. IR and IGF-1R bind to their peptide agonists triggering metabolic and cell growth responses. In contrast, IRR, despite sharing with them a strong sequence homology, has no peptide-like agonist but can be activated by mildly alkaline media. The spatial structure and activation mechanisms of IRR have not been established yet. The present work represents the first account of a structural analysis of a predimerized receptor-like tyrosine kinase by high-resolution atomic force microscopy in their basal and activated forms. Our data suggest that in neutral media, inactive IRR has two conformations, where one is symmetrical and highly similar to the inactive Lambda/U-shape of IR and IGF-1R ectodomains, whereas the second is drop-like and asymmetrical resembling the IRR ectodomain in solution. We did not observe complexes of IRR intracellular catalytic domains of the inactive receptor forms. At pH 9.0, we detected two presumably active IRR conformations, G-shaped and T-shaped. Both of conformations demonstrated formation of the complex of their intracellular catalytic domains responsible for autophosphorylation. The existence of two active IRR forms correlates well with the previously described positive cooperativity of the IRR activation. In conclusion, our data provide structural insights into the molecular mechanisms of alkali-induced IRR activation under mild native conditions that could be valuable for interpretation of results of IR and IGF-IR structural studies.

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.

Background: The insulin receptor-related receptor (IRR) was originally discovered due to its high homology to the other family members (insulin receptor and insulin-like growth factor 1 receptor). We determined that IRR can be activated by mildly alkaline extracellular media and has typical features of the ligand-receptor interaction, including its specificity and dose-dependence. Since pH-sensitive properties of IRR are determined by its ectodomain; therefore, we chose as an option to study the soluble extracellular domain IRR. Methods: The investigation carried out in Titan Krios 60-300 TEM/STEM (FEI, USA) CryoEM, equipped with direct electron detector Falcon II (FEI, USA) and Cs image corrector (CEOS, Germany), at an accelerating voltage of 300 kV. Data processing and 3D reconstruction were carried out using computing resources of the Federal Collective Usage Center Complex for Simulation and Data Processing for Mega-Science Facilities at NRC “Kurchatov Institute.” Results: The obtained 2D classifications of particles of the ectodomain IRR at a neutral pH form several 3D models. This indicates that the ectodomain has several possible conformations, which is consistent with our previously obtained data using SEC-SAXS and AFM. In the future, additional careful data processing is required, as well as studies of the IRR ectodomain in mildly alkaline pH. Conclusion: In this study, we presented the structural characteristics of the IRR ectodomain obtained by CryoEM. These results are an important step towards understanding the mechanism of functioning of the IRR.