[Show abstract][Hide abstract] ABSTRACT: IRR (insulin receptor-related receptor) is a receptor tyrosine kinase belonging to the insulin receptor family, which also includes insulin receptor and IGF-IR receptor. We have previously shown that IRR is activated by extracellular fluid with pH > 7.9 and regulates excess alkali excretion in the body. We performed a bioinformatic analysis of the pH-sensitive potential of all three members of the insulin receptor family of various animal species (from frog to man) and their chimeras with swapping of different domains in the extracellular region. An analysis using the AcalPred program showed that insulin receptor family proteins are divided into two classes: one class with the optimal working pH in the acidic medium (virtually all insulin receptor and insulin-like growth factor receptor orthologs, except for the IGF-IR ortholog from Xenopus laevis) and the second class with the optimal working pH in the alkaline medium (all IRR orthologs). The program had predicted that the most noticeable effect on the pH-sensitive property of IRR would be caused by the replacement of the L1 and C domains in its extracellular region, as well as the replacement of the second and third fibronectin repeats. It had also been assumed that replacement of the L2 domain would have the least significant effect on the alkaline sensitivity of IRR. To test the in silico predictions, we obtained three constructs with swapping of the L1C domains, the third L2 domain, and all three domains L1CL2 of IRR with similar domains of the insulin-like growth factor receptor. We found that replacement of the L1C and L1CL2 domains reduces the receptor's ability to be activated with alkaline pH, thus increasing the half-maximal effective concentration by about 100%. Replacement of the L2 domain increased the half-maximal effective concentration by 40%. Thus, our results indicate the high predictive potential of the AcalPred algorithm, not only for the pH-sensitive enzymes, but also for pH-sensitive receptors.
Acta Naturae 04/2015; 7(2):80-6. · 0.87 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Insulin receptor-related receptor (IRR) is a member of the insulin receptor (IR) family that works as an extracellular alkali sensor with positive cooperativity. The pH sensing property of IRR is defined by its extracellular region and involves multiple domains. We have previously demonstrated the primary role of L1C domains and identified potentially important amino acid residues within these domains. In this study, we addressed the roles of L2 and FnIII domains. Within the L2 domain, five amino acid residues (M406, V407, D408, P436 and V437) were identified as IRR-specific by performing a species conservation analysis of the IR family. Single-point mutations of these five residues to alanine produced either little or no negative effect on IRR pH-sensing activity. However, the triple mutation of M406, V407 and D408 (MVD) showed a strong negative effect, with a 4 fold decrease in IRR activity as estimated by in vitro autophosphorylation assay of solubilized receptors. The analysis of this mutant in intact cells revealed the absence of positive cooperativity. Unexpectedly, the double mutation of vicinal P436 and V437 (PV) exhibited a significant positive effect in the in vitro assay and partial positive cooperativity in the whole-cell assay. The role of FnIII domains was addressed by analyzing chimeras of IRR and IR. When the IRR FnIII domains were swapped with those of IR in different combinations, the activity was significantly reduced and positive cooperativity eliminated. However, two mutants with the targeted C-terminal part of IRR alpha subunit that lies within FnIII-2 domain and have been shown to be important for insulin binding by IR, appeared to be as active as wild-type IRR. On the basis of available data, we propose that IRR activation involves two separate centers of pH-dependent rearrangements that act synergistically to induce a major conformational change in the IRR molecule, resulting in internal kinase domains rapprochement and autophosphorylation.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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 it 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
Acta Naturae 01/2013; 5(3):62-73. · 0.87 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Acta Naturae 10/2011; 3(4):114-7. · 0.87 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: TPR-containing Rab8b-interacting protein (TRIP8b) is a brain-specific hydrophilic cytosolic protein that contains tetratricopeptide repeats (TPRs). Previous studies revealed interaction of this protein via its TPR-containing domain with Rab8b small GTPase, hyperpolarization-activated cyclic nucleotide-regulated channel (HCN) channels and G protein-coupled receptor calcium-independent receptor of α-latrotoxin. We identified clathrin as a major component of eluates from the TRIP8b affinity matrix. In the present study, by in vitro-binding analysis we demonstrate a direct interaction between clathrin and TRIP8b. The clathrin-binding site was localized in the N-terminal (non-TPR containing) part of the TRIP8b molecule that contains two short motifs involved in the clathrin binding. In transfected HEK293 cells, co-expression of HCN1 with TRIP8b resulted in translocation of the channels from the cell surface to large intracellular puncta where both TRIP8b and clathrin were concentrated. These puncta co-localized partially with an early endosome marker and strongly overlapped with lysosome staining reagent. When HCN1 was co-expressed with a clathrin-non-binding mutant of TRIP8b, clathrin did not translocate to HCN1 and TRIP8b-containing puncta, suggesting that TRIP8b interacts with HCN and clathrin independently. We found TRIP8b present in the fraction of clathrin-coated vesicles purified from brain tissues. Stripping the clathrin coat proteins from the vesicles with Tris alkaline buffer resulted in concomitant release of TRIP8b. Our data suggest complex regulatory functions of TRIP8b in neuronal endocytosis through independent interaction with membrane proteins and components of the clathrin coat.
Journal of Neurochemistry 07/2011; 118(6):988-98. DOI:10.1111/j.1471-4159.2011.07384.x · 4.24 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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 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.
[Show abstract][Hide abstract] ABSTRACT: CIRL-1 also called latrophilin 1 or CL belongs to the family of adhesion G protein-coupled receptors (GPCRs). As all members of adhesion GPSR family CIRL-1 consists of two heterologous subunits, extracellular hydrophilic p120 and heptahelical membrane protein p85. Both CIRL-1 subunits are encoded by one gene but as a result of intracellular proteolysis of precursor, mature receptor has two-subunit structure. It was also shown that a minor portion of the CIRL-1 receptor complexes dissociates, producing the soluble receptor ectodomain, and this dissociation is due to the second cleavage at the site between the site of primary proteolysis and the first transmembrane domain. Recently model of independent localization p120 and p85 on the cell surface was proposed. In this article we evaluated the amount of p120-p85 complex still presented on the cellular membrane and confirmed that on cell surface major amount of mature CIRL-1 presented as a p120-p85 subunit complex.
Biochemical and Biophysical Research Communications 10/2010; 402(4):658-62. DOI:10.1016/j.bbrc.2010.10.078 · 2.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Calcium-independent receptor of alpha-latrotoxin (CIRL-1) is an adhesion G protein-coupled receptor implicated in the regulation of exocytosis. CIRL-1 biosynthesis involves constitutive proteolytic processing that takes place in the endoplasmic reticulum, requires the receptor's GPS domain, and yields heterologous two-subunit receptor complexes. It was proposed that the GPS-directed cleavage is based on cis-autoproteolysis. In this study, we demonstrate that activators of protein kinase C - PMA and ionomycin, can inhibit the cleavage of CIRL-1 precursor in transfected cells. Both reagents also downregulate trafficking of CIRL-1 to the cell surface that results in accumulation of the uncleaved receptor precursor inside the cells. Experiments with a non-cleavable soluble mutant of CIRL-1 showed that the downregulation of the receptor trafficking is independent of its cleavage. Our data suggest that the GPS proteolysis of CIRL-1 is not a purely autocatalytic process and may involve auxiliary proteins or factors that become available in the course of CIRL-1 trafficking.
[Show abstract][Hide abstract] ABSTRACT: Monosiga brevicollis is classified into the class Choanoflagellate (collar flagellates). These unicellular eukaryotes show the closest phylogenetic relationship to the metazoans. Most studies of choanoflagellates are performed in attempt to solve the key problem of evolution—how the transition from unicellular to multicellular organisms occurred. Analysis of M. brevicollis RNA and DNA showed that choanoflagellates, in contrast to other unicellular organisms, can express proteins that are homologous to proteins of metazoans (in particular, cadherins, C-type lectins, G protein-coupled receptors, and components of tyrosine kinase signaling cascade) . In addition, it was shown that M. brevicollis genome contains immunoglobulin domains as well as α -integrin and collagen domains, which are required for cell‐cell interaction and immune system function in animals . Thus, choanoflagellates are the simplest living system for studying the primary functions of proteins of multicellular organisms that are involved in cell-cell interaction and signal transduction. In this study, we cloned cDNA and determined the complete amino acid sequence of the MB7TM2 receptor from M. brevicollis —a homologue of the CIRL receptor, an adhesion GPS-containing G protein-coupled secretin-type receptor that was discovered by us earlier. The mini-family of the G protein-coupled receptors CIRL comprises natural hybrids of proteins of two classes—signaling receptors and cell adhesion molecules. CIRL-1 is the acceptor of the presynaptic neurotoxin α -latrotoxin and is presumably involved in the regulation of neuronal exocytosis [3, 4]. Characteristic features of these G protein-coupled secretin-type receptors are a two-subunit structure, an extended extracellular N-terminal part containing cell adhesion modules, and the presence of seven transmembrane segments and a G protein-coupled receptor proteolysis site (GPS domain). In the GPS domain, the precursor molecule undergoes proteolysis to form two subunits of the receptor. Genomes of mammals, insects, and worms, contain genes for approximately 30 adhesion G protein-coupled secretin-type receptors, which contain the GPS domain . Currently, endogenous agonists of adhesion G protein-coupled receptors are unknown. In view of this, studies of the structural‐functional characteristics of the simplest homologues of these receptors are of interest since they help to clarify the fundamental properties of adhesion G protein-coupled receptors. Complete sequencing of M. brevicollis genome allowed us to discover a fragment that is homologous to the GPS domain of the CIRL receptor. To clone the fulllength cDNA of the putative ortholog CIRL, we obtained total cDNA of M. brevicollis . The M. brevicollis strain ATCC 50154 (obtained from the American Type Culture Collection) was cultured at room temperature in ATCC medium 1525 supplemented with living bacteria Enterobacter aerogenes (strain ATCC 13048) to a density of 10 6 –10 7 cells/ml. Total RNA was isolated according to Chomczynski ; polyadenylated mRNA was purified on oligo-dT-agarose (Invitrogen, United States). cDNA was obtained by reverse transcription using the Moloney murine leukemia virus reverse transcriptase (M-MuLV RT; Fermentas, Lithuania) and the oligo- dT 18 primer.
[Show abstract][Hide abstract] ABSTRACT: CIRL (the calcium-independent receptor of alpha-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 noncovalently 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 noncleaved 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.
[Show abstract][Hide abstract] ABSTRACT: A thorough analysis of proteins capable of interacting with presynaptic receptors of α-latrotoxin was carried out. The protein
components of receptor complexes were purified from rat brain membranes by the affinity chromatography on immobilized α-latrotoxin
and antibodies to the cytoplasmic moiety of the calciumindependent receptor of α-latrotoxin (CIRL) and analyzed then by mass
spectrometry. Several proteins were identified, with structural proteins, intracellular signal proteins, and proteins involved
in the endocytosis and transport of synaptic vesicles being among them.
[Show abstract][Hide abstract] ABSTRACT: Calcium-independent receptor of latrotoxin (CIRL) is an orphan heptahelical receptor implicated in regulation of exocytosis. To characterize molecular mechanisms of CIRL functioning, we searched for its intracellular partners using the yeast two-hybrid SR system with the cytoplasmic C-terminal fragment of CIRL as bait. One of the interacting proteins was identified as TRIP8b, a putative cytosolic adapter protein with multiple tetratricopeptide repeats. To understand functional significance of CIRL-TRIP8b interaction, we further isolated TRIP8b-interacting proteins by affinity chromatography of brain extracts on immobilized recombinant TRIP8b. Sixteen proteins were identified by mass spectrometry in the purified preparations. Clathrin and subunits of AP2 complex appeared to be the major TRIP8b-interacting proteins. Our data suggest a role of TRIP8b in receptor-mediated endocytosis.
[Show abstract][Hide abstract] ABSTRACT: Autoantibody-mediated tissue destruction is among the main features of organ-specific autoimmunity. This report describes "an antibody enzyme" (abzyme) contribution to the site-specific degradation of a neural antigen. We detected proteolytic activity toward myelin basic protein (MBP) in the fraction of antibodies purified from the sera of humans with multiple sclerosis (MS) and mice with induced experimental allergic encephalomyelitis. Chromatography and zymography data demonstrated that the proteolytic activity of this preparation was exclusively associated with the antibodies. No activity was found in the IgG fraction of healthy donors. The human and murine abzymes efficiently cleaved MBP but not other protein substrates tested. The sites of MBP cleavage determined by mass spectrometry were localized within immunodominant regions of MBP. The abzymes could also cleave recombinant substrates containing encephalytogenic MBP(85-101) peptide. An established MS therapeutic Copaxone appeared to be a specific abzyme inhibitor. Thus, the discovered epitope-specific antibody-mediated degradation of MBP suggests a mechanistic explanation of the slow development of neurodegeneration associated with MS.
Proceedings of the National Academy of Sciences 02/2006; 103(2):281-6. DOI:10.1073/pnas.0509849103 · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The calcium-independent receptor of alpha-latrotoxin (CIRL), a neuronal cell surface receptor implicated in the regulation of exocytosis, is a natural chimera of the cell adhesion protein and the G protein-coupled receptor (GPCR). In contrast with canonic GPCRs, CIRL consists of two heterologous non-covalently bound subunits, p120 and p85, due to endogenous proteolytic processing of the receptor precursor in the endoplasmic reticulum. Extracellularly oriented p120 contains hydrophilic cell adhesion domains, whereas p85 resembles a generic GPCR. We determined that the site of the CIRL cleavage is located within a juxtamembrane Cys- and Trp-rich domain of the N-terminal extracellular region of CIRL. Mutations in this domain make CIRL resistant to the cleavage and impair its trafficking. Therefore, we have named it GPS for G protein-coupled receptor proteolysis site. The GPS motif is found in homologous adhesion GPCRs and thus defines a novel receptor family. We postulate that the proteolytic processing and two-subunit structure is a common characteristic feature in the family of GPS-containing adhesion GPCRs.
[Show abstract][Hide abstract] ABSTRACT: The calcium-independent receptor of α-latrotoxin (CIRL), a neuronal cell surface receptor implicated in the regulation of
exocytosis, is a natural chimera of the cell adhesion protein and the G protein-coupled receptor (GPCR). In contrast with
canonic GPCRs, CIRL consists of two heterologous non-covalently bound subunits, p120 and p85, due to endogenous proteolytic
processing of the receptor precursor in the endoplasmic reticulum. Extracellularly oriented p120 contains hydrophilic cell
adhesion domains, whereas p85 resembles a generic GPCR. We determined that the site of the CIRL cleavage is located within
a juxtamembrane Cys- and Trp-rich domain of the N-terminal extracellular region of CIRL. Mutations in this domain make CIRL
resistant to the cleavage and impair its trafficking. Therefore, we have named it GPS for G protein-coupled receptor proteolysis site. The GPS motif is found in homologous adhesion GPCRs and thus defines a novel receptor family. We postulate that the proteolytic
processing and two-subunit structure is a common characteristic feature in the family of GPS-containing adhesion GPCRs.
Journal of Biological Chemistry 11/2002; 277(48):46518-46526. · 4.57 Impact Factor