Alexander G. Petrenko

Dissecting signaling and functions of adhesion G protein-coupled receptors.

G protein–coupled receptors (GPCRs) comprise an expanded superfamily of receptors in the human genome. Adhesion class G protein–coupled receptors (adhesion‐GPCRs) form the second largest class of GPCRs. Despite the abundance, size, molecular structure, and functions in facilitating cell and matrix contacts in a variety of organ systems, adhesion‐GPCRs are by far the most poorly understood GPCR class. Adhesion‐GPCRs possess a unique molecular structure, with extended N‐termini containing various adhesion domains. In addition, many adhesion‐GPCRs are autoproteolytically cleaved into an N‐terminal fragment (NTF, NT, α‐subunit) and C‐terminal fragment (CTF, CT, β‐subunit) at a conserved GPCR autoproteolysis–inducing (GAIN) domain that contains a GPCR proteolysis site (GPS). These two features distinguish adhesion‐GPCRs from other GPCR classes. Though active research on adhesion‐GPCRs in diverse areas, such as immunity, neuroscience, and development and tumor biology has been intensified in the recent years, the general biological and pharmacological properties of adhesion‐GPCRs are not well known, and they have not yet been used for biomedical purposes. The “6th International Adhesion‐GPCR Workshop,” held at the Institute of Physiology of the University of Würzburg on September 6–8, 2012, assembled a majority of the investigators currently actively pursuing research on adhesion‐GPCRs, including scientists from laboratories in Europe, the United States, and Asia. The meeting featured the nascent mechanistic understanding of the molecular events driving the signal transduction of adhesion‐GPCRs, novel models to evaluate their functions, and evidence for their involvement in human disease.

Insulin receptor-related receptor as an extracellular pH sensor involved in the regulation of acid-base balance.

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.

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

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. 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.

Regulation of CIRL-1 proteolysis and trafficking

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 receptors 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.

Mapping of alkali-sensing sites of the insulin receptor-related receptor. The role of L2 and fibronectin domains.

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.

Identification of proteins in complexes with α-latrotoxin receptors

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.

Association of the subunits of the calcium-independent receptor of α-latrotoxin

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.

Control of Adhesion GPCR Function Through Proteolytic Processing

Proteolytic processing events in adhesion GPCRs. aGPCRs can undergo multiple autoproteolytic (red asterisks) and proteolytic processing events by exogenous proteases (yellow asterisks) that may be involved in signaling events of the receptors. Proteolytic processing is an unusual property of adhesion family G protein-coupled receptors (aGPCRs) that was observed upon their cloning and biochemical characterization.Ever since, much effort has been dedicated to delineate the mechanisms and requirements for cleavage events in the control of aGPCR function. Most notably, all aGPCRs possess a juxtamembrane protein fold, the GPCR autoproteolysis-inducing (GAIN) domain, which operates as an autoprotease for many aGPCR homologs investigated thus far. Analysis of its autoproteolytic reaction, the consequences for receptor fate and function, and the allocation of physiological effects to this peculiar feature of aGPCRs has occupied the experimental agenda of the aGPCR field and shaped our current understanding of the signaling properties and cell biological effects of aGPCRs. Interestingly, individual aGPCRs may undergo additional proteolytic steps, one of them resulting in shedding of the entire ectodomain that is secreted and can function independently. Here, we summarize the current state of knowledge on GAIN domain-mediated and GAIN domain-independent aGPCR cleavage events and their significance for the pharmacological and cellular actions of aGPCRs. Further, we compare and contrast the proteolytic profile of aGPCRs with known signaling routes that are governed through proteolysis of surface molecules such as the Notch and ephrin pathways.

Association of Cell Adhesion Molecules Contactin-6 and Latrophilin-1 Regulates Neuronal Apoptosis

[This corrects the article on p. 143 in vol. 9, PMID: 28018171.].

Analysis of structural determinants of alkali sensor IRR positive cooperativity

160 Insulin receptorrrelated receptor (IRR) is a recepp tor tyrosine kinase and belongs to the insulin receptor family, which includes the insulin receptor (IR) itself and the insulinnlike growth factor receptor (IGFFIR). We have previously shown that IRR receptor is acti vated when the pH of the extracellular fluid increases above 8.0. Thus it may serve as an extracellular alkaline pH sensor in the body. Notably, the cooperativity of the receptor activation with an IRR agonist is positive, whereas for the insulin receptor it is negative. In this study, we analyzed the ability of a chimeric IRR molee cule containing three domains of the IGFFIR receptor in the extracellular region to be activated by alkaline pH. This chimeric receptor retains the ability to be activated by alkaline pH, but the cooperativity of actii vation is greatly reduced. The insulin receptor family consists of the insulin receptor (IR), insulinnlike growth factor receptor (IGFFIR), and insulin receptorrrelated receptor (IRR). All three receptors are highly homologous [1, 2]. The extracellular NNterminal part of the αsubunit of the three receptors contains domains L1 and L2 (leucinee rich), between which domain C (furinnlike, cysteinee rich) is located. The tyrosine kinase domain is located in the cytoplasmic moiety of βsubunit. It was assumed that the duplication and segregation of the genes coding for IGFFIR and IRR occurred evolutionarily later than the segregation o the insulin receptor gene [2]. For this reason, the degree of homology between IGFFIR and IRR is somewhat higher than between IR and IRR [1]. The binding of the peptide ligand to the extracelluu lar part of IR or IGFFIR causes conformational changes that result in autophosphorylation of the tyrosine residues located in the cytoplasmic tyrosine kinase domain. It was shown by various methods that the functional regions responsible for ligand binding to IR and IGFFIR are located in the extracellular domains of L1 and C (for review, see [3]). We have shown that IRR, unlike its homologues, can be actii vated by an increase in the extracellular fluid pH to more than 8.0 rather by a natural protein ligand [4, 5]. In mice with a targeted inactivation of the insrr, encoding the IRR receptor, the regulation of acid– base balance was disturbed [8]. We have shown that the major sites that determine the pH sensitivity are located in the first two domains L1 and C [5]. Moreover, unlike the homologous IR and …