LRRC8 proteins share a common ancestor with pannexins, and may form hexameric channels involved in cell-cell communication

Leucine-rich repeat-containing 8 (LRRC8) proteins are composed of four transmembrane helices and 17 leucine-rich repeats (LRR). Although LRRC8 proteins have been associated with important processes, like maturation of B cells or adipocyte differentiation, their biology and molecular function are largely unknown. We found that LRRC8 proteins originated from the combination of a pannexin and an LRR domain (most likely related to the SHOC2, LAP, RSU1 and LRRIQ4 protein families) before the diversification of chordates. We propose that, like pannexins, LRRC8 proteins form hexameric channels, which participate in cell-cell communication processes. According to the inferred topological model, and contrary to what was previously assumed, the six LRR domains are located in the cytoplasm, and could participate in the organisation of signalling cascades. By compiling available proteomics and gene expression data, and on the basis of the LRRC8 proposed hexameric channel structure, we present clues to the function of this family.     

Cisplatin activates volume sensitive LRRC8 channel mediated currents in Xenopus oocytes

LRRC8 proteins have been shown to underlie the ubiquitous volume regulated anion channel (VRAC). VRAC channels are composed of the LRRC8A subunit and at least one among the LRRC8B-E subunits. In addition to their role in volume regulation, LRRC8 proteins have been implicated in the uptake of chemotherapeutic agents. We had found that LRRC8 channels can be conveniently expressed in Xenopus oocytes, a system without endogenous VRAC activity. The fusion with fluorescent proteins yielded constitutive activity for A/C, A/D and A/E heteromers. Here we tested the effect of the anticancer drug cisplatin on LRRC8A-VFP/8E-mCherry and LRRC8A-VFP/8D-mCherry co-expressing oocytes. Incubation with cisplatin dramatically activated currents for both subunit combinations, confirming that VRAC channels provide an uptake pathway for cisplatin and that intracellular cisplatin accumulation strongly activates the channels. Thus, specific activators of LRRC8 proteins might be useful tools to counteract chemotherapeutic drug resistance.     

Biophysics and Structure-Function Relationships of LRRC8-Formed Volume-Regulated Anion Channels

Volume-regulated anion channels (VRACs) are key players in regulatory volume decrease of vertebrate cells by mediating the extrusion of chloride and organic osmolytes. They play additional roles in various physiological processes beyond their role in osmotic volume regulation. VRACs are formed by heteromers of LRRC8 proteins; LRRC8A (also called SWELL1) is an essential subunit that combines with any of its paralogs, LRRC8B–E, to form hexameric VRAC complexes. The subunit composition of VRACs determines electrophysiological characteristics of their anion transport such as single-channel conductance, outward rectification, and depolarization-dependent inactivation kinetics. In addition, differently composed VRACs conduct diverse substrates, such as LRRC8D enhancing VRAC permeability to organic substances like taurine or cisplatin. Here, after a recapitulation of the biophysical properties of VRAC-mediated ion and osmolyte transport, we summarize the insights gathered since the molecular identification of VRACs. We describe the recently solved structures of LRRC8 complexes and discuss them in terms of their structure-function relationships. These studies open up many potential avenues for future research.     

Specific and essential but not sufficient roles of LRRC8A in the activity of volume-sensitive outwardly rectifying anion channel (VSOR)

The broadly expressed volume-sensitive outwardly rectifying anion channel (VSOR, also called VRAC) plays essential roles in cell survival and death. Recent findings have suggested that LRRC8A is a core component of VSOR in human cells. In the present study, VSOR currents were found to be largely reduced by siRNA against LRRC8A in mouse C127 cells as well. In contrast, LRRC8A knockdown never affected activities of 4 other types of anion channel activated by acid, Ca²⁺, patch excision or cAMP. While cisplatin-resistant KCP-4 cells poorly expressed endogenous VSOR activity, molecular expression levels of LRRC8A, LRRC8D and LRRC8E were indistinguishable between VSOR-deficient KCP-4 cells and the parental VSOR-rich KB cells. Furthermore, overexpression of LRRC8A alone or together with LRRC8D or LRRC8E in KCP-4 cells failed to restore VSOR activity. These results show that deficiency of VSOR currents in KCP-4 cells is not due to insufficient expression of the LRRC8A/D/E gene, suggesting an essential involvement of some other factor(s), and indicate that further study is required to better understand the complexities of the molecular determinants of VSOR, including the precise role of LRRC8 proteins.     

Subunit composition of VRAC channels determines substrate specificity and cellular resistance to Pt‐based anti‐cancer drugs

Although platinum‐based drugs are widely used chemotherapeutics for cancer treatment, the determinants of tumor cell responsiveness remain poorly understood. We show that the loss of subunits LRRC8A and LRRC8D of the heteromeric LRRC8 volume‐regulated anion channels (VRACs) increased resistance to clinically relevant cisplatin/carboplatin concentrations. Under isotonic conditions, about 50% of cisplatin uptake depended on LRRC8A and LRRC8D, but neither on LRRC8C nor on LRRC8E. Cell swelling strongly enhanced LRRC8‐dependent cisplatin uptake, bolstering the notion that cisplatin enters cells through VRAC. LRRC8A disruption also suppressed drug‐induced apoptosis independently from drug uptake, possibly by impairing VRAC‐dependent apoptotic cell volume decrease. Hence, by mediating cisplatin uptake and facilitating apoptosis, VRAC plays a dual role in the cellular drug response. Incorporation of the LRRC8D subunit into VRAC substantially increased its permeability for cisplatin and the cellular osmolyte taurine, indicating that LRRC8 proteins form the channel pore. Our work suggests that LRRC8D‐containing VRACs are crucial for cell volume regulation by an important organic osmolyte and may influence cisplatin/carboplatin responsiveness of tumors.     

Distinct contributions of LRRC8A and its paralogs to the VSOR anion channel from those of the ASOR anion channel

Volume- and acid-sensitive outwardly rectifying anion channels (VSOR and ASOR) activated by swelling and acidification exhibit voltage-dependent inactivation and activation time courses, respectively. Recently, LRRC8A and some paralogs were shown to be essentially involved in the activity and inactivation kinetics of VSOR currents in human colonic HCT116 cells. In human cervix HeLa cells, here, inactivation of VSOR currents was found to become accelerated by RNA silencing only of LRRC8A but never decelerated by that of any LRRC8 isoform. These data suggest that LRRC8A is associated with the deceleration mechanism of VSOR inactivation, while none of LRRC8 members is related to the acceleration mechanism. Activation kinetics of ASOR currents was unaffected by knockdown of any LRRC8 family member. Double, triple and quadruple gene-silencing studies indicated that combinatory expression of LRRC8A with LRRC8D and LRRC8C is essential for VSOR activity, whereas none of LRRC8 family members is involved in ASOR activity.     

LRRC8 involved in B cell development belongs to a novel family of leucine-rich repeat proteins

In a previous study, we isolated a novel gene, LRRC8 (leucine-rich repeat-containing 8), in a girl with congenital agammaglobulinemia. We have now identified four unknown LRRC8-like genes, named TA-LRRP, AD158, LRRC5, and FLJ23420. Their predicted structures are very similar to each other, and highly conserved between humans and the mouse. All five genes encode proteins consisting of 16 extracellular leucine-rich repeats (LRRs), all of which have four transmembrane regions except for FLJ23420. These genes belong to a novel family, designated the LRRC8 family, within the superfamily of LRR proteins. TA-LRRP, AD158, and LRRC5 might be implicated in proliferation and activation of lymphocytes and monocytes.     

Integrins and other cell adhesion molecules

Cell-cell and cell-substratum interactions are mediated through several different families of receptors. In addition to targeting cell adhesion to specific extracellular matrix proteins and ligands on adjacent cells, these receptors influence many diverse processes including cellular growth, differentiation, junction formation, and polarity. Several families of adhesion receptors have been identified. These include: 1) the integrins, heterodimeric molecules that function both as cell-substratum and cell-cell adhesion receptors; 2) the adhesion molecules of the immunoglobulin superfamily, which are involved in cell-cell adhesion and especially important during embryo-genesis, wound healing, and the inflammatory response; 3) the cadherins, developmentally regulated, calcium-dependent homophilic cell-cell adhesion proteins; 4) the LEC-CAMs, cell adhesion molecules with lectin-like domains that mediate white blood cell/endothelial cell adhesion; and 5) homing receptors that target lymphocytes to specific lymphoid tissue. In this review we summarize recent data describing the structure and function of some of these cell adhesion molecules (with special emphasis on the integrin family) and discuss the possible role of these molecules in development, inflammation, wound healing, coagulation, and tumor metastasis.     

IQGAP1 as signal integrator: Ca2+, calmodulin,Cdc42 and the cytoskeleton

A family of proteins known as IQGAPs have been identified in yeast, amebas and mammals. IQGAPs are multidomain molecules that contain several protein-interacting motifs which mediate binding to target proteins. Mammalian IQGAP1 is a component of signaling networks that are integral to maintaining cytoskeletal architecture and cell-cell adhesion. Published data suggest that IQGAP1 is a scaffolding protein that modulates cross-talk among diverse pathways in complex regulatory circuits. These pathways include modulating the actin cytoskeleton, mediating signaling by Rho family GTPases and calmodulin, regulating E-cadherin and beta-catenin function and organizing microtubules.     

Rac1 Recruits the Adapter Protein CMS/CD2AP to Cell-Cell Contacts

Rac1 is a member of the Rho family of small GTPases, which regulate cell adhesion and migration through their control of the actin cytoskeleton. Rho-GTPases are structurally very similar, with the exception of a hypervariable domain in the C terminus. Using peptide-based pulldown assays in combination with mass spectrometry, we previously showed that the hypervariable domain in Rac1 mediates specific protein-protein interactions. Most recently, we found that the Rac1 C terminus associates to the ubiquitously expressed adapter protein CMS/CD2AP. CD2AP is critical for the formation and maintenance of a specialized cell-cell contact between kidney podocyte foot processes, the slit diaphragm. Here, CD2AP links the cell adhesion protein nephrin to the actin cytoskeleton. In addition, CMS/CD2AP binds actin-regulating proteins, such as CAPZ and cortactin, and has been implicated in the internalization of growth factor receptors. We found that CD2AP specifically interacts with the C-terminal domain of Rac1 but not with that of other Rho family members. Efficient interaction between Rac1 and CD2AP requires both the proline-rich domain and the poly-basic region in the Rac1 C terminus, and at least two of the three N-terminal SH3 domains of CD2AP. CD2AP co-localizes with Rac1 to membrane ruffles, and small interfering RNA-based experiments showed that CD2AP links Rac1 to CAPZ and cortactin. Finally, expression of constitutive active Rac1 recruits CD2AP to cell-cell contacts in epithelial cells, where we found CD2AP to participate in the control of the epithelial barrier function. These data identify CD2AP as a novel Rac1-associated adapter protein that participates in the regulation of epithelial cell-cell contact.     

The p24 family of transmembrane proteins at the interface between endoplasmic reticulum and Golgi apparatus

Summary The p24 family of small transmembrane proteins was discovered recently in yeast and mammalian cells, and some of its members have been implicated in biosynthetic protein transport. The p24 proteins are proposed to act on transport vesicles as receptors for coat and/or cargo, but their precise function(s) remain controversial. Here, we describe this protein family, and we review the available experimental data concerning their localization and function. Finally, we hypothesize about a possible role of p24 proteins in organelle morphogenesis.Abbreviations CGN cis-Golgi network - COP coat protein - ER endoplasmic reticulum - VSV-G vesicular stomatitis virus glycoprotein G     

Cellular mechanisms of netrin function: long-range and short-range actions.

Netrins are secreted proteins that direct axon extension and cell migration during neural development. They are bifunctional cues that act as an attractant for some cell types and as a repellent for others. Several lines of evidence suggest that two classes of receptors, the deleted in colorectal cancer (DCC) family and the UNC-5 family, mediate the attractant and repellent response to netrin. Although netrins were first identified as diffusible long-range cues for developing axons, recent findings provide evidence that they also function as short-range cues close to the surface of the cells that produce them. This short-range function of netrin contributes to guiding neurite outgrowth and mediating cell-cell interactions during development and perhaps also in adults.     

SNARE complex-mediated degranulation in mast cells

Mast cell function and dysregulation is important in the development and progression of allergic and autoimmune disease. Identifying novel proteins involved in mast cell function and disease progression is the first step in the design of new therapeutic strategies. Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are a family of proteins demonstrated to mediate the transport and fusion of secretory vesicles to the membrane in mast cells, leading to the subsequent release of the vesicle cargo through an exocytotic mechanism. The functional role[s] of specific SNARE family member complexes in mast cell degranulation has not been fully elucidated. Here, we review recent and historical data on the expression, formation and localization of various SNARE proteins and their complexes in murine and human mast cells. We summarize the functional data identifying the key SNARE family members that appear to participate in mast cell degranulation. Furthermore, we discuss the utilization of RNA interference (RNAi) methods to validate SNARE function and the use of siRNA as a therapeutic approach to the treatment of inflammatory disease. These studies provide an overview of the specific SNARE proteins and complexes that serve as novel targets for the development of new therapies to treat allergic and autoimmune disease.     

Regulators of cell volume: The structural and functional properties of anion channels of the LRRC8 family

Members of the LRRC8 family participate in the response of vertebrate cells to osmotic changes in their environment. These proteins form heteromeric assemblies composed of the obligatory subunit LRRC8A and at least one of the other four homologs, which together function as anion-selective channels with distinct properties that are activated upon cell-swelling. The hexameric complexes share a conserved architecture consisting of a membrane-inserted pore domain with an ion permeation path located at the axis of symmetry and cytoplasmic leucine-rich repeat domains that regulate the open probability of the channel. In this review, we summarize the current understanding of structure–function relationships of these unusual ion channels whose mechanisms are, despite their large physiological importance, still poorly understood.     

Epithelial cell shape: cadherins and small GTPases

Cadherins are cell-cell adhesion receptors that are essential for the establishment of the epithelial cell shape and maintenance of the differentiated epithelial phenotype. In order to show efficient adhesion, cadherin receptors require an association with actin filaments and the activity of RHO proteins. The RHO family of small GTPases is primarily involved in the reorganization of the cytoskeleton. In different cell types, each member of the family can induce specific types of organization of actin filaments: stress fibers (Rho), lamellae/ruffles (Rac), or filopodia (Cdc42). This review focuses on how the function of small GTPases may impinge on the regulation of cadherin-dependent adhesion. In particular, it discusses the impact that the above cytoskeletal structures induced by RHO proteins have on the development of epithelial morphology. Finally, the participation of small GTPase-interacting proteins is considered during the remodeling of cell shape that follows cell-cell contact formation.     

Comparison of class A and D G protein-coupled receptors: common features in structure and activation

All G protein-coupled receptors (GPCRs) share a common seven TM helix architecture and the ability to activate heterotrimeric G proteins. Nevertheless, these receptors have widely divergent sequences with no significant homology. We present a detailed structure-function comparison of the very divergent Class A and D receptors to address whether there is a common activation mechanism across the GPCR superfamily. The Class A and D receptors are represented by the vertebrate visual pigment rhodopsin and the yeast alpha-factor pheromone receptor Ste2, respectively. Conserved amino acids within each specific receptor class and amino acids where mutation alters receptor function were located in the structures of rhodopsin and Ste2 to assess whether there are functionally equivalent positions or regions within these receptors. We find several general similarities that are quite striking. First, strongly polar amino acids mediate helix interactions. Their mutation generally leads to loss of function or constitutive activity. Second, small and weakly polar amino acids facilitate tight helix packing. Third, proline is essential at similar positions in transmembrane helices 6 and 7 of both receptors. Mapping the specific location of the conserved amino acids and sites of constitutively active mutations identified conserved microdomains on transmembrane helices H3, H6, and H7, suggesting that there are underlying similarities in the mechanism of the widely divergent Class A and Class D receptors.     

Protein transport machineries for precursor translocation across the inner mitochondrial membrane

The mitochondrial inner membrane has a central function for the energy metabolism of the cell. The respiratory chain generates a proton gradient across the inner mitochondrial membrane, which is used to produce ATP by the F1Fo-ATPase. To maintain the electrochemical gradient, the inner membrane represents an efficient permeability barrier for small molecules. Nevertheless, metabolites as well as polypeptide chains need to be transported across the inner membrane while the electrochemical gradient is retained. While specialized metabolite carrier proteins mediate the transport of small molecules, dedicated protein translocation machineries in the inner mitochondrial membrane (so called TIM complexes) transport precursor proteins across the inner membrane. Here we describe the organization of the TIM complexes and discuss the current models as to how they mediate the posttranslational import of proteins across and into the inner mitochondrial membrane.     

A virus-encoded cell-cell fusion machine dependent on surrogate adhesins

The reovirus fusion-associated small transmembrane (FAST) proteins function as virus-encoded cellular fusogens, mediating efficient cell-cell rather than virus-cell membrane fusion. With ectodomains of only approximately 20-40 residues, it is unclear how such diminutive viral fusion proteins mediate the initial stages (i.e. membrane contact and close membrane apposition) of the fusion reaction that precede actual membrane merger. We now show that the FAST proteins lack specific receptor-binding activity, and in their natural biological context of promoting cell-cell fusion, rely on cadherins to promote close membrane apposition. The FAST proteins, however, are not specifically reliant on cadherin engagement to mediate membrane apposition as indicated by their ability to efficiently utilize other adhesins in the fusion reaction. Results further indicate that surrogate adhesion proteins that bridge membranes as close as 13 nm apart enhance FAST protein-induced cell-cell fusion, but active actin remodelling is required for maximal fusion activity. The FAST proteins are the first example of membrane fusion proteins that have specifically evolved to function as opportunistic fusogens, designed to exploit and convert naturally occurring adhesion sites into fusion sites. The capacity of surrogate, non-cognate adhesins and active actin remodelling to enhance the cell-cell fusion activity of the FAST proteins are features perfectly suited to the structural and functional evolution of these fusogens as the minimal fusion component of a virus-encoded cellular fusion machine. These results also provide a basis for reconciling the rudimentary structure of the FAST proteins with their capacity to fuse cellular membranes.     

Transport between the cytoplasm and the nucleus

Summary Active transport of proteins and RNAs across the nuclear-pore complex (NPC) is mediated by a family of related transport receptors which shuttle between the cytoplasm and the nucleoplasm. A number of import and export pathways have been described. Some transport substrates require adapters which mediate association with certain transporters. The transport receptors specifically bind to a recognition signal within the transport substrate or adapter, pass the NPC in one direction, and deliver their cargo to the other side of the nuclear envelope. The Ran GTPase is the crucial regulator of bidirectional transport. Ran-modulating proteins establish an asymmetric intracellular distribution of Ran. As a result, Ran is mainly bound to GTP in the nucleus and to GDP in the cytoplasm. Evidently, RanGTP regulates binding and release of the transport substrates by binding to the transport receptors in the nucleus as well as the transport direction across the NPC. However, little is known about the molecular mechanism of translocation through the NPC.