Extracellular Ligand-binding Domain Research Articles

Novel SEC61G–EGFR Fusion Gene in Pediatric Ependymomas Discovered by Clonal Expansion of Stem Cells in Absence of Exogenous Mitogens

The basis for molecular and cellular heterogeneity in ependymomas of the central nervous system is not understood. This study suggests a basis for this phenomenon in the selection for mitogen-independent (MI) stem-like cells with impaired proliferation but increased intracranial tumorigenicity. MI ependymoma cell lines created by selection for EGF/FGF2-independent proliferation exhibited constitutive activation of EGFR, AKT, and STAT3 and sensitization to the antiproliferative effects of EGFR tyrosine kinase inhibitors (TKI). One highly tumorigenic MI line harbored membrane-bound, constitutively active, truncated EGFR. Two EGFR mutants (ΔN566 and ΔN599) were identified as products of intrachromosomal rearrangements fusing the 3' coding portion of the EGFR gene to the 5'-UTR of the SEC61G, yielding products lacking the entire extracellular ligand-binding domain of the receptor while retaining the transmembrane and tyrosine kinase domains. EGFR TKI efficiently targeted ΔN566/ΔN599-mutant-mediated signaling and prolonged the survival of mice bearing intracranial xenografts of MI cells harboring these mutations. RT-PCR sequencing of 16 childhood ependymoma samples identified SEC61G-EGFR chimeric mRNAs in one infratentorial ependymoma WHO III, arguing that this fusion occurs in a small proportion of these tumors. Our findings demonstrate how in vitro culture selections applied to genetically heterogeneous tumors can help identify focal mutations that are potentially pharmaceutically actionable in rare cancers. Cancer Res; 77(21); 5860-72. ©2017 AACR.

HRAS, EGFR, MET, and RON Genes Are Recurrently Activated by Provirus Insertion in Liver Tumors Induced by the Retrovirus Myeloblastosis-Associated Virus 2.

Myeloblastosis-associated virus 2 (MAV-2) is a highly tumorigenic simple avian retrovirus. Chickens infected in ovo with MAV-2 develop tumors in the kidneys, lungs, and liver with a short latency, less than 8 weeks. Here we report the results of molecular analyses of MAV-2-induced liver tumors that fall into three classes: hepatic hemangiosarcomas (HHSs), intrahepatic cholangiocarcinomas (ICCs), and hepatocellular carcinomas (HCCs). Comprehensive inverse PCR-based screening of 92 chicken liver tumors revealed that in ca. 86% of these tumors, MAV-2 provirus had integrated into one of four gene loci: HRAS, EGFR, MET, and RON Insertionally mutated genes correlated with tumor type: HRAS was hit in HHSs, MET in ICCs, RON mostly in ICCs, and EGFR mostly in HCCs. The provirus insertions led to the overexpression of the affected genes and, in the case of EGFR and RON, also to the truncation of exons encoding the extracellular ligand-binding domains of these transmembrane receptors. The structures of truncated EGFR and RON closely mimic the structures of oncogenic variants of these genes frequently found in human tumors (EGFRvIII and sfRON).IMPORTANCE These data describe the mechanisms of oncogenesis induced in chickens by the MAV-2 retrovirus. They also show that molecular processes converting cellular regulatory genes to cancer genes may be remarkably similar in chickens and humans. We suggest that the MAV-2 retrovirus-based model can complement experiments performed using mouse models and provide data that could translate to human medicine.

A large-scale expression strategy for multimeric extracellular protein complexes using Drosophila S2 cells and its application to the recombinant expression of heterodimeric ligand-binding domains of taste receptor.

Many of the extracellular proteins or extracellular domains of plasma membrane proteins exist or function as homo- or heteromeric multimer protein complexes. Successful recombinant production of such proteins is often achieved by co-expression of the components using eukaryotic cells via the secretory pathway. Here we report a strategy addressing large-scale expression of hetero-multimeric extracellular domains of plasma membrane proteins and its application to the extracellular domains of a taste receptor. The target receptor consists of a heterodimer of T1r2 and T1r3 proteins, and their extracellular ligand binding domains (LBDs) are responsible for the perception of major taste substances. However, despite the functional importance, recombinant production of the heterodimeric proteins has so far been unsuccessful. We achieved the successful preparation of the heterodimeric LBD by use of Drosophila S2 cells, which have a high secretory capacity, and by the establishment of a stable high-expression clone producing both subunits at a comparable level. The method overcame the problems encountered in the conventional transient expression of the receptor protein in insect cells using baculovirus or vector lipofection, which failed in the proper heterodimer production because of the biased expression of T1r3LBD over T1r2LBD. The large-scale expression methodology reported here may serve as one of the considerable strategies for the preparation of multimeric extracellular protein complexes.

Unitary Properties of AMPA Receptors with Reduced Desensitization

Wild-type AMPA receptors display a characteristic rapidly desensitizing phenotype. Many studies point to the dimer interface between pairs of extracellular ligand binding domains as the key region controlling the rate at which the receptors desensitize. However, mutations at the extracellular end of the pore-forming regions (near the putative ion channel gate) have also been shown to alter desensitization. Here we report the behavior of single GluA4 receptors carrying one of two mutations that greatly reduce desensitization at the level of ensemble currents: the dimer interface mutation L484Y and the Lurcher mutation (A623T, GluA4-Lc) in the extracellular end of M3 (the second true transmembrane helix). Analysis of unitary currents in patches with just one active receptor showed that each mutation greatly prolongs bursts of openings without prolonging the apparent duration of individual openings. Each mutation decreases the frequency with which individual receptors visit desensitized states, but both mutant receptors still desensitize multiple times per second. Cyclothiazide (CTZ) reduced desensitization of wild-type receptors and both types of mutant receptor. Analysis of shut-time distributions revealed a form of short-lived desensitization that was resistant to CTZ and was especially prominent for GluA4-Lc receptors. Despite reducing desensitization of GluA4 L484Y receptors, CTZ decreased the amplitude of ensemble currents through GluA2 and GluA4 LY receptor mutants. Single-channel analysis and comparison of the GluA2 L483Y ligand binding domain dimer in complex with glutamate with and without CTZ is consistent with the conclusion that CTZ binding to the dimer interface prevents effects of the LY mutation to modulate receptor activation, resulting in a reduction in the prevalence of large-conductance substates that accounts for the decrease in ensemble current amplitudes. Together, the results show that similar nondesensitizing AMPA-receptor phenotypes of population currents can arise from distinct underlying molecular mechanisms that produce different types of unitary activity.

Identification of LRP-1 as an endocytosis and recycling receptor for β1-integrin in thyroid cancer cells.

LRP-1 is a large endocytic receptor mediating the clearance of various molecules from the extracellular matrix. LRP-1 was reported to control focal adhesion turnover to optimize the adhesion-deadhesion balance to support invasion. To better understand how LRP-1 coordinates cell-extracellular matrix interface, we explored its ability to regulate cell surface integrins in thyroid carcinomas. Using an antibody approach, we demonstrated that β1-integrin levels were increased at the plasma membrane under LRP1 silencing or upon RAP treatment, used as LRP-1 antagonist. Our data revealed that LRP-1 binds with both inactive and active β1-integrin conformations and identified the extracellular ligand-binding domains II or IV of LRP-1 as sufficient to bind β1-integrin. Using a recombinant β1-integrin, we demonstrated that LRP-1 acts as a regulator of β1-integrin intracellular traffic. Moreover, RAP or LRP-1 blocking antibodies decreased up to 36% the number of β1-integrin-containing endosomes. LRP-1 blockade did not significantly affect the levels of β1-integrin-containing lysosomes while decreasing localization of β1-integrin within Rab-11 positive vesicles. Overall, we identified an original molecular process in which LRP-1 acts as a main regulator of β1-integrin internalization and recycling in thyroid cancer cells.

Activation of the EGF Receptor by Ligand Binding and Oncogenic Mutations: The "Rotation Model".

The epidermal growth factor receptor (EGFR) plays vital roles in cellular processes including cell proliferation, survival, motility, and differentiation. The dysregulated activation of the receptor is often implicated in human cancers. EGFR is synthesized as a single-pass transmembrane protein, which consists of an extracellular ligand-binding domain and an intracellular kinase domain separated by a single transmembrane domain. The receptor is activated by a variety of polypeptide ligands such as epidermal growth factor and transforming growth factor α. It has long been thought that EGFR is activated by ligand-induced dimerization of the receptor monomer, which brings intracellular kinase domains into close proximity for trans-autophosphorylation. An increasing number of diverse studies, however, demonstrate that EGFR is present as a pre-formed, yet inactive, dimer prior to ligand binding. Furthermore, recent progress in structural studies has provided insight into conformational changes during the activation of a pre-formed EGFR dimer. Upon ligand binding to the extracellular domain of EGFR, its transmembrane domains rotate or twist parallel to the plane of the cell membrane, resulting in the reorientation of the intracellular kinase domain dimer from a symmetric inactive configuration to an asymmetric active form (the “rotation model”). This model is also able to explain how oncogenic mutations activate the receptor in the absence of the ligand, without assuming that the mutations induce receptor dimerization. In this review, we discuss the mechanisms underlying the ligand-induced activation of the preformed EGFR dimer, as well as how oncogenic mutations constitutively activate the receptor dimer, based on the rotation model.

Abstract MIP-048: SHORT-FORM RON KINASE AS A NOVEL THERAPEUTIC TARGET IN OVARIAN CANCER

Abstract BACKGROUND: Although 70–80% of women respond to standard platinum-based chemotherapy, a majority of patients will develop recurrent platinum-resistant disease. Novel treatment approaches directed against key oncogenic drivers and/or the use of novel drug combinations to prevent/overcome platinum resistance are promising opportunities to combat this disease. sfRon is an understudied, alternative isoform of the full-length Ron receptor tyrosine kinase, the latter of which has been investigated in cancer for several years. Unlike full-length Ron, the sfRon protein lacks the extracellular ligand-binding domain, but organizes into a constitutively active transmembrane protein. Our in vitro and in vivo studies indicate that sfRon expression is associated with the acquisition of aggressive and invasive ovarian tumor phenotype. Such data carries additional importance because sfRon does not require ligand binding for activity, it may not be targetable using standard approaches such as antibody inhibitors. Instead, our data suggest that inhibition of Ron kinase activity, which would block both Ron and sfRon, might have great therapeutic benefit in ovarian tumors that express sfRon. RESULTS: Our lab has recently made the striking and unexpected discovery that sfRon expression is necessary for development of carcinogen-driven ovarian tumors in mice. Our study involved treating wild type mice vs. mice with a specific genetic deletion of sfRon (ΔsfRon) with DMBA, a known carcinogen. The study revealed that lack of sfRon completely protected the mice from ovarian cancer. The frequency of ovarian tumors was 25% in wild-type mice vs. 0% in ΔsfRon mice (p<0.0011). An increased understanding of how sfRon contributes to ovarian tumor development and progression is critical. Based on our findings supporting the role of sfRon in ovarian cancer, we aimed to investigate the expression of sfRon in primary ovarian samples. Our studies using ovarian tumors from patients revealed that sfRon becomes overexpressed in several different subtypes of ovarian cancer, with no expression in healthy ovarian tissue. Our in vitro studies revealed that introduction of sfRon into OVCAR3 cells is associated with the acquisition of aggressive and invasive tumor phenotype through the induction of EMT, increased proliferation and migration and decreased cell adhesion. The in vivo studies revealed the profound effect of sfRon expression on ovarian cancer growth and spreading to abdominal cavity. Our further studies using a large cohort of high-grade serous ovarian cancer (HG-SOC) patients demonstrated that Ron and/or sfRon are expressed in 86/126 (68%) of all cases. Statistical analysis revealed that significantly more patients in the cisplatin resistant group express high levels of Ron receptors, compared to patients in the cisplatin sensitive group, which suggest, that high levels of Ron and/or sfRon could contribute to cisplatin resistance. CONCLUSION: Our data implicate sfRon in ovarian cancer pathogenesis, and indicate that Ron kinase inhibitors targeting both Ron and sfRon may not only suppress ovarian tumor progression, but also have potential to overcome resistance to chemotherapy. These data could have significant impact on future clinical trials for both Ron kinase inhibitors and platinum compounds, which has high potential to impact patients in the short-term. Citation Format: Magdalena Bieniasz, Katherine Moxley, Luyao Wang, Alana L. Welm. SHORT-FORM RON KINASE AS A NOVEL THERAPEUTIC TARGET IN OVARIAN CANCER [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr MIP-048.

Structural basis for perception of diverse chemical substances by T1r taste receptors

The taste receptor type 1 (T1r) family perceives ‘palatable’ tastes. These receptors function as T1r2-T1r3 and T1r1-T1r3 heterodimers to recognize a wide array of sweet and umami (savory) tastes in sugars and amino acids. Nonetheless, it is unclear how diverse tastes are recognized by so few receptors. Here we present crystal structures of the extracellular ligand-binding domains (LBDs), the taste recognition regions of the fish T1r2-T1r3 heterodimer, bound to different amino acids. The ligand-binding pocket in T1r2LBD is rich in aromatic residues, spacious and accommodates hydrated percepts. Biophysical studies show that this binding site is characterized by a broad yet discriminating chemical recognition, contributing for the particular trait of taste perception. In contrast, the analogous pocket in T1r3LBD is occupied by a rather loosely bound amino acid, suggesting that the T1r3 has an auxiliary role. Overall, we provide a structural basis for understanding the chemical perception of taste receptors.

Positive Modulatory Interactions of NMDA Receptor GluN1/2B Ligand Binding Domains Attenuate Antagonists Activity.

N-methyl D-aspartate receptors (NMDAR) play crucial role in normal brain function and pathogenesis of neurodegenerative and psychiatric disorders. Functional tetra-heteromeric NMDAR contains two obligatory GluN1 subunits and two identical or different non-GluN1 subunits that include six different gene products; four GluN2 (A–D) and two GluN3 (A–B) subunits. The heterogeneity of subunit combination facilities the distinct function of NMDARs. All GluN subunits contain an extracellular N-terminal Domain (NTD) and ligand binding domain (LBD), transmembrane domain (TMD) and an intracellular C-terminal domain (CTD). Interaction between the GluN1 and co-assembling GluN2/3 subunits through the LBD has been proven crucial for defining receptor deactivation mechanisms that are unique for each combination of NMDAR. Modulating the LBD interactions has great therapeutic potential. In the present work, by amino acid point mutations and electrophysiology techniques, we have studied the role of LBD interactions in determining the effect of well-characterized pharmacological agents including agonists, competitive antagonists, and allosteric modulators. The results reveal that agonists (glycine and glutamate) potency was altered based on mutant amino acid sidechain chemistry and/or mutation site. Most antagonists inhibited mutant receptors with higher potency; interestingly, clinically used NMDAR channel blocker memantine was about three-fold more potent on mutated receptors (N521A, N521D, and K531A) than wild type receptors. These results provide novel insights on the clinical pharmacology of memantine, which is used for the treatment of mild to moderate Alzheimer's disease. In addition, these findings demonstrate the central role of LBD interactions that can be exploited to develop novel NMDAR based therapeutics.

Mutational Analysis at Intersubunit Interfaces of an Anionic Glutamate Receptor Reveals a Key Interaction Important for Channel Gating by Ivermectin.

The broad-spectrum anthelmintic drug ivermectin (IVM) activates and stabilizes an open-channel conformation of invertebrate chloride-selective glutamate receptors (GluClRs), thereby causing a continuous inflow of chloride ions and sustained membrane hyperpolarization. These effects suppress nervous impulses and vital physiological processes in parasitic nematodes. The GluClRs are pentamers. Homopentameric receptors assembled from the Caenorhabditis elegans (C. elegans) GluClα (GLC-1) subunit can inherently respond to IVM but not to glutamate (the neurotransmitter). In contrast, heteromeric GluClα/β (GLC-1/GLC-2) assemblies respond to both ligands, independently of each other. Glutamate and IVM bind at the interface between adjacent subunits, far away from each other; glutamate in the extracellular ligand-binding domain, and IVM in the ion-channel pore periphery. To understand the importance of putative intersubunit contacts located outside the glutamate and IVM binding sites, we introduced mutations at intersubunit interfaces, between these two binding-site types. Then, we determined the effect of these mutations on the activation of the heteromeric mutant receptors by glutamate and IVM. Amongst these mutations, we characterized an α-subunit point mutation located close to the putative IVM-binding pocket, in the extracellular end of the first transmembrane helix (M1). This mutation (αF276A) moderately reduced the sensitivity of the heteromeric GluClαF276A/βWT receptor to glutamate, and slightly decreased the receptor subunits’ cooperativity in response to glutamate. In contrast, the αF276A mutation drastically reduced the sensitivity of the receptor to IVM and significantly increased the receptor subunits’ cooperativity in response to IVM. We suggest that this mutation reduces the efficacy of channel gating, and impairs the integrity of the IVM-binding pocket, likely by disrupting important interactions between the tip of M1 and the M2-M3 loop of an adjacent subunit. We hypothesize that this physical contact between M1 and the M2-M3 loop tunes the relative orientation of the ion-channel transmembrane helices M1, M2 and M3 to optimize pore opening. Interestingly, pre-exposure of the GluClαF276A/βWT mutant receptor to subthreshold IVM concentration recovered the receptor sensitivity to glutamate. We infer that IVM likely retained its positive modulation activity by constraining the transmembrane helices in a preopen orientation sensitive to glutamate, with no need for the aforementioned disrupted interactions between M1 and the M2-M3 loop.

Co-expression of BirA with biotin bait achieves in vivo biotinylation of overexpressed stable N-glycosylated sRAGE in transgenic silkworms

Here, we demonstrated the expression of the N-glycosylated extracellular ligand binding domain of receptor for advanced glycation end products (sRAGE) in middle silk glands (MSGs) of transgenic silkworms using the GAL4/UAS system. Over 1 mg of sRAGE was obtained from one transgenic silkworm. sRAGE purified from the silkworm exhibited good stability and maintained specific ligand-binding ability. In addition, N-glycan analysis of sRAGE revealed that N-glucan completely lacked potentially allergenic fucose. Moreover, co-expression of biotin ligase (BirA) with C-terminal BioEase-tagged sRAGE in MSGs resulted in efficient biotinylation of sRAGE after addition of biotin bait. C-terminal biotinylated sRAGE could be immobilized onto a solid surface in one direction through binding to streptavidin without any loss of ability. The dissociation constant of sRAGE with fructose-BSA, a typical RAGE ligand, was 7.25 × 10−7 M, consistent with that on the mammalian cell surface. Thus, we developed a novel, innovative silkworm expression system for efficient expression of recombinant sRAGE, which could serve as a basis for the elucidation of RAGE-ligand interactions and facilitate the search for new ligands and inhibitors.

Trapping of ivermectin by a pentameric ligand-gated ion channel upon open-to-closed isomerization

Ivermectin (IVM) is a broad-spectrum anthelmintic drug used to treat human parasitic diseases like river blindness and lymphatic filariasis. By activating invertebrate pentameric glutamate-gated chloride channels (GluCl receptors; GluClRs), IVM induces sustained chloride influx and long-lasting membrane hyperpolarization that inhibit neural excitation in nematodes. Although IVM activates the C. elegans heteromeric GluClα/β receptor, it cannot activate a homomeric receptor composed of the C. elegans GluClβ subunits. To understand this incapability, we generated a homopentameric α7-GluClβ chimeric receptor that consists of an extracellular ligand-binding domain of an α7 nicotinic acetylcholine receptor known to be potentiated by IVM, and a chloride-selective channel domain assembled from GluClβ subunits. Application of IVM prior to acetylcholine inhibited the responses of the chimeric α7-GluClβR. Adding IVM to activated α7-GluClβRs, considerably accelerated the decline of ACh-elicited currents and stabilized the receptors in a non-conducting state. Determination of IVM association and dissociation rate constants and recovery experiments suggest that, following initial IVM binding to open α7-GluClβRs, the drug induces a conformational change and locks the ion channel in a closed state for a long duration. We further found that IVM also inhibits the activation by glutamate of a homomeric receptor assembled from the C. elegans full-length GluClβ subunits.

The evolution of the natural killer complex; a comparison between mammals using new high-quality genome assemblies and targeted annotation

Natural killer (NK) cells are a diverse population of lymphocytes with a range of biological roles including essential immune functions. NK cell diversity is in part created by the differential expression of cell surface receptors which modulate activation and function, including multiple subfamilies of C-type lectin receptors encoded within the NK complex (NKC). Little is known about the gene content of the NKC beyond rodent and primate lineages, other than it appears to be extremely variable between mammalian groups. We compared the NKC structure between mammalian species using new high-quality draft genome assemblies for cattle and goat; re-annotated sheep, pig, and horse genome assemblies; and the published human, rat, and mouse lemur NKC. The major NKC genes are largely in the equivalent positions in all eight species, with significant independent expansions and deletions between species, allowing us to propose a model for NKC evolution during mammalian radiation. The ruminant species, cattle and goats, have independently evolved a second KLRC locus flanked by KLRA and KLRJ, and a novel KLRH-like gene has acquired an activating tail. This novel gene has duplicated several times within cattle, while other activating receptor genes have been selectively disrupted. Targeted genome enrichment in cattle identified varying levels of allelic polymorphism between the NKC genes concentrated in the predicted extracellular ligand-binding domains. This novel recombination and allelic polymorphism is consistent with NKC evolution under balancing selection, suggesting that this diversity influences individual immune responses and may impact on differential outcomes of pathogen infection and vaccination.

Structural Rearrangements in GABA

GABAA receptors (GABARs) are allosteric proteins that convert a chemical signal (GABA binding) to an electrical signal (opening of an integral ion-conducting channel). They mediate inhibition in the brain and are the therapeutic targets for a variety of drugs such as benzodiazepines (BZDs), barbiturates, neuroactive steroids, volatile and intravenous anesthetics. For these receptors, binding of neurotransmitter in the extracellular ligand-binding domain triggers opening of an intrinsic ion channel more than 50A away in the transmembrane domain of the receptor. While recent structures of prokaryotic and eukaryotic pLGICs solved in detergent micelles have shed light on their molecular architecture, a full understanding of pLGIC function requires knowledge of the protein motions that govern their conformational transitions in physiological lipids. Comparing closed versus open channel structures of prokaryotic and eukaryotic pLGICs predict that agonist-mediated channel opening is associated with a quaternary twisting and a radial un-blooming (inward tilting) of the extracellular binding domain (ECD). Using a luminescence resonance energy transfer (LRET) approach that allows us to measure structural motions in full length heteropentameric GABARs in live cells, we are examining if, and how, the ECD moves in response to agonist-dependent gating transitions and allosteric drug binding. We found that GABA decreased the distances between fluorescent probes attached to the ECD of both the alpha and beta GABAR subunits, whereas binding of a competitive antagonist had no effect consistent with receptor activation promoting a global inward tilting of the ECD. Surprisingly, a BZD positive modulator that binds in the ECD but does not open the ion channel induced similar decreases in LRET-based distances as GABA raising the intriguing idea that the inward motion may reflect a low affinity to high affinity binding site transition in the ECD versus a closed to open channel transition.

Effect of GABRA1 Cytoplasmic Peptide on a Regulatory Protein

The pentameric neurotransmitter-gated chloride channel, GABA (type A) receptor, is well-recognized for phasic and tonic neuronal inhibition. The phasic response at the synapse is due to receptors typically containing two alpha, two beta, and one gamma subunit. Half of all GABA (type A) receptors in adult human brain have the alpha (type 1), or alpha-1, subunit (gene ID: GABRA1). Each consists of an extracellular ligand-binding domain and four transmembrane helices (TM1-4), both of which are highly conserved, as well as a large intracellular loop domain (ILD), whose structure is unknown. The human receptor is crystallized without the ILD, which connects TM3 and TM4, that is engineered out. The topology and function of the 86-residue alpha-1 ILD remains to be fully elucidated. GAPDH (d-glyceraldehyde-3-phosphate dehydrogenase) phosphorylates specific ILD residues, regulating receptor function. In this study, we examined the effect of a synthesized ILD peptide (residues 335 to 364 from GABRA1) on the oxidoreductase activity of GAPDH. The custom peptide, containing the first 30 cytosolic residues, was pre-incubated with GAPDH, and enzyme activity measured by NADH absorbance (340nm) upon oxidation of the substrate d-glyceraldehyde. When pre-incubated for 1hr at 20degC, the ILD peptide inhibited enzyme activity in a concentration-dependent manner showing an exponential non-linear function (y=a∗e ˆ (b/x+c)). The half-maximum inhibition occurred at a [peptide] = 8.7micromolar. When pre-incubated for 48hr at 4degC, the ILD peptide further enhanced inhibition of oxidoreductase activity, suggesting that hydrophobic forces play a role in the interaction between the ILD peptide and GAPDH. Molecular modeling experiments indicate that the ILD binding site on GAPDH is in close proximity to the phosphatidylserine-binding site on GAPDH. The findings may contribute to our understanding of receptor function and point to possible treatments in conditions, such as Alzheimer's dementia, where GABA receptor pharmacology is shown to play a role.

In Silico Prediction of Ligand-Binding Sites of Plant Receptor Kinases Using Conservation Mapping.

Plasma membrane-bound plant receptor-like kinases (RLKs) can be categorized based on their ligand-binding extracellular domain. The largest group encompasses RLKs having ectodomains with leucine-rich repeats (LRRs). The LRR-RLKs can further be assigned to classes mainly based on the number of LRRs. Many of the receptors of the classes X and XI with more than 20 LRRs are activated by small secreted peptide ligands. To understand how peptide signaling works, it is of interest to identify the amino acids of the receptor that are directly involved in ligand interaction. Such residues have most likely been conserved over evolutionary time and can therefore be predicted to be conserved in receptor orthologues of different plant species. Here we present an in silico method to identify such residues. This involves a simplified method for identification of orthologues and a web-based program for identifying the most conserved amino acids aside from the leucines that structure the ectodomain. The method has been validated for the LRR-RLKs HAESA (HAE) and PHYTOSULFOKINE RECEPTOR1 (PSKR1) for which conservation-mapping results closely matched recent structure-based identification of ligand and co-receptor-interacting residues.

The Sam Domain of EphA2 Receptor and its Relevance to Cancer: A Novel Challenge for Drug Discovery?

Eph receptors play important functions in developmental processes and diseases and among them EphA2 is well known for its controversial role in cancer. Drug discovery strategies are mainly centered on EphA2 extracellular ligand-binding domain however, the receptor also contains a largely unexplored cytosolic Sam (Sterile alpha motif) domain at the C-terminus. EphA2-Sam binds the Sam domain from the lipid phosphatase Ship2 and the first Sam domain of Odin. Sam-Sam interactions may be important to regulate ligand-induced receptor endocytosis and degradation i.e., processes that could be engaged against tumor malignancy. We critically analyzed literature related to a) Eph receptors with particular emphasis on EphA2 and its role in cancer, b) Sam domains, c) heterotypic Sam-Sam interactions involving EphA2-Sam. While literature data indicate that binding of EphA2-Sam to Ship2-Sam should largely generate pro-oncogenic effects in cancer cells, the correlation between EphA2- Sam/Odin-Sam1 complex and the disease is unclear. Recently a few linear peptides encompassing binding interfaces from either Ship2-Sam and Odin-Sam1 have been characterized but failed to efficiently block heterotypic Sam-Sam interactions involving EphA2-Sam due to the lack of a native like fold. Molecule antagonists of heterotypic EphA2-Sam associations could work as potential anticancer agents or be implemented as tools to further clarify receptor functions and eventually validate its role as a novel target in the field of anti-cancer drug discovery. Due to the failure of linear peptides there is a crucial need for novel approaches, based on cyclic or helical molecules, to target Sam-Sam interfaces.

Positive selection in cathelicidin host defense peptides: adaptation to exogenous pathogens or endogenous receptors?

The cause of adaptive protein evolution includes internal (for example, co-evolution of ligand-receptor pairs) and external (for example, adaptation to different ecological niches) mechanisms. Host defense peptides (HDPs) are a class of vertebrate-specific cationic antimicrobial peptides evolving under positive selection. Besides their antibiotic activity, HDPs also exert an effect on multiple host immune cells, thus providing an ideal model to study selective agents driving their evolution. On the basis of a combination of computational and experimental approaches, we studied the evolution of LL-37-type HDPs in mammals, the mature peptide of cathelicidin CAP18 (herein termed CAP18-MP) and investigated the driving force behind the evolution. Using codon-substitution maximum likelihood models, we analyzed CAP18-MPs in 40 species belonging to nine mammalian Orders and identified four positively selected sites (PSSs) that all are located on two terminal unordered regions of CAP18-MPs. Grafting the two positively selected regions of human or whale CAP18-MP to the α-helical scaffold of a rabbit homolog (substituting its corresponding parts) led to no alterations in antibacterial activity, spectrum and action mode. Likewise, further deletion of the two terminal regions did not alter its functional features. Evolutionary conservation analysis of mammalian FPR2, a receptor known to interact with the C-terminal positively selected region of LL-37, revealed high evolutionary variability in its ligand-binding extracellular loop domains, matching sequence diversity of the unordered regions in CAP18-MPs. This is the first report describing that the signature of positive selection of cathelicidins is not associated with their direct bactericidal activity, but rather with the evolutionary variability of their endogenous receptors.

The E Loop of the Transmitter Binding Site Is a Key Determinant of the Modulatory Effects of Physostigmine on Neuronal Nicotinic α4β2 Receptors.

Physostigmine is a well known inhibitor of acetylcholinesterase, which can also activate, potentiate, and inhibit acetylcholine receptors, including neuronal nicotinic receptors comprising α4 and β2 subunits. We have found that the two stoichiometric forms of this receptor differ in the effects of physostigmine. The form containing three copies of α4 and two of β2 was potentiated at low concentrations of acetylcholine chloride (ACh) and physostigmine, whereas the form containing two copies of α4 and three of β2 was inhibited. Chimeric constructs of subunits indicated that the presence of inhibition or potentiation depended on the source of the extracellular ligand binding domain of the subunit. Further sets of chimeric constructs demonstrated that a portion of the ACh binding domain, the E loop, is a key determinant. Transferring the E loop from the β2 subunit to the α4 subunit resulted in strong inhibition, whereas the reciprocal transfer reduced inhibition. To control the number and position of the incorporated chimeric subunits, we expressed chimeric constructs with subunit dimers. Surprisingly, incorporation of a subunit with an altered E loop had similar effects whether it contributed either to an intersubunit interface containing a canonical ACh binding site or to an alternative interface. The observation that the α4 E loop is involved suggests that physostigmine interacts with regions of subunits that contribute to the ACh binding site, whereas the lack of interface specificity indicates that interaction with a particular ACh binding site is not the critical factor.

Gephyrin-binding peptides visualize postsynaptic sites and modulate neurotransmission.

γ-Aminobutyric acid type A and glycine receptors are the major mediators of fast synaptic inhibition in the human central nervous system and are established drug targets. However, all drugs targeting these receptors bind to the extracellular ligand-binding domain of the receptors, which inherently is associated with perturbation of the basic physiological action. Here we pursue a fundamentally different approach, by instead targeting the intracellular receptor-gephyrin interaction. First, we defined the gephyrin peptide-binding consensus sequence, which facilitated the development of gephyrin super-binding peptides and later effective affinity probes for the isolation of native gephyrin. Next, we demonstrated that fluorescent super-binding peptides could be used to directly visualize inhibitory postsynaptic sites for the first time in conventional and super-resolution microscopy. Finally, we demonstrate that the gephyrin super-binding peptides act as acute intracellular modulators of fast synaptic inhibition by modulating receptor clustering, thus being conceptually novel modulators of inhibitory neurotransmission.