Intracellular Catalytic Domain Research Articles

Biophysical analysis of the membrane-proximal Venus Flytrap domain of ESAG4 receptor-like adenylate cyclase from Trypanosoma brucei

The protozoan parasite Trypanosoma brucei possesses a large family of transmembrane receptor-like adenylate cyclases (RACs), primarily located to the flagellar surface and involved in sensing of the extracellular environment. RACs exhibit a conserved topology characterized by a large N-terminal extracellular moiety harbouring two Venus Flytrap (VFT) bilobate structures separated from an intracellular catalytic domain by a single transmembrane helix. RAC activation, which typically occurs under mild acid stress, requires the dimerization of the intracellular catalytic domain. The occurrence of VFT domains in the RAC’s extracellular moiety suggests their potential responsiveness to extracellular ligands in the absence of stress, although no such ligands have been identified so far. Herein we report the biophysical characterization of the membrane-proximal VFT2 domain of a bloodstream form-specific RAC called ESAG4, whose ectodomain 3D structure is completely unknown. The paper describes an AlphaFold2-based optimisation of the expression construct, enabling facile and high-yield recombinant production and purification of the target protein. Through an interdisciplinary approach combining various biophysical methods, we demonstrate that the optimised VFT2 domain obtained by recombination is properly folded and behaves as a monomer in solution. The latter suggests a ligand-binding capacity independent of dimerization, unlike typical mammalian VFT receptors, as guanylate cyclase. In silico VFT2 genomic analyses shows divergence among cyclase isoforms, hinting at ligand specificity. Taken together this improved procedure enabling facile and high-yield recombinant production and purification of the target protein could benefit researchers studying trypanosomal RAC VFT domains but also any trypanosome domain with poorly defined boundaries. Additionally, our findings support the stable monomeric VFT2 domain as a useful tool for future structural investigations and ligand screening.

Identification of Two Insulin Receptors from the Swimming Crab Portunus trituberculatus: Molecular Characterization, Expression Analysis, and Interactions with Insulin-Like Androgenic Gland Hormone.

AbstractThe insulin-like androgenic gland hormone is a crucial sexual regulator that is involved in the masculine sexual differentiation of crustaceans. As an insulin-like peptide, the insulin-like androgenic gland hormone has been proposed to act through the insulin receptor-mediated pathway. The present study cloned and characterized two insulin receptors (PtIR1 and PtIR2) from the swimming crab Portunus trituberculatus hallmarked with a conserved intracellular tyrosine kinase catalytic domain and several other typical insulin receptor domains in their deduced amino acid sequences. Both insulin receptors were predominately expressed in the testis and the insulin-like androgenic gland hormone-producing organ androgenic gland. Their testicular expression during the annual cycle suggested that they may play critical roles in spermatogenesis. By using the protein colocalization analysis in HEK293 cells, interactions of PtIAG with the two PtIRs were further confirmed. In addition, the insulin receptor antagonist was found to attenuate the stimulatory effects of androgenic gland homogenate on the phosphorylated MAPK levels in testis explants, suggesting that the insulin receptor-dependent MAPK pathway may be essential for insulin-like androgenic gland hormone functions.

Clustering of phosphatase RPTPα promotes Src signaling and the arthritogenic action of synovial fibroblasts.

Receptor-type protein phosphatase α (RPTPα) promotes fibroblast-dependent arthritis and fibrosis, in part, by enhancing the activation of the kinase SRC. Synovial fibroblasts lining joint tissue mediate inflammation and tissue damage, and their infiltration into adjacent tissues promotes disease progression. RPTPα includes an ectodomain and two intracellular catalytic domains (D1 and D2) and, in cancer cells, undergoes inhibitory homodimerization, which is dependent on a D1 wedge motif. Through single-molecule localization and labeled molecule interaction microscopy of migrating synovial fibroblasts, we investigated the role of RPTPα dimerization in the activation of SRC, the migration of synovial fibroblasts, and joint damage in a mouse model of arthritis. RPTPα clustered with other RPTPα and with SRC molecules in the context of actin-rich structures. A known dimerization-impairing mutation in the wedge motif (P210L/P211L) and the deletion of the D2 domain reduced RPTPα-RPTPα clustering; however, it also unexpectedly reduced RPTPα-SRC association. The same mutations also reduced recruitment of RPTPα to actin-rich structures and inhibited SRC activation and cellular migration. An antibody against the RPTPα ectodomain that prevented the clustering of RPTPα also inhibited RPTPα-SRC association and SRC activation and attenuated fibroblast migration and joint damage in arthritic mice. A catalytically inactivating RPTPα-C469S mutation protected mice from arthritis and reduced SRC activation in synovial fibroblasts. We conclude that RPTPα clustering retains it to actin-rich structures to promote SRC-mediated fibroblast migration and can be modulated through the extracellular domain.

Identification of tetracycline combinations as EphB1 tyrosine kinase inhibitors for treatment of neuropathic pain

Previous studies have demonstrated that the synaptic EphB1 receptor tyrosine kinase is a major mediator of neuropathic pain, suggesting that targeting the activity of this receptor might be a viable therapeutic option. Therefore, we set out to determine if any FDA-approved drugs can act as inhibitors of the EphB1 intracellular catalytic domain. An in silico screen was first used to identify a number of tetracycline antibiotics which demonstrated potential docking to the ATP-binding catalytic domain of EphB1. Kinase assays showed that demeclocycline, chlortetracycline, and minocycline inhibit EphB1 kinase activity at low micromolar concentrations. In addition, we cocrystallized chlortetracycline and EphB1 receptor, which confirmed its binding to the ATP-binding domain. Finally, in vivo administration of the three-tetracycline combination inhibited the phosphorylation of EphB1 in the brain, spinal cord, and dorsal root ganglion (DRG) and effectively blocked neuropathic pain in mice. These results indicate that demeclocycline, chlortetracycline, and minocycline can be repurposed for treatment of neuropathic pain and potentially for other indications that would benefit from inhibition of EphB1 receptor kinase activity.

Activity-dependent conformational transitions of the insulin receptor–related receptor

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

RPTPα phosphatase activity is allosterically regulated by the membrane-distal catalytic domain

Receptor-type protein tyrosine phosphatase α (RPTPα) is an important positive regulator of SRC kinase activation and a known promoter of cancer growth, fibrosis, and arthritis. The domain structure of RPTPs comprises an extracellular region, a transmembrane helix, and two tandem intracellular catalytic domains referred to as D1 and D2. The D2 domain of RPTPs is believed to mostly play a regulatory function; however, no regulatory model has been established for RPTPα-D2 or other RPTP-D2 domains. Here, we solved the 1.8 Å resolution crystal structure of the cytoplasmic region of RPTPα, encompassing D1 and D2, trapped in a conformation that revealed a possible mechanism through which D2 can allosterically inhibit D1 activity. Using a D2-truncation RPTPα variant and mutational analysis of the D1/D2 interfaces, we show that D2 inhibits RPTPα phosphatase activity and identified a 405PFTP408 motif in D1 that mediates the inhibitory effect of D2. Expression of the gain-of-function F406A/T407A RPTPα variant in HEK293T cells enhanced SRC activation, supporting the relevance of our proposed D2-mediated regulation mechanism in cell signaling. There is emerging interest in the development of allosteric inhibitors of RPTPs but a scarcity of validated allosteric sites for RPTPs. The results of our study not only shed light on the regulatory role of RPTP-D2 domains, but also provide a potentially useful tool for the discovery of chemical probes targeting RPTPα and other RPTPs.

Mutations in NlInR1 affect normal growth and lifespan in the brown planthopper Nilaparvata lugens

The brown planthopper (BPH) Nilaparvata lugens contains two insulin receptor homologues, designated NlInR1 and NlInR2. NlInR1 is strikingly homologous to the typical InR in insects and vertebrates, containing a ligand-activated intracellular tyrosine kinase catalytic domain. Herein, we report an optimized CRISPR/Cas9 system to induce mutations in the NlInR1 locus in BPH, consisting of a Cas9 plasmid that is specifically expressed in the germline via the Nlvasa promoter and versatile sgRNA expression plasmids under the control of the U6 promoter. We systematically evaluated the efficiency of injection mix compositions and demonstrated an appropriate combination of Cas9/sgRNA to target essential genes. Furthermore, we showed that homozygous mutants for the NlInR1 gene are early embryonic lethal, whereas heterozygous mutants grow more slowly, exhibit a severe reduction in body weight and wing size and live longer than the wild type. Interestingly, the severity of the mutant phenotype was different when targeting distinct important domains of the NlInR1 locus. The severity of the mutant phenotype is similar to that of insulin/insulin-like growth factor (IGF) signaling pathway deficiencies in vertebrates, suggesting a conserved function of NlInR1 in the regulation of development and longevity. Global expression profiling suggests that NlInR1 regulates many cellular processes in BPH, including insulin resistance, phototransduction, metabolism, endocytosis, longevity, biosynthesis and protein processing. Our results also pave the way for understanding the precise molecular mechanism of insulin signaling in wing polyphenism in insects.

501P - Oncogenic mutations at the dimer interface of EGFR lead to formation of covalent homo-dimers and allosteric activation of the kinase domain: A mechanism which alters the selectivity profile of oncogenic EGFR

BackgroundMutation of either the intracellular catalytic domain or the extracellular domain of the receptor for epidermal growth factor (EGFR) drives oncogenicity. Extracellular domain EGFR mutations are highly expressed in patients with glioblastoma. Despite clinical success with targeting EGFR catalytic site mutants, no drugs have proven effective in glioblastoma patients expressing extracellular EGFR mutations. MethodsHerein, we define the molecular mechanism for oncogenic activation of families of extracellular EGFR mutations and reveal how this mechanism renders current generation small molecule ATP-site inhibitors ineffective. We define these types of lesions as allosteric oncogenic mutations. ResultsWe demonstrate that a group of the most commonly expressed extracellular domain EGFR mutants expressed in glioblastomas is activated by disulfide-bond mediated covalent homodimerization, collectively referred to as locked dimerization (LoDi-EGFR oncogenes). Strikingly, current generation small molecules binding to the active kinase conformation potently inhibit catalytic site mutants, but induce covalent dimerization and activate LoDi-EGFR receptors, manifesting in paradoxical acceleration of proliferation. Herein we describe the discovery of novel ATP competitive small molecules that potently (<50nM) inhibit the family of LoDi-EGFR oncogenes expressed in GBM, and importantly, achieve selectivity versus WT-EGFR of greater than 10-fold. ConclusionsThese data demonstrate how the locked-dimer mechanism of EGFR oncogenesis has profound impact on the activity of small molecules acting at the distal catalytic site, providing further evidence for “inside-out” allosteric signaling in EGFR. This provides a mechanistic understanding for the failure of current generation EGFR inhibitors to effectively treat LoDi-EGFR mutants in GBM and sets guidelines for the discovery and development of novel selective LoDi-EGFR inhibitors. Legal entity responsible for the studyBlack Diamond Therapeutics. FundingBlack Diamond Therapeutics. DisclosureE. Buck: Leadership role, Shareholder / Stockholder / Stock options, Full / Part-time employment: Black Diamond Therapeutics. M. O’Connor: Leadership role, Shareholder / Stockholder / Stock options, Full / Part-time employment: Black Diamond Therapeutics. A. Flohr: Leadership role, Shareholder / Stockholder / Stock options: Black Diamond Therapeutics. R. Iacone: Leadership role, Shareholder / Stockholder / Stock options: Black Diamond Therapeutics. A.V. Mayweg: Leadership role, Shareholder / Stockholder / Stock options, Officer / Board of Directors: Black Diamond Therapeutics. D.M. Epstein: Leadership role, Shareholder / Stockholder / Stock options, Officer / Board of Directors: Black Diamond Therapeutics. All other authors have declared no conflicts of interest.

Abstract LB-111: Epidermal growth factor receptor oncogenes expressed in glioblastoma are activated as covalent dimers and exhibit unique pharmacology

Abstract Mutation of either the intracellular catalytic domain or the extracellular domain of the receptor for epidermal growth factor (EGFR) drives oncogenicity. Extracellular domain EGFR mutations are highly expressed in patients with glioblastoma. Despite clinical success with targeting EGFR catalytic site mutants, no drugs have proven effective in glioblastoma patients expressing extracellular EGFR mutations. Herein, we define the molecular mechanism for oncogenic activation of families of extracellular EGFR mutations and reveal how this mechanism renders current generation small molecule ATP-site inhibitors ineffective. We demonstrate that a group of the most commonly expressed extracellular domain EGFR mutants expressed in glioblastomas is activated by disulfide-bond mediated covalent homodimerization, collectively referred to as locked dimerization (LoDi-EGFR oncogenes). Strikingly, current generation small molecules binding to the active kinase conformation potently inhibit catalytic site mutants, but induce covalent dimerization and activate LoDi-EGFR receptors, manifesting in paradoxical acceleration of proliferation. These data demonstrate how the locked-dimer mechanism of EGFR oncogenesis has profound impact on the activity of small molecules acting at the distal catalytic site, providing further evidence for “inside-out” allosteric signaling in EGFR. This provides a mechanistic understanding for the failure of current generation EGFR inhibitors to effectively treat LoDi-EGFR mutants in GBM and sets guidelines for discovery of selective LoDi-EGFR inhibitors. Citation Format: Matthew O'Connor, Theodore Nicolaides, Jie Zhang, Alexander Flohr, Roberto Iacone, Alexander V. Mayweg, David M. Epstein, Elizabeth Buck. Epidermal growth factor receptor oncogenes expressed in glioblastoma are activated as covalent dimers and exhibit unique pharmacology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-111.

Catalytically active guanylyl cyclase-B requires glycosylation and mutations that inhibit this process cause dwarfism

Background C-type natriuretic peptide (CNP) is a paracrine factor that stimulates long bone growth, axonal path finding and inhibits meiosis in the oocyte [1]. The biologic signaling receptor for CNP is guanylyl cyclase (GC)-B, also known at NPRB or NPR2 [2]. GC-B is a homo-oligomer, possibly a dimer, containing a glycosylated extracellular ligand-binding domain, a single membrane-spanning region, and intracellular kinase homology domain (KHD), dimerization domain and C-terminal GC catalytic domain. Phosphorylation of the region leading into and at the beginning of the kinase homology domain is required for CNP activation of GC-B and dephosphorylation inactivates the enzyme [3]. Homozygous inactivating mutations in GC-B result in Acromesomelic Dysplasia, Type Maroteaux (AMDM) dwarfism [4-6], and heterozygous inactivating mutations in GC-B cause non-pathological reductions in stature [7]. Conversely, genetic mutations that increase GC-B activity result in skeletal overgrowth [8-10]. More than fifteen inactivating missense mutations in GC-B have been identified in humans. These mutations are randomly distributed from the N-terminus (P32) to the C-terminus (G959A) of the enzyme, consistent with two potential mechanisms. The first involves multiple processes like disruption of CNP or GTP binding to the extracellular or catalytic domains, respectively. The second more general mechanism involves conformational changes in secondary, tertiary or quaternary structure that preclude catalytic domain formation or activation. Previous investigators reported that 11 out of 12 [1], 2 out of 3 [2] or 1 out of 2 [3] missense mutations inhibited the transport of GC-B to the cell surface due to defective cellular trafficking and retention in the ER as indicated by reduced immunofluorescence imaging. Thus, the current hypothesis is that AMDM mutations inactivate GCB by disrupting intracellular trafficking. Here, we report that four intracellular GC-B mutants known to cause AMDM dwarfism bind CNP on the cell surface but have dramatically reduced catalytic activity.

Linaclotide-induced electrolyte secretion in human and rat colon: Ussing chamber studies

Background Linaclotide is a guanylyl cyclase C (GC-C) agonist approved for treatment of adult patients with irritable bowel syndrome with constipation (IBS-C). Linaclotide binding to the extracellular receptor domain of GC-C activates the intracellular catalytic domain of GC-C, resulting in the generation of cyclic GMP (cGMP). cGMP, via a pathway leading to activation of the cystic fibrosis transmembrane conductance regulator (CFTR) ion channel, stimulates Cland HCO3-secretion and concomitant inhibition of Na+ absorption by sodium/hydrogen exchanger 3 (NHE3), driving the efflux of water into the lumen and accelerating bowel transit. However, the effect of linaclotide on electrolyte transport in the lower gastrointestinal tract has not yet been characterized. This study specifically examined the effect of linaclotide on electrolyte transport in the colon. We used an in vitro Ussing chamber assay to measure the secretion of electrolytes in human ascending colon segments and in rat proximal colon in response to linaclotide treatment.

Structure-function analyses of tyrosine phosphatase PTP69D in giant fiber synapse formation of Drosophila.

PTP69D is a receptor protein tyrosine phosphatase (RPTP) with two intracellular catalytic domains (Cat1 and Cat2) and has been shown to play a role in axon guidance of embryonic motoneurons as well as targeting of photoreceptor neurons in the visual system of Drosophila melanogaster. Here, we characterized the developmental role of PTP69D in the giant fiber (GF) neurons, two interneurons in the central nervous system (CNS) that control the escape response of the fly. Our studies revealed that PTP69D has a function in synaptic terminal growth in the CNS. We found that missense mutations in the first immunoglobulin (Ig) domain and in the Cat1 domain, present in Ptp69D10 and Ptp69D20 mutants, respectively, did not affect axon guidance or targeting but resulted in stunted terminal growth of the GFs. Cell autonomous rescue experiments demonstrated a function for the Cat1 and the first Ig domain of PTP69D in the GFs but not in its postsynaptic target neurons. In addition, complementation studies and structure-function analyses revealed that for GF terminal growth Cat1 function of PTP69D requires the immunoglobulin and the Cat2 domains, but not the fibronectin III or the membrane proximal region domains. In contrast, the fibronectin III but not the immunoglobulin domains were previously shown to be essential for axon targeting of photoreceptor neurons. Thus, our studies uncover a novel role for PTP69D in synaptic terminal growth in the CNS that is mechanistically distinct from its function in photoreceptor targeting.

Expression, purification, and characterization of human osteoclastic protein-tyrosine phosphatase catalytic domain in Escherichia coli

Osteoclastic protein tyrosine phosphatase (PTP-oc) is a structurally unique transmembrane protein tyrosine phosphatase (PTP) that contains only a relatively small intracellular PTP catalytic domain, does not have an extracellular domain, and lacks a signal peptide proximal to the NH2 terminus. The present study reports the expression, purification, and characterization of the intracellular catalytic domain of PTP-oc (ΔPTP-oc). ΔPTP-oc was expressed in Escherichia coli cells as a fusion with a six-histidine tag and was purified via nickel affinity chromatography. When with para-nitrophenylphosphate (p-NPP) as a substrate, ΔPTP-oc exhibited classical Michaelis–Menten kinetics. Its responses to temperature and ionic strength were similar to those of other PTPs. The optimal pH value of ΔPTP-oc is approximately 7.0, unlike other PTPs, whose optimal pH values are approximately 5.0.

Intracellular catalytic domain of symbiosis receptor kinase hyperactivates spontaneous nodulation in absence of rhizobia.

Symbiosis Receptor Kinase (SYMRK), a member of the Nod factor signaling pathway, is indispensible for both nodule organogenesis and intracellular colonization of symbionts in rhizobia-legume symbiosis. Here, we show that the intracellular kinase domain of a SYMRK (SYMRK-kd) but not its inactive or full-length version leads to hyperactivation of the nodule organogenic program in Medicago truncatula TR25 (symrk knockout mutant) in the absence of rhizobia. Spontaneous nodulation in TR25/SYMRK-kd was 6-fold higher than rhizobia-induced nodulation in TR25/SYMRK roots. The merged clusters of spontaneous nodules indicated that TR25 roots in the presence of SYMRK-kd have overcome the control over both nodule numbers and their spatial position. In the presence of rhizobia, SYMRK-kd could rescue the epidermal infection processes in TR25, but colonization of symbionts in the nodule interior was significantly compromised. In summary, ligand-independent deregulated activation of SYMRK hyperactivates nodule organogenesis in the absence of rhizobia, but its ectodomain is required for proper symbiont colonization.

Membrane pacman: small steps for the voltage‐sensitive phosphatases

The voltage-sensitive phosphatases (VSPs) are a fascinating family of membrane-associated enzymes consisting of a voltage-sensing domain (VSD), similar to that found in canonical voltage-gated channels, and an intracellular catalytic domain. Although found in every eukaryotic organism, their functions are not at all clear. Since many of these phosphatases catalyse the degradation of important signalling lipids such as phosphatidylinositol 4,5-bisphosphate (PIP2), they serve several important functions. For example, they control sperm mobility and play a role in depolarization-induced calcium transients upon egg fertilization. It has been speculated that they are important in the control of cell proliferation and organ differentiation; however, our understanding of the physiological roles of these proteins is still developing (Villalba-Galea, 2012). Voltage-dependent control of enzymatic activity in VSPs was first demonstrated by Murata et al. (2005). It was also shown that changes in voltage produce charge movement mediated by the VSD that can be recorded as transient sensing currents, related to the gating currents of voltage-dependent ion channels. One of the major questions still needing clarification is what is the mechanism by which the VSD controls the enzymatic activity. Progress in this area of research has been rapid and the article by Sakata and Okamura in this issue of The Journal of Physiology (Sakata & Okamura, 2014) represents an important advance into this problem. In the case of the tetrameric voltage-gated ion channels, four VSDs are coupled to the opening of a single pore domain, which can be thought of as the analogue of the catalytic domain in VSPs. It has been known for some time that membrane depolarization causes outward movement of the VSDs and that this is coupled to opening of the channel pore through a specialized linker domain. For these proteins, the electromechanical coupling process is strict, meaning that all four VSDs have to be in an activated position for the pore to be open and this relationship can be evidenced experimentally because charge movement, in the form of gating or sensing currents, precedes channel opening at all voltages. It also means that spontaneous or voltage-independent opening of the channel pore is exceedingly improbable. An important similar question for the VSPs relates to the interplay between VSD movements, as reported by the transient sensing current, and the activity of the phosphatase domain. Does the VSD need to be fully activated upon depolarization for the enzymatic activity to take place? The X-ray structures of the catalytic domain suggest a direct and graded control of the catalytic domain by voltage through activation of the VSD, but this point still requires clarification (Kohout et al. 2010). Sakata & Okamura (2014) now shed light on this matter in their novel studies that make use of a clever combination of diverse mutants of the Danio rerio (zebrafish) phosphatase, known as Dr-VSP. Their present findings demonstrate that the phosphatase activity is linked to VSD movement in a graded fashion. In other words, the voltage sensor domain does not need to be fully activated to trigger the enzymatic activity. The experiments in this work thus are consistent with a coupling between charge movement and enzyme activity that is different from what has been observed in voltage-gated potassium ion channels. These data suggest instead that, in contrast to voltage-gated channels, an allosteric coupling model could explain the relationship between activation of the voltage sensor and the catalytic domain of Dr-VSP (Fig. 1). In such a mechanism, both the resting and the activated state(s) of the voltage sensor can drive the activation of the phosphatase domain, although with different efficiencies. If this is the case, one immediate prediction of an allosteric coupling model is that there should be some phosphatase activity at negative potentials. In reality such spontaneous and voltage-independent activity might be very difficult to detect. Another consequence of the allosteric mechanism is that charge movement should have different voltage dependence when the phosphatase is in the active or the inactive conformation. Sakata & Okamura (2014) have thus opened the gates for such future experiments, which will enhance our understanding of the inner workings of these mesmerizing proteins. The resting states of the voltage-sensing domains, Ri, are occupied at negative voltages, while depolarization shifts the equilibrium towards activated states, Ai. The orange arrows represent the charge-moving transitions that give rise to the sensing current. Canonical voltage-gated channels follow the path enclosed in grey, in which the open state is reached only after all charge-moving transitions take place, while the voltage-sensitive phosphatase Dr-VSP can access the activated (catalytic) states, Ai, from any of the resting states, Ri. None declared.

A Functional Genetic Variant (N521D) in Natriuretic Peptide Receptor 3 Is Associated with Diastolic Dysfunction: The Prevalence of Asymptomatic Ventricular Dysfunction Study

ObjectivesTo evaluate the impact of a functional genetic variant in the natriuretic peptide clearance receptor, NPR3, on circulating natriuretic peptides (NPs) and myocardial structure and function in the general community.BackgroundNPR3 plays an important role in the clearance of NPs and through direct signaling mechanisms modulates smooth muscle cell function and cardiac fibroblast proliferation. A NPR3 nonsynonymous single nucleotide polymorphism (SNP) rs2270915, resulting in a N521D substitution in the intracellular catalytic domain that interacts with Gi could affect receptor function. Whether this SNP is associated with alterations in NPs levels and altered cardiac structure and function is unknown.MethodsDNA samples of 1931 randomly selected residents of Olmsted County, Minnesota were genotyped. Plasma NT-proANP1-98, ANP1-28, proBNP1-108, NT-proBNP1-76, BNP1-32 and BNP3-32 levels were measured. All subjects underwent comprehensive echocardiography.ResultsGenotype frequencies for rs2270915 were as follows: (A/A 60%, A/G 36%, G/G 4%). All analyses performed were for homozygotes G/G versus wild type A/A plus the heterozygotes A/G. Diastolic dysfunction was significantly more common (p = 0.007) in the homozygotes G/G (43%) than the A/A+A/G (28%) group. Multivariate regression adjusted for age, sex, body mass index and hypertension demonstrated rs2270915 to be independently associated with diastolic dysfunction (odds ratio 1.94, p = 0.03). There was no significant difference in NPs levels between the 2 groups suggesting that the clearance function of the receptor was not affected.ConclusionsA nonsynonymous NPR3 SNP is independently associated with diastolic dysfunction and this association does not appear to be related to alterations in circulating levels of natriuretic peptides.

The Linker Pivot in Ci-VSP: The Key to Unlock Catalysis

In the voltage-sensitive phosphatase Ci-VSP, conformational changes in the transmembrane voltage sensor domain (VSD) are transduced to the intracellular catalytic domain (CD) leading to its dephosphorylation activity against membrane-embedded phosphoinositides. The linker between both domains is proposed to be crucial for the VSD-CD coupling. With a combined approach of electrophysiological measurements on Xenopus oocytes and molecular dynamics simulations of a Ci-VSP model embedded in a lipid bilayer, we analyzed how conformational changes in the linker mediate the interaction between the CD and the activated VSD. In this way, we identified specific residues in the linker that interact with well-defined amino acids in one of the three loops forming the active site of the protein, named TI loop. With our results, we shed light into the early steps of the coupling process between the VSD and the CD, which are based on fine-tuned electrostatic and hydrophobic interactions between the linker, the membrane and the CD.

Pharmacology and clinical potential of guanylyl cyclase C agonists in the treatment of ulcerative colitis

Agonists of the transmembrane intestinal receptor guanylyl cyclase C (GCC) have recently attracted interest as promising human therapeutics. Peptide ligands that can specifically induce GCC signaling in the intestine include endogenous hormones guanylin and uroguanylin, diarrheagenic bacterial enterotoxins (ST), and synthetic drugs linaclotide, plecanatide, and SP-333. These agonists bind to GCC at intestinal epithelial surfaces and activate the receptor's intracellular catalytic domain, an event initiating discrete biological responses upon conversion of guanosine-5'-triphosphate to cyclic guanosine monophosphate. A principal action of GCC agonists in the colon is the promotion of mucosal homeostasis and its dependent barrier function. Herein, GCC agonists are being developed as new medications to treat inflammatory bowel diseases, pathological conditions characterized by mucosal barrier hyperpermeability, abnormal immune reactions, and chronic local inflammation. This review will present important concepts underlying the pharmacology and therapeutic utility of GCC agonists for patients with ulcerative colitis, one of the most prevalent inflammatory bowel disease disorders.

Revealing the Relationship between Fibroblast Growth Factor Receptor-Like 1 (FGFRL1) and Free Zinc in Pancreatic Beta-Cells using Quantitative Fluorescence Microscopy

Type 2 diabetes is characterized by a loss of normal pancreatic beta-cell function and mass. In beta-cells, tyrosine kinase Fibroblast Growth Factors Receptors (FGFRs) modulate insulin processing, fatty acid metabolism and cell survival. However, a deeper understanding of mechanisms that regulate function of these receptors will be necessary to use this pathway therapeutically. We have recently identified beta-cell expression of Fibroblast Growth Factor Receptor-like 1 (FGFRL1), a newly-identified member of the FGFR family. FGFRL1 shares the canonical extracellular domain of FGFRs but uniquely exhibits a short C-terminal histidine-rich zinc-binding domain rather than intracellular catalytic kinase domains. Zinc is a second messenger normally found at picomolar concentration in the cytosol that mediates phosphatase activity to regulate Mitogen-Activated Protein Kinase (MAPK) signaling. We determined that FGFRL1 co-localizes with insulin secretory granules where zinc accumulates at micromolar concentrations. We therefore postulate that FGFRL1 alters beta-cell MAPK signaling by chelating zinc and regulating its intracellular concentration. To measure intercellular zinc, we imaged living murine beta-cells expressing full-length and truncated fluorescent protein variants of FGFRL1 co-labeled with cell-permeable zinc indicators FluoZin-3 and RhodZin-3. Our data confirm that FGFRL1 reduces free intracellular zinc via the unique histidine-rich region. We further show that zinc induces FGFRL1 receptor dimerization at the cell membrane using homo-fluorescence resonance energy transfer (Homo-FRET) imaging. Dimerization of FGFRL1 and association with insulin secretory granules suggest receptor activity is tightly regulated by zinc and likely associated with glucose-stimulated insulin secretion. Elucidating novel signaling mechanisms that regulate FGFR-activity in beta-cells will improve our understanding of how this pathway can be used therapeutically to treat diabetes.

EJ-3 - Targeting the her Family in Head and Neck Cancer: A Road with Hurdles

ABSTRACT The epidermal growth factor receptor (EGFR) is playing a role in the multistep carcinogenesis of squamous cell carcinoma of the head and neck (SCCHN) and its signaling promotes SCCHN growth and progression. EGFR is over expressed in the vast majority (>90%) of H&N cases and a high expression is associated with an unfavorable prognosis. Therefore, EGFR is a rational therapeutic target. In practice, both monoclonal antibodies (MAbs), which bind to the outer domain, and small molecule oral tyrosine kinase inhibitors (TKIs) that target the intracellular catalytic domain of the EGFR are being used. Cetuximab (Cet) is the only targeted agent that got approval for the treatment of SCCHN from the regulatory agencies of Europe and the United States, both in locoregionally advanced disease in association with radiation (RT) and in recurrent/metastatic disease in combination with platinum/5-FU-based chemotherapy (in United States also as single agent in second line) and is still under active investigation in this disease. Unfortunately, several important questions are still unanswered (e.g. is RT + Cet equal in efficacy to RT + cisplatin (P)?, is maintenance therapy essential and for how long?, what are the adequate markers to predict response to Cet?, etc). Moreover, some results have been disappointing (e.g. PRT + Cet is not superior to PRT alone) and some applications are still investigational (e.g. Cet + induction chemotherapy and Cet in the postoperative setting). Interestingly, the results of trials involving other EGFR-directed MAbs, i.e. zalutumumab, panitumumab and nimotuzumab, so far have been consistent with the cetuximab data, but failed their primary end points. The results with EGFR-directed TKIs overall have been disappointing. Dual TKIs of both EGFR and HER/2 (the preferred dimerization partner of EGFR) have shown some promise, as well as newer irreversible HER-family blockers, such as afatinib and PF-299804. Both afatinib and PF-299804 have also demonstrated preclinical activity against tumor cells bearing a truncated EGFR protein, lacking the extracellular ligand-binding domain (EGFR variant III). Despite the hurdles, targeting the HER has a place in SCCHN treatment.