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ABSTRACT: P2X3 and P2X2+3 receptors are present on sensory neurons, where they contribute not only to transient nociceptive responses, but also to
hypersensitivity underlying pathological pain states elicited by nerve injuries. Increased signalling through P2X3 and P2X2+3 receptors may arise from an increased routing to the plasma membrane and/or gain of function of pre-existing receptors. An
obvious effector mechanism for functional modulation is protein kinase C (PKC)-mediated phosphorylation, since all P2X family
members share a conserved consensus sequence for PKC, TXR/K, within the intracellularly located N-terminal domain. Contradictory
reports have been published regarding the exact role of this motif. In the present study, we confirm that site-directed elimination
of the potential phosphor-acceptor threonine or the basic residue in the P+2 position of the TXR/K sequence accelerates desensitization
of P2X2 receptors and abolishes P2X3 receptor function. Moreover, the PKC activator phorbol 12-myristate 13-acetate increased P2X3 (but not P2X2) receptor-mediated currents. Biochemically, however, we were unable to demonstrate by various experimental approaches a direct
phosphorylation of wild-type P2X2 and P2X3 receptors expressed in both Xenopus laevis oocytes and HEK293 cells. In conclusion, our data support the view that the TXR/K motif plays an important role in P2X function
and that phorbol 12-myristate 13-acetate is capable of modulating some P2X receptor subtypes. The underlying mechanism, however,
is unlikely to involve direct PKC-mediated P2X receptor phosphorylation.
Purinergic Signalling 04/2012; 3(4):377-388. · 3.16 Impact Factor
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Ralf Hausmann,
Mandy Bodnar,
Ronja Woltersdorf,
Haihong Wang,
Martin Fuchs,
Nanette Messemer,
Ying Qin,
Janka Günther,
Thomas Riedel,
Marcus Grohmann,
Karen Nieber, Günther Schmalzing,
Patrizia Rubini,
Peter Illes
[show abstract]
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ABSTRACT: The aim of the present experiments was to clarify the subunit stoichiometry of P2X2/3 and P2X2/6 receptors, where the same subunit (P2X2) forms a receptor with two different partners (P2X3 or P2X6). For this purpose, four non-functional Ala mutants of the P2X2, P2X3, and P2X6 subunits were generated by replacing single, homologous amino acids particularly important for agonist binding. Co-expression of these mutants in HEK293 cells to yield the P2X2 WT/P2X3 mutant or P2X2 mutant/P2X3 WT receptors resulted in a selective blockade of agonist responses in the former combination only. In contrast, of the P2X2 WT/P2X6 mutant and P2X2 mutant/P2X6 WT receptors, only the latter combination failed to respond to agonists. The effects of α,β-methylene-ATP and 2-methylthio-ATP were determined by measuring transmembrane currents by the patch clamp technique and intracellular Ca(2+) transients by the Ca(2+)-imaging method. Protein labeling, purification, and PAGE confirmed the assembly and surface trafficking of the investigated WT and WT/mutant combinations in Xenopus laevis oocytes. In conclusion, both electrophysiological and biochemical investigations uniformly indicate that one subunit of P2X2 and two subunits of P2X3 form P2X2/3 heteromeric receptors, whereas two subunits of P2X2 and one subunit of P2X6 constitute P2X2/6 receptors. Further, it was shown that already two binding sites of the three possible ones are sufficient to allow these receptors to react with their agonists.
Journal of Biological Chemistry 02/2012; 287(17):13930-43. · 4.77 Impact Factor
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ABSTRACT: P2X7 receptors (P2X7Rs) are nonselective cation channels that are opened by the binding of extracellular ATP and are involved in the modulation of epithelial secretion, inflammation and nociception. Here, we investigated the effect of extracellular anions on channel gating and permeation of human P2X7Rs (hP2X7Rs) expressed in Xenopus laevis oocytes. Two-microelectrode voltage-clamp recordings showed that ATP-induced hP2X7R-mediated currents increased when extracellular chloride was substituted by the organic anions glutamate or aspartate and decreased when chloride was replaced by the inorganic anions nitrate, sulfate or iodide. ATP concentration-response comparisons revealed that substitution of chloride by glutamate decreased agonist efficacy, while substitution by iodide increased agonist efficacy at high ATP concentrations. Meanwhile, the ATP potency remained unchanged. Activation of the hP2X7R at low ATP concentrations via the high-affinity ATP effector site was not affected by the replacement of chloride by glutamate or iodide. To analyze the anion effect on the hP2X7R at the single-molecule level, we performed single-channel current measurements using the patch-clamp technique in the outside-out configuration. Chloride substitution did not affect the single-channel conductance, but the probability that the P2X7R channel was open increased when chloride was replaced by glutamate and decreased when chloride was replaced by iodide. This effect was due to an influence of the anions on the mean closed times of the hP2X7R channel. We conclude that hP2X7R channels are not anion-permeable in physiological Na+-based media and that external anions allosterically affect ion channel opening in the fully ATP4-liganded P2X7R through an extracellular anion binding site.
Biochimica et Biophysica Acta 08/2011; 1808(12):2913-22. · 4.66 Impact Factor
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ABSTRACT: The intravenous anesthetic propofol acts as a positive allosteric modulator of glycine (GlyRs) and γ-aminobutyric acid type A (GABAARs) receptors. Although the role of transmembrane residues is recognized, little is known about the involvement of other regions in the modulatory effects of propofol. Therefore, the influence of the large intracellular loop in propofol sensitivity of both receptors was explored.
The large intracellular loop of α1 GlyRs and α1β2 GABAARs was screened using alanine replacement. Sensitivity to propofol was studied using patch-clamp recording in HEK293 cells transiently transfected with wild type or mutant receptors.
Alanine mutation of a conserved phenylalanine residue within the α1 large intracellular loop significantly reduced propofol enhancement in both GlyRs (360 ± 30 vs. 75 ± 10%, mean ± SEM) and GABAARs (361 ± 49% vs. 80 ± 23%). Remarkably, propofol-hyposensitive mutant receptors retained their sensitivity to other allosteric modulators such as alcohols, etomidate, trichloroethanol, and isoflurane. At the single-channel level, the ability of propofol to increase open probability was significantly reduced in both α1 GlyR (189 ± 36 vs. 22 ± 13%) and α1β2 GABAAR (279 ± 29 vs. 29 ± 11%) mutant receptors.
In this study, it is demonstrated that the large intracellular loop of both GlyR and GABAAR has a conserved single phenylalanine residue (F380 and F385, respectively) that influences its sensitivity to propofol. Results suggest a new role of the large intracellular loop in the allosteric modulation of two members of the Cys-loop superfamily. Thus, these data provide new insights into the molecular framework behind the modulation of inhibitory ion channels by propofol.
Anesthesiology 06/2011; 115(3):464-73. · 5.36 Impact Factor
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ABSTRACT: N-Glycosylation is normally a co-translational process that occurs as soon as a nascent and unfolded polypeptide chain has emerged ~12 residues into the lumen of the endoplasmic reticulum (ER). Here, we describe the efficient utilization of an N-glycosylation site engineered within the luminal extreme C-terminal residues of distinct integral membrane glycoproteins, a native ER resident protein and an engineered secreted protein. This N-glycan addition required that the acceptor asparagine within an Asn-Trp-Ser sequon be located at least four residues away from the C-terminus of the polypeptide and, in the case of membrane proteins, at least 13 residues away from the lumenal side of the transmembrane segment. Pulse-chase assays revealed that the natural N-glycans of the proteins studied were attached co-translationally, whereas C-terminal N-glycosylation occurred post-translocationally within a time frame of hours in Xenopus laevis oocytes and minutes in human embryonic kidney 293 (HEK293) cells. In oocyte and HEK cell expression systems, affinity tag-driven C-terminal N-glycosylation may facilitate the determination of orientation of the C-terminal tail of membrane proteins relative to the membrane.
Glycobiology 02/2011; 21(9):1147-60. · 3.58 Impact Factor
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ABSTRACT: Evaluation and optimization of anthraquinone derivatives related to Reactive Blue 2 at P2X2 receptors yielded the first potent and selective P2X2 receptor antagonists. The compounds were tested for inhibition of ATP (10 μM) mediated currents in Xenopus oocytes expressing the rat P2X2 receptor. The most potent antagonists were sodium 1-amino-4-[3-(4,6-dichloro[1,3,5]triazine-2-ylamino)phenylamino]-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate (63, PSB-10211, IC(50) 86 nM) and disodium 1-amino-4-[3-(4,6-dichloro[1,3,5]triazine-2-ylamino)-4-sulfophenylamino]-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate (57, PSB-1011, IC(50) 79 nM). Compound 57 exhibited a competitive mechanism of action (pA(2) 7.49). It was >100-fold selective versus P2X4, P2X7, and several investigated P2Y receptor subtypes (P2Y(2,4,6,12)); selectivity versus P2X1 and P2X3 receptors was moderate (>5-fold). Compound 57 was >13-fold more potent at the homomeric P2X2 than at the heteromeric P2X2/3 receptor. Several anthraquinone derivatives were found to act as positive modulators of ATP effects at P2X2 receptors, for example, sodium 1-amino-4-(3-phenoxyphenylamino)-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate (51, PSB-10129, EC(50) 489 nM), which led to about a 3-fold increase in the ATP-elicited current.
Journal of Medicinal Chemistry 01/2011; 54(3):817-30. · 4.80 Impact Factor
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Mandy Bodnar,
Haihong Wang,
Thomas Riedel,
Stefan Hintze,
Erzsebet Kato,
Ghada Fallah,
Helke Gröger-Arndt,
Rashid Giniatullin,
Marcus Grohmann,
Ralf Hausmann, Günther Schmalzing,
Peter Illes,
Patrizia Rubini
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ABSTRACT: Homomeric P2X3 receptors are present in sensory ganglia and participate in pain perception. Amino acid (AA) residues were replaced in the four supposed nucleotide binding segments (NBSs) of the human (h) P2X3 receptor by alanine, and these mutants were expressed in HEK293 cells and Xenopus laevis oocytes. Patch clamp and two-electrode voltage clamp measurements as well as the Ca(2+) imaging technique were used to compare the concentration-response curves of the selective P2X1,3 agonist α,β-methylene ATP obtained at the wild-type P2X3 receptor and its NBS mutants. Within these NBSs, certain Gly (Gly-66), Lys (Lys-63, Lys-176, Lys-284, Lys-299), Asn (Asn-177, Asn-279), Arg (Arg-281, Arg-295), and Thr (Thr-172) residues were of great importance for a full agonist response. However, the replacement of further AAs in the NBSs by Ala also appeared to modify the amplitude of the current and/or [Ca(2+)](i) responses, although sometimes to a minor degree. The agonist potency decrease was additive after the simultaneous replacement of two adjacent AAs by Ala (K65A/G66A, F171A/T172A, N279A/F280A, F280A/R281A) but was not altered after Ala substitution of two non-adjacent AAs within the same NBS (F171A/N177A). SDS-PAGE in the Cy5 cell surface-labeled form demonstrated that the mutants appeared at the cell surface in oocytes. Thus, groups of AAs organized in NBSs rather than individual amino acids appear to be responsible for agonist binding at the hP2X3 receptor. These NBSs are located at the interface of the three subunits forming a functional receptor.
Journal of Biological Chemistry 01/2011; 286(4):2739-49. · 4.77 Impact Factor
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Christian Wolf,
Christiane Rosefort,
Ghada Fallah,
Matthias U Kassack,
Alexandra Hamacher,
Mandy Bodnar,
Haihong Wang,
Peter Illes,
Achim Kless,
Gregor Bahrenberg, Günther Schmalzing,
Ralf Hausmann
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ABSTRACT: P2X2 receptors are members of the ATP-gated P2X family of cation channels, and they participate in neurotransmission in sympathetic ganglia and interneurons. Here, we identified 7,7'-(carbonylbis(imino-3,1-phenylenecarbonylimino-3,1-(4-methyl-phenylene)carbonylimino))bis(1-methoxy-naphthalene-3,6-disulfonic acid) tetrasodium salt (NF770) as a nanomolar-potent competitive P2X2 receptor antagonist within a series of 139 suramin derivatives. Three structural determinants contributed to the inhibition of P2X2 receptors by NF770: 1) a "large urea" structure with two symmetric phenylenecarbonylimino groups; 2) attachment of the naphthalene moiety in position 7,7'; and 3) the specific position of two sulfonic acid groups (3,3'; 6,6') and of one methoxy group (1,1') at the naphthalene moiety. This structure-activity relationship was interpreted using a rat P2X2 homology model based on the crystal structure of the closed zebrafish P2X4 receptor. Docking of the suramin derivatives into the modeled ATP-binding pocket provides a uniform explanation for the observed differences in inhibitory potencies. Changes in the chemical structure that increase the inhibitory potency of the suramin derivatives improved the spatial orientation within the ATP-binding pocket to allow for stronger polar interactions of functional groups with Gly72, Glu167, or Arg290. Gly72 is responsible for the orientation of the methoxy group close to Arg290 or Glu167. Combined mutational and functional analysis confirmed that residues Gly72 and Glu167 are as important for ATP binding as Arg290, the ATP-binding role of which has been shown in previous studies. The in silico prediction of Gly72 and Glu167 as ATP-binding residues strongly supports the validity of our homology docking.
Molecular pharmacology 12/2010; 79(4):649-61. · 4.53 Impact Factor
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ABSTRACT: TMEM16A/anoctamin-1 has been identified as a protein with the classic properties of a Ca(2+)-activated chloride channel. Here, we used blue native polyacrylamide gel electrophoresis (BN-PAGE) and chemical cross-linking to assess the quaternary structure of the mouse TMEM16A(a) and TMEM16A(ac) splice variants as well as a genetically concatenated TMEM16A(a) homodimer. The constructs carried hexahistidyl (His) tags to allow for their purification using a nondenaturing metal affinity resin. Neither His-tagging nor head-to-tail concatenation of two copies of TMEM16A(a) noticeably affected Ca(2+)-induced measured macroscopic Cl(-) currents compared with the wild-type TMEM16A(a) channel. The digitonin-solubilized, nondenatured TMEM16A(a) protein migrated in the BN-PAGE gel as a homodimer, as judged by comparison with the concatenated TMEM16A(a) homodimer and channel proteins of known oligomeric structures (e.g. the voltage-gated Cl(-) channel CLC-1). Cross-linking with glutaraldehyde corroborated the homodimeric structure of TMEM16A(a). The TMEM16A(a) homodimer detected in Xenopus laevis oocytes and HEK 293 cells dissociated into monomers following denaturation with SDS, and reducing versus nonreducing SDS-PAGE provided no evidence for the presence of intersubunit disulfide bonds. Together, our data demonstrate that the Ca(2+)-activated chloride channel member TMEM16A shares an obligate homodimeric architecture with the hCLC-1 channel.
Molecular & Cellular Proteomics 10/2010; 10(2):M110.004697. · 7.40 Impact Factor
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Elena Adinolfi,
Maria Cirillo,
Ronja Woltersdorf,
Simonetta Falzoni,
Paola Chiozzi,
Patrizia Pellegatti,
Maria Giulia Callegari,
Doriana Sandonà,
Fritz Markwardt, Günther Schmalzing,
Francesco Di Virgilio
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ABSTRACT: P2X7 is the largest member of the P2X subfamily of purinergic receptors. A typical feature is the carboxyl tail, which allows formation of a large pore. Recently a naturally occurring truncated P2X7 splice variant, isoform B (P2X7B), has been identified. Here we show that P2X7B expression in HEK293 cells, a cell type lacking endogenous P2X receptors, mediated ATP-stimulated channel activity but not plasma membrane permeabilization, raised endoplasmic reticulum Ca(2+) content, activated the transcription factor NFATc1, increased the cellular ATP content, and stimulated growth. In addition, P2X7B-transfected HEK293 cells (HEK293-P2X7B), like most tumor cells, showed strong soft agar-infiltrating ability. When coexpressed with full-length P2X7 (P2X7A), P2X7B coassembled with P2X7A into a heterotrimer and potentiated all known responses mediated by this latter receptor. P2X7B mRNA was found to be widely distributed in human tissues, especially in the immune and nervous systems, and to a much higher level than P2X7A. Finally, P2X7B expression was increased on mitogenic stimulation of peripheral blood lymphocyte. Altogether, these data show that P2X7B is widely expressed in several human tissues, modulates P2X7A functions, participates in the control of cell growth, and may help understand the role of the P2X7 receptor in the control of normal and cancer cell proliferation.
The FASEB Journal 05/2010; 24(9):3393-404. · 5.71 Impact Factor
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ABSTRACT: Cys-loop receptors are pentameric ligand-gated ion channels (pLGICs) that mediate fast synaptic transmission. Here functional pentameric assembly of truncated fragments comprising the ligand-binding N-terminal ectodomains and the first three transmembrane helices, M1-M3, of both the inhibitory glycine receptor (GlyR) alpha1 and the 5HT(3)A receptor subunits was found to be rescued by coexpressing the complementary fourth transmembrane helix, M4. Alanine scanning identified multiple aromatic residues in M1, M3 and M4 as key determinants of GlyR assembly. Homology modeling and molecular dynamics simulations revealed that these residues define an interhelical aromatic network, which we propose determines the geometry of M1-M4 tetrahelical packing such that nascent pLGIC subunits must adopt a closed fivefold symmetry. Because pLGIC ectodomains form random nonstoichiometric oligomers, proper pentameric assembly apparently depends on intersubunit interactions between extracellular domains and intrasubunit interactions between transmembrane segments.
Nature Structural & Molecular Biology 12/2009; 17(1):90-8. · 12.71 Impact Factor
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ABSTRACT: Small- to medium-sized neurons in the dorsal root ganglion (DRG) convey nociceptive information to the spinal cord. The co-expression of TRPV1 receptors (sensitive to vanilloids, heat and acidic pH) with P2X(3) receptors (sensitive to extracellular ATP) has been found in many DRG neurons. We investigated whether the co-activation of these two receptor classes in small-diameter cells leads to a modulation of the resulting current responses shaping the intensity of pain sensation. The whole-cell patch clamp method was used to record agonist-induced currents in cultured rat DRG neurons and in HEK293 cells transfected with the respective wild-type recombinant receptors or their mutants. Co-immunoprecipitation studies were used to demonstrate the physical association of TRPV1 and P2X(3) receptors. At a negative holding potential, the P2X(3) receptor agonist alpha,beta-meATP induced less current in the presence of the TRPV1 agonist capsaicin than that in its absence. This inhibitory interaction was not changed at a positive holding potential, in a Ba(2+)-containing superfusion medium, or when the buffering of intrapipette Ca(2+) was altered. The C-terminal truncation at Glu362 of P2X(3) receptors abolished the TRPV1/P2X(3) cross-talk in the HEK293 expression system. Co-immunoprecipitation studies with polyclonal antibodies generated against TRPV1 and P2X(3) showed a visible signal in HEK293 cells transfected with both receptors. It is concluded that the two pain-relevant receptors TRPV1 and P2X(3) interact with each other in an inhibitory manner probably by a physical association established by a motif located at the C-terminal end of the P2X(3) receptor distal to Glu362.
Pain 03/2009; 143(1-2):26-36. · 5.78 Impact Factor
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ABSTRACT: The current understanding about ethanol effects on the ligand-gated ion channel (LGIC) superfamily has been restricted to identify potential binding sites within transmembrane (TM) domains in the Cys-loop family. Here, we demonstrate a key role of the TM3-4 intracellular loop and G betagamma signaling for potentiation of glycine receptors (GlyRs) by ethanol. We discovered 2 motifs within the large intracellular loop of the GlyR alpha(1) subunit that are critical for the actions of pharmacological concentrations of ethanol. Significantly, the sites were ethanol-specific because they did not alter the sensitivity to general anesthetics, neurosteroids, or longer n-alcohols. Furthermore, G betagamma scavengers selectively attenuated the ethanol effects on recombinant and native neuronal GlyRs. These results show a selective mechanism for low-ethanol concentration effects on the GlyR and provide a mechanism on ethanol pharmacology, which may be applicable to other LGIC members. Moreover, these data provide an opportunity to develop new genetically modified animal models and novel drugs to treat alcohol-related medical concerns.
Proceedings of the National Academy of Sciences 01/2009; 105(51):20523-8. · 9.68 Impact Factor
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ABSTRACT: The SLC26 gene family encodes multifunctional transport proteins in numerous tissues and organs. Some paralogs function as anion exchangers, others as anion channels, and one, prestin (SLC26A5), represents a membrane-bound motor protein in outer hair cells of the inner ear. At present, little is known about the molecular basis of this functional diversity. We studied the subunit stoichiometry of one bacterial, one teleost, and two mammalian SLC26 isoforms expressed in Xenopus laevis oocytes or in mammalian cells using blue native PAGE and chemical cross-linking. All tested SLC26s are assembled as dimers composed of two identical subunits. Co-expression of two mutant prestins with distinct voltage-dependent capacitances results in motor proteins with novel electrical properties, indicating that the two subunits do not function independently. Our results indicate that an evolutionarily conserved dimeric quaternary structure represents the native and functional state of SLC26 transporters.
Journal of Biological Chemistry 03/2008; 283(7):4177-88. · 4.77 Impact Factor
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ABSTRACT: P2X(3) and P2X(2+3) receptors are present on sensory neurons, where they contribute not only to transient nociceptive responses, but also to hypersensitivity underlying pathological pain states elicited by nerve injuries. Increased signalling through P2X(3) and P2X(2+3) receptors may arise from an increased routing to the plasma membrane and/or gain of function of pre-existing receptors. An obvious effector mechanism for functional modulation is protein kinase C (PKC)-mediated phosphorylation, since all P2X family members share a conserved consensus sequence for PKC, TXR/K, within the intracellularly located N-terminal domain. Contradictory reports have been published regarding the exact role of this motif. In the present study, we confirm that site-directed elimination of the potential phosphor-acceptor threonine or the basic residue in the P+2 position of the TXR/K sequence accelerates desensitization of P2X(2) receptors and abolishes P2X(3) receptor function. Moreover, the PKC activator phorbol 12-myristate 13-acetate increased P2X(3) (but not P2X(2)) receptor-mediated currents. Biochemically, however, we were unable to demonstrate by various experimental approaches a direct phosphorylation of wild-type P2X(2) and P2X(3) receptors expressed in both Xenopus laevis oocytes and HEK293 cells. In conclusion, our data support the view that the TXR/K motif plays an important role in P2X function and that phorbol 12-myristate 13-acetate is capable of modulating some P2X receptor subtypes. The underlying mechanism, however, is unlikely to involve direct PKC-mediated P2X receptor phosphorylation.
Purinergic Signalling 10/2007; 3(4):377-88. · 3.16 Impact Factor
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ABSTRACT: Gephyrin is a bifunctional modular protein that, in neurons, clusters glycine receptors and gamma-aminobutyric acid, type A receptors in the postsynaptic membrane of inhibitory synapses. By x-ray crystallography and cross-linking, the N-terminal G-domain of gephyrin has been shown to form trimers and the C-terminal E-domain dimers, respectively. Gephyrin therefore has been proposed to form a hexagonal submembranous lattice onto which inhibitory receptors are anchored. Here, crystal structure-based substitutions at oligomerization interfaces revealed that both G-domain trimerization and E-domain dimerization are essential for the formation of higher order gephyrin oligomers and postsynaptic gephyrin clusters. Insertion of the alternatively spliced C5' cassette into the G-domain inhibited clustering by interfering with trimerization, and mutation of the glycine receptor beta-subunit binding region prevented the localization of the clusters at synaptic sites. Together our findings show that domain interactions mediate gephyrin scaffold formation.
Journal of Biological Chemistry 03/2007; 282(8):5625-32. · 4.77 Impact Factor
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[show abstract]
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ABSTRACT: Gephyrin is a bifunctional modular protein that, in neurons, clusters glycine receptors and γ-aminobutyric acid, type A receptors
in the postsynaptic membrane of inhibitory synapses. By x-ray crystallography and cross-linking, the N-terminal G-domain of
gephyrin has been shown to form trimers and the C-terminal E-domain dimers, respectively. Gephyrin therefore has been proposed
to form a hexagonal submembranous lattice onto which inhibitory receptors are anchored. Here, crystal structure-based substitutions
at oligomerization interfaces revealed that both G-domain trimerization and E-domain dimerization are essential for the formation
of higher order gephyrin oligomers and postsynaptic gephyrin clusters. Insertion of the alternatively spliced C5′ cassette
into the G-domain inhibited clustering by interfering with trimerization, and mutation of the glycine receptor β-subunit binding
region prevented the localization of the clusters at synaptic sites. Together our findings show that domain interactions mediate
gephyrin scaffold formation.
Journal of Biological Chemistry 02/2007; 282(8):5625-5632. · 4.77 Impact Factor
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ABSTRACT: Although fusion proteins of the extracellular parts of receptor subunits termed cytokine traps turned out to be promising cytokine inhibitors for anti-cytokine therapies, their mode of action has not been analyzed. We developed a fusion protein consisting of the ligand binding domains of the IL-6 receptor subunits IL-6Ralpha and gp130 that acts as a highly potent IL-6 inhibitor. Gp130 is a shared cytokine receptor also used by the IL-6-related cytokines oncostatin M and leukemia inhibitory factor. In this study, we have shown that the IL-6 receptor fusion protein (IL-6-RFP) is a specific IL-6 inhibitor that does not block oncostatin M or leukemia inhibitory factor. We characterized the complex of IL-6-RFP and fluorescently labeled IL-6 (YFPIL-6) by blue native PAGE and gel filtration. A 2-fold molar excess of IL-6-RFP over IL-6 was sufficient to entirely bind IL-6 in a complex with IL-6-RFP. As shown by treatment with urea and binding competition experiments, the complex of IL-6 and IL-6-RFP is more stable than the complex of IL-6, soluble IL-6Ralpha, and soluble gp130. By live cell imaging, we have demonstrated that YFP-IL-6 bound to the surface of cells expressing gp130-CFP is removed from the plasma membrane upon the addition of IL-6-RFP. The apparent molecular mass of the IL-6.IL-6-RFP complex determined by blue native PAGE and gel filtration suggests that IL-6 is trapped in a structure analogous to the native hexameric IL-6 receptor complex. Thus, fusion of the ligand binding domains of heteromeric receptors leads to highly specific cytokine inhibitors with superior activity compared with the separate soluble receptors.
Journal of Biological Chemistry 02/2007; 282(2):1238-48. · 4.77 Impact Factor
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[show abstract]
[hide abstract]
ABSTRACT: Although fusion proteins of the extracellular parts of receptor subunits termed cytokine traps turned out to be promising
cytokine inhibitors for anti-cytokine therapies, their mode of action has not been analyzed. We developed a fusion protein
consisting of the ligand binding domains of the IL-6 receptor subunits IL-6Rα and gp130 that acts as a highly potent IL-6
inhibitor. Gp130 is a shared cytokine receptor also used by the IL-6-related cytokines oncostatin M and leukemia inhibitory
factor. In this study, we have shown that the IL-6 receptor fusion protein (IL-6-RFP) is a specific IL-6 inhibitor that does
not block oncostatin M or leukemia inhibitory factor. We characterized the complex of IL-6-RFP and fluorescently labeled IL-6
(YFPIL-6) by blue native PAGE and gel filtration. A 2-fold molar excess of IL-6-RFP over IL-6 was sufficient to entirely bind
IL-6 in a complex with IL-6-RFP. As shown by treatment with urea and binding competition experiments, the complex of IL-6
and IL-6-RFP is more stable than the complex of IL-6, soluble IL-6Rα, and soluble gp130. By live cell imaging, we have demonstrated
that YFP-IL-6 bound to the surface of cells expressing gp130-CFP is removed from the plasma membrane upon the addition of
IL-6-RFP. The apparent molecular mass of the IL-6·IL-6-RFP complex determined by blue native PAGE and gel filtration suggests
that IL-6 is trapped in a structure analogous to the native hexameric IL-6 receptor complex. Thus, fusion of the ligand binding
domains of heteromeric receptors leads to highly specific cytokine inhibitors with superior activity compared with the separate
soluble receptors.
Journal of Biological Chemistry 01/2007; 282(2):1238-1248. · 4.77 Impact Factor
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ABSTRACT: Functional homomeric and heteromeric ATP-gated P2X receptor channels have been shown to display a characteristic trimeric architecture. Of the seven different isoforms (designated P2X(1)-P2X(7)), P2X(5) occurs in humans primarily as a non-functional variant lacking the C-terminal end of the ectodomain and the outer half of the second transmembrane domain. We show that this truncated variant, which results from the splice-skipping of exon 10, is prone to subunit aggregation because the residual transmembrane domain 2 is too short to insert into the membrane. Alleviation of the negative hydrophobic mismatch by the addition of a stretch of moderately hydrophobic residues enabled formation of a second membrane-spanning domain and strictly parallel homotrimerization. Systematic mutagenesis identified only one transmembrane domain 2 residue, Asp(355), which supported homotrimerization in a side chain-specific manner. Our results indicate that transmembrane domain 2 formation contributes 2-fold to hP2X(5) homotrimerization by tethering the end of the ectodomain to the membrane, thereby topologically restricting conformational mobility, and by intramembrane positioning of Asp(355). While transmembrane domain 2 appears to favor assembly by enabling productive subunit interactions in the ectodomain, Asp(355) seems to assist by simultaneously driving intramembrane helix interactions. Overall, these results indicate a complex interplay between topology, helix-helix interactions, and oligomerization to achieve a correctly folded structure.
Journal of Biological Chemistry 01/2007; 281(51):39561-72. · 4.77 Impact Factor
