WT Receptors Research Articles

Abstract 2472: Effective ER antagonists resist conformational restrictions imposed by somatic mutation but do not correlate with effects on receptor stability in live cells

Abstract Approximately 50% of luminal breast cancer patients become resistant to first-line endocrine therapies after an average of 5 years. Acquired activating mutations to the ESR1 ligand binding domain enable hormone therapy resistance in approximately 25-40% of these patients. Competitive estrogen antagonists function by maintaining the receptor in a transcriptionally inactive state by forcing estrogen receptor alpha ligand binding domain (ERα LBD) helix 12 into the AF-2 cleft to prevent the docking of LXXLL-containing transcriptional coactivators. Multiple next-generation ERα antagonists that function as molecular degraders of the receptor have been described and shown activity in breast cancers harboring the activating ESR1 mutations. To understand whether ERα antagonism correlates with receptor stability, we examined a comprehensive panel of structurally diverse antagonists in live breast cancer cell assays using WT, Y537S, and D538G-mutant receptors. We found that potent antagonists are able to both stabilize and degrade the receptor and further demonstrate that the D538G mutation reduces turnover with ICI due to specific defects in receptor ubiquitination and SUMOylation. High resolution x-ray crystal structures of WT and Y537S ERα LBD in complex with stabilizing and degrading antiestrogens suggest that modulators that enforce the classical antagonist-like helix 12 conformation in the mutant receptor achieve the greatest activity in transcriptional assays, regardless of their effects on receptor stability Citation Format: David Hosfield, Nan-Sheng Li, Ross Han, Muriel Laine, Sandra Weber, Madaline Sauvage, Sylvie Madar, Geoffrey Greene, Sean Fanning. Effective ER antagonists resist conformational restrictions imposed by somatic mutation but do not correlate with effects on receptor stability in live cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2472.

Proline Residues Contribute to Efficient GABAp Receptor Function.

GABAp receptors are homomeric pentameric ligand-gated ion channels (pLGICs) and are useful for probing the molecular details of the mechanism of action in this important protein family. Here, we explore the role of proline (Pro) residues by creating mutant receptors, expressing them in HEK293 cells, and using fluorescent membrane potential sensitive dye to monitor receptor activity. The data revealed that 3 of the Pro-to-alanine substitutions resulted in nonfunctional receptors (one in the Cys-loop, one in loop A and one in the β2-β3 loop), 7 resulted in increased EC50 values, and the remaining 13 resulted in receptors with properties similar to WT receptors. Further exploration of the nonfunctional mutants using expression in Xenopus laevis oocytes and whole-cell voltage-clamp electrophysiology, incorporating both canonical and noncanonical amino acids, revealed that the Pro in the Cys-loop had a preference for analogues with a high intrinsic cis bias, the Pro in loop A required a ring, and the β2-β3 loop Pro contributes to expression. The data from the Cys-loop Pro are consistent with those from other pLGICs, while those of the loop A Pro and some of the other Pros surprisingly differ. Thus, overall, the data clarify the roles of many of the Pros in the GABAp receptor and also suggest that caution must be applied in using data from one receptor to understand molecular details of all pLGICs.

The molecular determinants of neurosteroid binding in the GABA(A) receptor

Neurosteroids positively modulate GABA-A receptor (GABAAR) channel activity by binding to a transmembrane domain intersubunit site. Understanding the interactions in this site that determine neurosteroid binding and its effect is essential for the design of neurosteroid-based therapeutics. Using photo-affinity labeling and an ELIC-α1GABAAR chimera, we investigated the impact of mutations (Q242L, Q242W and W246L) within the intersubunit site on neurosteroid binding. These mutations, which abolish the thermal stabilizing effect of allopregnanolone on the chimera, reduce neither photolabeling within the intersubunit site nor competitive prevention of labeling by allopregnanolone. Instead, these mutations change the orientation of neurosteroid photolabeling. Molecular docking of allopregnanolone in WT and Q242W receptors confirms that the mutation favors re-orientation of allopregnanolone within the binding pocket. Collectively, the data indicate that mutations at Gln242 or Trp246 that eliminate neurosteroid effects do not eliminate neurosteroid binding within the intersubunit site, but significantly alter the preferred orientation of the neurosteroid within the site. The interactions formed by Gln242 and Trp246 within this pocket play a vital role in determining the orientation of the neurosteroid that may be necessary for its functional effect.

The GluN2B-Glu413Gly NMDA receptor variant arising from a de novo GRIN2B mutation promotes ligand-unbinding and domain opening.

N-methyl-D-aspartate (NMDA) receptors are transmembrane glutamate-binding ion channels that mediate neurotransmission in mammals. NMDA receptor subunits are tetrameric complexes of GluN1 and GluN2A-D subunits, encoded by the GRIN gene family. Of these subunits, GluN2B is suggested to be required for normal development of the central nervous system. A mutation identified in a patient with developmental delay, E413G, resides in the GluN2B ligand-binding domain and substantially reduces glutamate potency by an unknown mechanism. GluN2B Gly413, though near the agonist, is not in van der Waals contact with glutamate. Visual analysis of the GluN2B structure with the E413G mutation modeled suggests that replacement of Glu with Gly at this position increases solvent access to the ligand-binding domain. This was confirmed by molecular modeling, which showed that the ligand is more mobile in GluN2B-E413G than WT GluN2B. Evaluation of agonist occupancy using random accelerated molecular dynamics (RAMD) simulations predicts that the glutamate exits the binding-site more rapidly for GluN2B-E413G than WT receptors. This analysis was extended to other binding-site mutations, which produced qualitative agreement between experimentally determined EC50 values, deactivation time constants, and ligand motion within the binding-site. Furthermore, long sub-microsecond molecular dynamics simulations of the bi-lobed ligand-binding domain revealed that it adopted a cleft-open ligand-free state more often for GluN2B-E413G than wild-type GluN2B. This is consistent with the idea that L-glutamate binding is altered such that the ligand-binding domain occupies the open-cleft conformation associated with the closed channel.

Distinct Modulation of Spontaneous and GABA-Evoked Gating by Flurazepam Shapes Cross-Talk Between Agonist-Free and Liganded GABAA Receptor Activity.

GABAA receptors (GABAARs) play a crucial inhibitory role in the CNS. Benzodiazepines (BDZs) are positive modulators of specific subtypes of GABAARs, but the underlying mechanism remains obscure. Early studies demonstrated the major impact of BDZs on binding and more recent investigations indicated gating, but it is unclear which transitions are affected. Moreover, the upregulation of GABAAR spontaneous activity by BDZs indicates their impact on receptor gating but the underlying mechanisms remain unknown. Herein, we investigated the effect of a BDZ (flurazepam) on the spontaneous and GABA-induced activity for wild-type (WT, α1β2γ2) and mutated (at the orthosteric binding site α1F64) GABAARs. Surprisingly, in spite of the localization at the binding site, these mutations increased the spontaneous activity. Flurazepam (FLU) upregulated this activity for mutants and WT receptors to a similar extent by affecting opening/closing transitions. Spontaneous activity affected GABA-evoked currents and is manifested as an overshoot after agonist removal that depended on the modulation by BDZs. We explain the mechanism of this phenomenon as a cross-desensitization of ligand-activated and spontaneously active receptors. Moreover, due to spontaneous activity, FLU-pretreatment and co-application (agonist + FLU) protocols yielded distinct results. We provide also the first evidence that GABAAR may enter the desensitized state in the absence of GABA in a FLU-dependent manner. Based on our data and model simulations, we propose that FLU affects agonist-induced gating by modifying primarily preactivation and desensitization. We conclude that the mechanisms of modulation of spontaneous and ligand-activated GABAAR activity concerns gating but distinct transitions are affected in spontaneous and agonist-evoked activity.

Glycine Receptor Oligomerization Characterized by Number and Brightness Analysis

Glycine receptors (GlyR) are ligand-gated chloride channels that mediate synaptic inhibition in the CNS. A loss of GlyR function by single site mutations is the major cause hyperekplexia, a rare neuromotor disorder that is characterized by exaggerated startle reflexes. The V280M mutation in the α1 subunit causes heperekplexia but, surprisingly, results in a gain of function of the receptor. The mutant receptor shows spontaneous activation and prolonged deactivation. It has been suggested that V280M might change GlyRs oligomerization and the formation of clusters. We tested this idea by using Number and Brightness (N&B) analysis to determine GlyR oligomerization. A SNAP tag was fused to the N-terminal of wt and V280M human α1 GlyR. Receptors were expressed in HEK-293 cells. Cells were incubated with the Atto-488 fluorescent probe that covalently labels SNAP tag-containing receptors. N&B analysis measures the variance in fluorescence as receptors diffuse on the surface of the basolateral cell membrane. Larger variance corresponds to larger diffusing entities - multimers of GlyRs. Our tentative stoichiometry assignment is monomer, dimer, 4-mer and 8-mer. Using outside-out patch clamp recordings of macroscopic currents, we found that the SNAP-tagged wt and V280M GlyRs had similar glycine-sensitivity and kinetics compared to the corresponding untagged receptors. Using fluorescence measurements, we found that SNAP-tagged wt receptors tended to form dimers and 4-mers as the dominant species. In the presence of 3 mM glycine ,monomers were predominant. Addition of 1 µM strychnine antagonized the effect of glycine. SNAP-tagged V280M receptors exhibit a higher level of oligomerization than wt receptors. In preliminary data from 12 cells, the 8-mer species was dominant. Further experiments are needed to determine whether changes in receptor clustering underlie the hyperekplexia phenotype of V280M α1 GlyRs.

Spontaneous activity, singly bound states and the impact of alpha1Phe64 mutation on GABAAR gating in the novel kinetic model based on the single-channel recordings

GABAA receptor is the primary mediator of inhibition in the adult mammalian brain. Our recent studies revealed that a classic gating scheme for GABAAR needed to be updated with an intermediate step (flipping) and that the α1Phe64 mutation at the GABA binding site affects this transition. However, description of flipping at the single-channel level remains incomplete. In particular, its role in singly-bound and spontaneous activity remains unknown. We have performed thus single-channel recordings over wide range of agonist concentration for wild-type α1β2γ2L receptors and α1Phe64 mutants. For WT receptors we observed relatively frequent brief spontaneous openings which were also present at low [GABA]. However, closed times distributions for spontaneous activity and at low [GABA] were clearly different indicating that a proportion of short-lived openings were due to liganded, most likely singly bound receptors. Increasing [GABA] resulted in prolongation of bursts and increased occurrence of bursts with long openings and short closures. Mutations of α1Phe64 residue dramatically affected the open and closed time distributions at high and saturating [GABA], especially in the case of cysteine mutants. However, this mutation weakly affected spontaneous or singly bound activity. Model fitting of our single-channel data led us to propose a novel and, to our knowledge, most complete GABAAR kinetic model in which flipping occurs in singly and doubly bound states. However, spontaneous activity did not reveal involvement of flipping. Moreover, we report that α1Phe64 mutation affects not only the flipping but also the opening/closing transitions indicating its generalized impact on the receptor gating.

Abstract 392: The Role of Scavenger Receptor-BI’s Transmembrane Domains in Cholesterol Transport: A "Locked Dimer" Strategy

Efficient reverse cholesterol transport requires interactions between high density lipoprotein (HDL) and its receptor, scavenger receptor-BI (SR-BI). SR-BI is an 82 kDa protein with a large extracellular domain anchored by two transmembrane domains (TMDs). Our lab recently solved the NMR structure of SR-BI’s C-terminal TMD (C-TMD), a region that mediates SR-BI dimerization. Further, FRET studies suggest HDL-induced movement between neighboring SR-BI monomers, which led to our hypothesis that flexibility between SR-BI TMDs facilitates cholesterol transport. Using structure-guided mutagenesis, we introduced cysteine residues into the C-TMD of full-length SR-BI to create “locked dimers” of the receptor. Total lysate and cell surface expression of WT-, A444C-, L451C-, or G453C-SR-BI were verified in transiently-transfected COS-7 cells by immunoblot analysis and flow cytometry, respectively. Based on the predicted orientation of sulfhydryl side chains relative to the putative dimerization motif, we used immunoblot analysis following electrophoresis under reducing/non-reducing conditions to confirm that A444C- and L451C-SR-BI, but not G453C-SR-BI, formed disulfide bonds. Compared to WT-SR-BI, the locked dimer mutants, A444C- and L451C-SR-BI, exhibited normal selective uptake of [ 3 H]-cholesteryl oleyl ether, despite slightly reduced [ 125 I]-HDL binding. SR-BI-mediated cholesterol efflux to HDL from cells pre-labeled with [ 3 H]-cholesterol was also unaltered by the presence of locked dimers. Finally, we investigated the ability of WT and mutant SR-BI receptors to alter accessibility of membrane free cholesterol to exogenous cholesterol oxidase (as judged by cholestenone levels). L451C- or G453C-SR-BI expression led to reduced cholestenone production compared to WT-SR-BI, suggesting that these mutants may be defective in reorganizing pools of membrane cholesterol. In conclusion, our preliminary data suggest that limiting conformational flexibility between TMDs by forcing locked dimers of SR-BI may not have a major impact on SR-BI-mediated cholesterol transport. However, locked dimers of SR-BI appear to affect the ability of SR-BI to modulate plasma membrane pools of free cholesterol, and this deserves further investigation.

Loop G in the GABAA receptor α1 subunit influences gating efficacy.

The functional importance of residues in loop G of the GABAA receptor has not been investigated. D43 and T47 in the α1 subunit are of particular significance as their structural modification inhibits activation by GABA. While the T47C substitution had no significant effect, non-conservative substitution of either residue (D43C or T47R) reduced the apparent potency of GABA. Propofol potentiated maximal GABA-evoked currents mediated by α1(D43C)β2γ2 and α1(T47R)β2γ2 receptors. Non-stationary variance analysis revealed a reduction in maximal GABA-evoked Popen , suggesting impaired agonist efficacy. Further analysis of α1(T47R)β2γ2 receptors revealed that the efficacy of the partial agonist THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridine-3-ol) relative to GABA was impaired. GABA-, THIP- and propofol-evoked currents mediated by α1(T47R)β2γ2 receptors deactivated faster than those mediated by α1β2γ2 receptors, indicating that the mutation impairs agonist-evoked gating. Spontaneous gating caused by the β2(L285R) mutation was also reduced in α1(T47R)β2(L285R)γ2 compared to α1β2(L285R)γ2 receptors, confirming that α1(T47R) impairs gating independently of agonist activation. The modification of cysteine residues (substituted for D43 and T47) by 2-aminoethyl methanethiosulfonate in the GABAA α1 subunit loop G is known to impair activation of α1β2γ2 receptors by GABA and propofol. While the T47C substitution had no significant effect, non-conservative substitution of either residue (D43C or T47R) reduced the apparent potency of GABA. Propofol (1μm), which potentiates sub-maximal but not maximal GABA-evoked currents mediated by α1β2γ2 receptors, also potentiated maximal currents mediated by α1(D43C)β2γ2 and α1(T47R)β2γ2 receptors. Furthermore, the peak open probabilities of α1(D43C)β2γ2 and α1(T47R)β2γ2 receptors were reduced. The kinetics of macroscopic currents mediated by α1(D43C)β2γ2 and α1(T47R)β2γ2 receptors were characterised by slower desensitisation and faster deactivation. Similar changes in macroscopic current kinetics, together with a slower activation rate, were observed with the loop D α1(F64C) substitution, known to impair both efficacy and agonist binding, and when the partial agonist THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridine-3-ol) was used to activate WT or α1(T47R)β2γ2 receptors. Propofol-evoked currents mediated by α1(T47R)β2γ2 and α1(F64C)β2γ2 receptors also exhibited faster deactivation than their WT counterparts, revealing that these substitutions impair gating through a mechanism independent of orthosteric binding. Spontaneous gating caused by the introduction of the β2(L285R) mutation was also reduced in α1(T47R)β2(L285R)γ2 compared to α1β2(L285R)γ2 receptors, confirming that α1(T47R) impairs gating independently of activation by any agonist. These findings implicate movement of the GABAA receptor α1 subunit's β1 strand during agonist-dependent and spontaneous gating. Immobilisation of the β1 strand may provide a mechanism for the inhibition of gating by inverse agonists such as bicuculline.

Conditional Expression of Mutant ELANE Produces Unfolded Protein Response but Fails to Promote Cell Death or Differentiation Block: What Is the Mechanism for Severe Congenital Neutropenia?

Introduction: Germline mutations in the neutrophil elastase gene ELANE cause ~60% of severe congenital neutropenia (SCN). SCN frequently transforms to acute myeloid leukemia concomitant with acquisition of a mutant, truncated CSF3R, e.g. CSF3R d715.Two hypotheses have been advanced to explain the neutropenia: mislocalization of ELANE or unfolded protein response (UPR) induced by mutant ELANE. We present here data that demonstrates UPR induction by mutant ELANE but fails to results in either enhanced cell death or block in neutrophilic differentiation.Methods: We expressed mutant ELANE G185R found in patients with SCN. We used the IL-3-dependent murine myeloid 32D cell line that can terminally differentiate into neutrophils. Using the 32D cell, we developed 32D cell lines that express a chimeric rabbit growth hormone receptor (rGHR)/CSF3R receptor to prevent crosstalk with endogenous murine CSF3R. We used 32D cells that express either the wt or mutant d715 chimeric receptors: rGHR/wtCSF3R or rGHR/d715 respectively. The goal of our study was to identify how mutant ELANE expression is tolerated in the presence of wt CSF3R or CSF3R d715 signaling. We wanted to determine the effect of ELANE expression on cell survival, proliferation, differentiation, and UPR in context of wt vs mutant CSF3R signaling. To stimulate cytoplasmic CSF3R signaling the cells are treated with human growth hormone (hGH). We generated: rGHR/wtCSF3R-wtELANE, rGHR/wtCSF3R-G185R, rGHR/d175-wtELANE, and rGHR/d715-G185R. Cell proliferation, viability and differentiation assays were performed ± hGH.Results: No differences were observed in viability or differentiation. Interestingly cells expressing the mutant ELANE G185R showed enhanced proliferation vs wt hELANE, observed only in the context of rGHR/wtCSF3R and not with rGHR/d715. Morphological and flow cytometry based differentiation assays identified cell lines expressing GHR/wtCSF3R showed terminal differentiation. However, as expected rGHR/d715 expressing cell lines did not differentiate. We next determined if UPR is observed in these cells. Quantitative reverse transcribed PCR (qPCR) and western blot analysis identified increased expression of BiP at mRNA level and CHOP at both mRNA and protein levels in cells expressing the mutant ELANE G185R. CHOP has been identified to be the pro-apoptotic component of the UPR. Thus we observed enhanced CHOP expression, but we observed a paradoxical increase in proliferation for cells expressing ELANE G185R. We next identified that only rGHR/wtCSF3R-G185R cells were sensitive to either tunicamycin or bortezomib treatment, potent inducers of UPR. Because these cell lines were stable transfectants and might have adapted to mutant ELANE expression, we next put wt or mutant ELANE in a doxycycline-inducible expression system and transduced parental 32D cells. Induction of ELANE was dose and time dependent. Similar to the stable expression system, phenotypic differences between ± doxycycline treatments were not observed even though an increase in mRNA of UPR mediators was detected: CHOP, BiP, GADD34, PERK ATF4, and ATF6. An interesting observation was that, although there was a pan increase in UPR protein mRNA, there was a reduction in XBP1 splicing. IRE-1 mRNA levels were also low or undetectable in our cell lines. This might explain reduced XBP1 splicing. However, when 32D parent cells were treated with thapsigargin, a potent inducer of UPR and cell death, an increase in XBP1 splicing was observed.Conclusions: Our observations suggest that UPR induction by mutant ELANE is not strong enough to promote cell death and that mutant ELANE causes SCN through an alternative mechanism.This work was supported by NIH R01R01HL128173. DisclosuresNo relevant conflicts of interest to declare.

Abstract P194: A Novel Signalosome in the Alpha1a-Adrenergic Receptor and its Genetic Variant Signaling Pathway

Objectives : Human α 1 adrenergic receptors (AR), members of G protein-coupled receptor superfamily (GPCR) regulate blood pressure via smooth muscle cell (SMC) proliferation and vasoconstriction. We showed that α 1a AR-G247R (247R) genetic variant, identified in a hypertensive patient, constitutively couples to β-arrestin1/MMP/EGFR transactivation pathway leading to hyperproliferation in fibroblasts, cardiomyoblasts and SMCs. A scaffolding protein spinophilin (SPL) serves as a docking site for many regulatory proteins including RGS2 (negative R egulators of G -protein S ignaling). SPL also binds 3 rd intracellular loop of some GPCRs. Hypothesis: differential interaction of 247R or α 1a AR-WT (WT) with SPL/RGS2/β-arrestin signalosome mediates unique signaling of 247R and hyperproliferation in cardiovascular cells. Methods: Receptor-protein interactions were determined by co-immunoprecipitation from HEK293 cells expressing HA-α 1 ARs and full length Myc-SPL, its fragments or Flag-RGS2. Protein levels were analyzed by Western. SPL knockdown or RGS2 overexpression was performed using SPL-specific or scrambled siRNA or Flag-RGS2. Results: Our results reveal that in SMCs or cardiomyoblasts 247R but not WT upregulates endogenous SPL. SPL exhibits stronger (~2-fold) interaction with WT compared to 247R or α 1b , recruits RGS2 and inhibits receptor signaling. In contrast, weak SPL-247R interaction diminishes RGS2 inhibitory effect permitting hyperproliferation of 247R cells. SPL knockdown inhibits 247R-induced proliferation by allowing RGS2 directly bind to 247R as observed with RGS2 overexpression. Overexpression of SPL with RGS2 restores hyperproliferation suggesting that in 247R cells SPL binds RGS2 preventing RGS2-247R interaction. Conclusions: We present SPL/RGS2/β-arrestin as a novel signalosome responsible for α 1a AR-247R genetic variant triggered hyperproliferation in different cardiovascular cells. We reveal that SPL regulates α 1a AR signaling by differentially binding WT or 247R receptors and recruiting RGS2 protein to the receptor. These novel findings unravel critical roles of SPL and RGS2 in α 1 AR signaling, as well as identify SPL as a potential novel target for treatment of α 1 AR-mediated cardiovascular disorders.

An atypical residue in the pore of Varroa destructor GABA-activated RDL receptors affects picrotoxin block and thymol modulation

GABA-activated RDL receptors are the insect equivalent of mammalian GABAA receptors, and play a vital role in neurotransmission and insecticide action. Here we clone the pore lining M2 region of the Varroa mite RDL receptor and show that it has 4 atypical residues when compared to M2 regions of most other insects, including bees, which are the major host of Varroa mites. We create mutant Drosophila RDL receptors containing these substitutions and characterise their effects on function. Using two electrode voltage clamp electrophysiology we show that one substitution (T6′M) ablates picrotoxin inhibition and increases the potency of GABA. This mutation also alters the effect of thymol, which enhances both insect and mammalian GABA responses, and is widely used as a miticide. Thymol decreases the GABA EC50 of WT receptors, enhancing responses, but in T6′M-containing receptors it is inhibitory. The other 3 atypical residues have no major effects on either the GABA EC50, the picrotoxin potency or the effect of thymol. In conclusion we show that the RDL 6′ residue is important for channel block, activation and modulation, and understanding its function also has the potential to prove useful in the design of Varroa-specific insecticidal agents.

Kinetics of leptin binding to the Q223R leptin receptor.

Studies in human populations and mouse models of disease have linked the common leptin receptor Q223R mutation to obesity, multiple forms of cancer, adverse drug reactions, and susceptibility to enteric and respiratory infections. Contradictory results cast doubt on the phenotypic consequences of this variant. We set out to determine whether the Q223R substitution affects leptin binding kinetics using surface plasmon resonance (SPR), a technique that allows sensitive real-time monitoring of protein-protein interactions. We measured the binding and dissociation rate constants for leptin to the extracellular domain of WT and Q223R murine leptin receptors expressed as Fc-fusion proteins and found that the mutant receptor does not significantly differ in kinetics of leptin binding from the WT leptin receptor. (WT: ka 1.76×106±0.193×106 M−1 s−1, kd 1.21×10−4±0.707×10−4 s−1, KD 6.47×10−11±3.30×10−11 M; Q223R: ka 1.75×106±0.0245×106 M−1 s−1, kd 1.47×10−4±0.0505×10−4 s−1, KD 8.43×10−11±0.407×10−11 M). Our results support earlier findings that differences in affinity and kinetics of leptin binding are unlikely to explain mechanistically the phenotypes that have been linked to this common genetic variant. Future studies will seek to elucidate the mechanism by which this mutation influences susceptibility to metabolic, infectious, and malignant pathologies.

ATP Binding Site Mutagenesis Reveals Different Subunit Stoichiometry of Functional P2X2/3 and P2X2/6 Receptors

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.

Nedd4-1 binds and ubiquitylates activated FGFR1 to control its endocytosis and function

Fibroblast growth factor receptor 1 (FGFR1) has critical roles in cellular proliferation and differentiation during animal development and adult homeostasis. Here, we show that human Nedd4 (Nedd4-1), an E3 ubiquitin ligase comprised of a C2 domain, 4 WW domains, and a Hect domain, regulates endocytosis and signalling of FGFR1. Nedd4-1 binds directly to and ubiquitylates activated FGFR1, by interacting primarily via its WW3 domain with a novel non-canonical sequence (non-PY motif) on FGFR1. Deletion of this recognition motif (FGFR1-Δ6) abolishes Nedd4-1 binding and receptor ubiquitylation, and impairs endocytosis of activated receptor, as also observed upon Nedd4-1 knockdown. Accordingly, FGFR1-Δ6, or Nedd4-1 knockdown, exhibits sustained FGF-dependent receptor Tyr phosphorylation and downstream signalling (activation of FRS2α, Akt, Erk1/2, and PLCγ). Expression of FGFR1-Δ6 in human embryonic neural stem cells strongly promotes FGF2-dependent neuronal differentiation. Furthermore, expression of this FGFR1-Δ6 mutant in zebrafish embryos disrupts anterior neuronal patterning (head development), consistent with excessive FGFR1 signalling. These results identify Nedd4-1 as a key regulator of FGFR1 endocytosis and signalling during neuronal differentiation and embryonic development.

A novel Polymorphism Linked to Epilepsy Encoding a Missense Mutation in the Pre-M1 Region of a6 Subunits Alters the Gating, but not Trafficking, of GABAa Receptors

Charged residues in the pre-M1 region of Cys-loop ligand-gated ion channels affect current activation, desensitization and deactivation, suggesting that these interface residues may be involved in coupling agonist binding to channel gating. Mutations that affect function and/or expression of GABAA receptors (GABARs) have been associated with idiopathic generalized epilepsies (IGEs). The Parallel Sequence Profiling of Ion Channels in Epilepsy Project at Baylor College of Medicine funded by NINDS and NHGRI identified a novel single nucleotide polymorphism encoding a missense mutation, Q237R, in the GABAR α6 subunit in a patient with an IGE (http://www.hgsc.bcm.tmc.edu/project-medseq-r-ionchannel.hgsc). Homology modeling suggested that Q237R is located within the β10-M1linker or “pre-M1” segment of the N-terminal α6 subunit extracellular domain where it contributes to GABAR subunit-subunit interfaces. We studied gating properties and surface expression of wild type (wt) α6β2γ2 and mutant α6(Q237R)β2γ2 receptors expressed in HEK293T cells. Transient currents were evoked using ultrafast application of 400-µsec pulses of saturating GABA (1 mM) applied to excised outside-out macropatches. Peak amplitudes of α6(Q237R)β2γ2 currents were not reduced but α6(Q237R)β2γ2 currents had slower activation (∼1.9 msec), slower and more desensitization (∼60 %), and faster deactivation (∼2-fold) than wt currents. On-cell α6(Q237R)β2γ2 and α6β2γ2 single channel currents had similar mean open durations, but opening and burst frequencies were significantly higher for mutant than for wt currents. We assessed surface and total cellular expression of wt and mutant receptors using flow cytometry. Co-expression of α6(Q237R) with β2 and γ2L subunits resulted in no significant reduction in total or surface expression of any subunit. We concluded that this mutation alters gating, but not trafficking, of α6β2γ2 GABARs.

Amino Acid Residues That Confer High Selectivity of the α6 Nicotinic Acetylcholine Receptor Subunit to α-Conotoxin MII[S4A,E11A,L15A

Nicotinic acetylcholine receptors (nAChRs) containing alpha3 and beta2 subunits are found in autonomic ganglia and mediate ganglionic transmission. The closely related alpha6 nAChR subtype is found in the central nervous system where changes in its level of expression are observed in Parkinson's disease. To obtain a ligand that discriminates between these two receptors, we designed and synthesized a novel analog ofalpha-conotoxin MII, MII[S4A,E11A,L15A], and tested it on nAChRs expressed in Xenopus oocytes. The peptide blocked chimeric alpha6/alpha3beta2beta3 nAChRs with an IC(50) of 1.2 nm; in contrast, its IC(50) on the closely related alpha3beta2 as well as non-alpha6 nAChRs was three orders of magnitude higher. We identified the residues in the receptors that are responsible for their differential sensitivity to the peptide. We constructed chimeras with increasingly longer fragments of the N-terminal ligand binding domain of the alpha3 subunit inserted into the homologous positions of the alpha6 subunit, and these were used to determine that the region downstream of the first 140 amino acids was involved. Further mutagenesis of this region revealed that the alpha6 subunit residues Glu-152, Asp-184, and Thr-195 were critical, and replacement of these three residues with their homologs from the alpha3 subunit increased the IC(50) of the peptide by >1000-fold. Conversely, when these key residues inalpha3 were replaced with those fromalpha6, the IC(50) decreased by almost 150-fold. Similar effects were seen with other alpha6-selective conotoxins, suggesting the general importance of thesealpha6 residues in conferring selective binding.

Phosphorylation of Liver X Receptor α Selectively Regulates Target Gene Expression in Macrophages

Dysregulation of liver X receptor alpha (LXRalpha) activity has been linked to cardiovascular and metabolic diseases. Here, we show that LXRalpha target gene selectivity is achieved by modulation of LXRalpha phosphorylation. Under basal conditions, LXRalpha is phosphorylated at S198; phosphorylation is enhanced by LXR ligands and reduced both by casein kinase 2 (CK2) inhibitors and by activation of its heterodimeric partner RXR with 9-cis-retinoic acid (9cRA). Expression of some (AIM and LPL), but not other (ABCA1 or SREBPc1) established LXR target genes is increased in RAW 264.7 cells expressing the LXRalpha S198A phosphorylation-deficient mutant compared to those with WT receptors. Surprisingly, a gene normally not expressed in macrophages, the chemokine CCL24, is activated specifically in cells expressing LXRalpha S198A. Furthermore, inhibition of S198 phosphorylation by 9cRA or by a CK2 inhibitor similarly promotes CCL24 expression, thereby phenocopying the S198A mutation. Thus, our findings reveal a previously unrecognized role for phosphorylation in restricting the repertoire of LXRalpha-responsive genes.

High-frequency HTR3B variant associated with major depression dramatically augments the signaling of the human 5-HT 3AB receptor

The 5-hydroxytryptamine-3 (5-HT3) receptor mediates the fast excitatory neurotransmission of serotonin and is known to mediate the nausea/emesis induced by radio/chemotherapy and anesthetics. A polymorphism encoding the variation Y129S in the 5-HT3B subunit exists in high frequency in the general population and has been shown to be inversely correlated to the incidence of major depression in women. We show that 5-HT3AB(Y129S) receptors exhibit a substantially increased maximal response to serotonin compared with WT receptors in two fluorescence-based cellular assays. In electrophysiological recordings, the deactivation and desensitization kinetics of the 5-HT3AB(Y129S) receptor are 20- and 10-fold slower, respectively, than those of the WT receptor. Single-channel measurements reveal a 7-fold-increased mean open time of 5-HT3AB(Y129S) receptors compared with WT receptors. The augmented signaling displayed by 5-HT3AB(Y129S) receptors may confer protection against the development of depression. The variant also may influence the development and/or treatment of nausea and other disorders involving 5-HT3 receptors. Thus, the impact of the high-frequency variant 5-HT3B(Y129S) on 5-HT3AB receptor signaling calls for a search for additional phenotypes, and the variant may thus aid in establishing the role of the 5-HT3AB receptor in pathophysiology.

Properties of GluR3 receptors tagged with GFP at the amino or carboxyl terminus

Anatomical visualization of neurotransmitter receptor localization is facilitated by tagging receptors, but this process can alter their functional properties. We have evaluated the distribution and properties of WT glutamate receptor 3 (GluR3) alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors (WT GluR3) and two receptors in which GFP was tagged to the amino terminus (GFP-GluR3) or to the carboxyl terminus (GluR3-GFP). Although the fluorescence in Xenopus oocytes was stronger in the vegetal hemisphere because of localization of internal structures (probable sites of production, storage or recycling of receptors), the insertion of receptors into the plasma membrane was polarized to the animal hemisphere. The fluorescence intensity of oocytes injected with GluR3-GFP RNA was approximately double that of oocytes injected with GFP-GluR3 RNA. Accordingly, GluR3-GFP oocytes generated larger kainate-induced currents than GFP-GluR3 oocytes, with similar EC(50) values. Currents elicited by glutamate, or AMPA coapplied with cyclothiazide, were also larger in GluR3-GFP oocytes. The glutamate- to kainate-current amplitude ratios differed, with GluR3-GFP being activated more efficiently by glutamate than the WT or GFP-GluR3 receptors. This pattern correlates with the slower decay of glutamate-induced currents generated by GluR3-GFP receptors. These changes were not observed when GFP was tagged to the amino terminus, and these receptors behaved like the WT. The antagonistic effects of 6-nitro-7-sulfamoylbenzo[f]quinoxaline-2,3-dione (NBQX) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) were not altered in any of the tagged receptors. We conclude that GFP is a useful and convenient tag for visualizing these proteins. However, the effects of different sites of tag insertion on receptor characteristics must be taken into account in assessing the roles played by these receptor proteins.