Functional and molecular distinction between recombinant rat GABAA receptor subtypes by Zn2+.

gamma-Aminobutyric acid receptor (GABAAR) channels in different neurons display heterogeneous functional properties. Molecular cloning revealed a large number of GABAAR subunits that assemble into GABAAR subtypes with different functional properties, suggesting that the subunit combination determines the functional properties of the receptor. In this study, the subunit composition of GABAARs is related to a functional distinction between Zn2(+)-sensitive and Zn2(+)-insensitive receptor subtypes. GABAARs reconstituted in transiently transfected fibroblasts from combinations of cDNAs encoding alpha and beta subunits are potently blocked by Zn2+. The presence of a gamma subunit in any combination with the other subunits leads to the formation of GABAARs that are almost insensitive to Zn2+. These data provide a structural correlate to the functional heterogeneity of the action of Zn2+ on GABAARs in native membranes and show that Zn2+ insensitivity of GABA-activated currents indicates the presence of a gamma-subunit in the assembled GABAAR channel.     

The M3/M4 cytoplasmic loop of the alpha1 subunit restricts GABAARs lateral mobility: a study using fluorescence recovery after photobleaching

A crucial problem in neurobiology is how neurons are able to maintain neurotransmitter receptors at specific membrane domains. The large structural heterogeneity of gamma aminobutyric acid receptors (GABAARs) led to the hypothesis that there could be a link between GABAAR gene diversity and the targeting properties of the receptor complex. Previous studies using Fluorescence Recovery After Photobleaching (FRAP) have shown a restricted mobility in GABAARs containing the alpha1 subunit. The M3/M4 cytoplasmic loop is the region of the alpha1 subunit with the lowest sequence homology to other subunits. Therefore, we asked whether the M3/M4 loop is involved in cytoskeletal anchoring and GABAAR clustering. A series of alpha1 chimeric subunits was constructed: alpha1CH (control subunit), alpha1CD (Cytoplasmic loop deleted), alpha1CD2, and alpha1CD3 (alpha1 with the M3/M4 loop from the alpha2 and alpha3 subunits, respectively). Our results using FRAP indicate an involvement of the M3/M4 cytoplasmic loop of the alpha1 subunit in controlling receptor lateral mobility. On the other hand, inmunocytochemical approaches showed that this domain is not involved in subunit targeting to the cell surface, subunit-subunit assembly, or receptor aggregation.     

A GABAergic system in airway epithelium is essential for mucus overproduction in asthma

Gamma-aminobutyric acid (GABA) is an important neurotransmitter that, through the subtype A GABA receptor (GABAAR), induces inhibition in the adult brain. Here we show that an excitatory, rather than inhibitory, GABAergic system exists in airway epithelial cells. Both GABAARs and the GABA synthetic enzyme glutamic acid decarboxylase (GAD) are expressed in pulmonary epithelial cells. Activation of GABAARs depolarized these cells. The expression of GAD in the cytosol and GABAARs in the apical membranes of airway epithelial cells increased markedly when mice were sensitized and then challenged with ovalbumin, an approach for inducing allergic asthmatic reactions. Similarly, GAD and GABAARs in airway epithelial cells of humans with asthma increased after allergen inhalation challenge. Intranasal application of selective GABAAR inhibitors suppressed the hyperplasia of goblet cells and the overproduction of mucus induced by ovalbumin or interleukin-13 in mice. These findings show that a previously unknown epithelial GABAergic system has an essential role in asthma.     

Evolution of GABA(A) receptor diversity in the human genome

Nowhere is the record of receptor evolution more accessible than in the organization of the 19 vertebrate genes coding for subunits of the major inhibitory neurotransmitter receptor in the central nervous system, the gamma-aminobutyric acid receptor (GABAAR). Co-expression of alpha, beta, and gamma subunit genes is necessary for the formation of a GABAAR that is potentiated by widely used anxiolytics, anticonvulsants, and hypnotics. The identification of alpha, beta, and gamma genes on chromosomes 4, 5, and 15 suggests that co-localization of a gamma gene with an alpha and beta may be important for brain function. We have now directly examined the organization of GABAAR subunit genes on human chromosomes. Estimates of physical distance using in situ hybridization to cells in interphase, and gene localization using hybridization to cells in metaphase demonstrate the existence of beta-alpha-alpha-gamma gene clusters in cytogenetic bands on chromosomes 4(p12) and 5(q34). Sequencing of PAC clones establishes intercluster conservation of a unique head-to-head configuration for alpha and beta genes on chromosomes 4, 5, and 15. Remarkably, phylogenetic tree analysis predicts the existence of a beta-alpha-gamma ancestral gene cluster in which internal duplication of an ancestral alpha was followed by cluster duplication, resulting in the relative chromosomal positions of modern GABAAR subunit genes in the human genome.     

The alpha1 subunit of GABAA receptor is repressed by c-myc and is pro-apoptotic

The c-myc oncoprotein plays a critical role in the regulation of cellular proliferation and apoptosis. To mediate these biological functions, a variety of target genes are activated or repressed by c-myc, but few genes have yet been identified that directly mediate c-myc's role in proliferation or apoptosis. During a screen for genes that are repressed by c-myc, we identified the alpha1 subunit of gamma aminobutyric acid receptor (GABAAR-alpha1) as a novel target of c-myc. GABAAR is the major inhibitory neurotransmitter receptor in the mammalian central nervous system and is involved in developmental events in the brain, such as neurite outgrowth, neuronal survival, neuronal migration, and proliferation. We show here that GABAAR-alpha1 expression is rapidly and directly repressed by c-myc. GABAAR-alpha1 expression is elevated in c-myc null cells and upregulation of GABAAR-alpha1 correlates with downregulation of c-myc protein expression during neuronal cell differentiation. We also show that overexpression of GABAAR-alpha1 causes apoptosis, which is blocked by the coexpression of Bcl-2 or Bcl-XL. Induction of apoptosis is specific for the alpha1 subunit, since neither the beta1 or beta2 subunits of GABAAR induced apoptosis. Derepression of GABAAR-alpha1 expression upon downregulation of c-myc represents a unique apoptotic mechanism and a distinct function for the alpha1 subunit, independent of its role as a component of the GABAAR in the plasma membrane. In addition, the regulation of GABAAR-alpha1 expression by c-myc provides a potential direct role for the Myc proteins in neurological processes and neurodegenerative disorders.     

Cytoskeletal protein radixin activates integrin alpha(M)beta(2) by binding to its cytoplasmic tail

Talin binding of integrins, via its band 4.1, ezrin, radixin, and moesin (FERM)-homologous domain, directly activates the integrin receptor. However, it is not known whether other FERM-containing proteins also possess such an integrin activating capability. We report here that radixin, one of the original FERM-domain proteins, binds to the membrane-proximal region of the integrin beta(2) but not alpha(M) cytoplasmic tail. Importantly, we show that radixin binding significantly enhances the adhesive activity of integrin alpha(M)beta(2). Given the distinct biological activities of radixin and talin, radixin may represent a novel talin-independent pathway for integrin activation under specific settings.     

Regionally selective effects of GABA on hypothalamic GABAA receptor mRNA in vitro

We tested whether GABAA receptor (R) subunit mRNA levels are homeostatically influenced by short-term exposure to GABA in two adjacent regions of the posterior hypothalamus. mRNA levels for seven GABAAR subunits and GABA-synthesizing enzyme (GAD) were quantified in the perifornical (PF) and dorsomedial (DM) hypothalamus following superfusion of slices for 90 min with a drug-free medium, GABA uptake blocker with or without GABAAR antagonist, gabazine, or GABAAR agonist with tetrodotoxin. Increasing endogenous GABA decreased mRNAs for all seven GABAAR subunits in the PF, and for three also in the DM, region; gabazine antagonized these effects in the PF region only and increased GAD-65 mRNA. Stimulation of GABAARs in the presence of tetrodotoxin decreased mRNA for one GABAAR subunit (beta1). We conclude that, in the PF region where GABA facilitates sleep, increased GABA release may limit GABAAR-mediated inhibition, whereas in the DM region, GABA-induced changes are mainly mediated by non-GABAA receptors.     

Functional regulation of GABAA receptors in nervous system pathologies

Inhibitory neurotransmission is primarily governed by γ-aminobutyric acid (GABA) type A receptors (GABAARs). GABAARs are heteropentameric ligand-gated channels formed by the combination of 19 possible subunits. GABAAR subunits are subject to multiple types of regulation, impacting the localization, properties, and function of assembled receptors. GABAARs mediate both phasic (synaptic) and tonic (extrasynaptic) inhibition. While the regulatory mechanisms governing synaptic receptors have begun to be defined, little is known about the regulation of extrasynaptic receptors. We examine the contributions of GABAARs to the pathogenesis of neurodevelopmental disorders, schizophrenia, depression, epilepsy, and stroke, with particular focus on extrasynaptic GABAARs. We suggest that extrasynaptic GABAARs are attractive targets for the treatment of these disorders, and that research should be focused on delineating the mechanisms that regulate extrasynaptic GABAARs, promoting new therapeutic approaches.     

Calcium/calmodulin-dependent kinase II phosphorylation of the GABAA receptor alpha1 subunit modulates benzodiazepine binding

gamma-Aminobutyric acid (GABA) is the primary neurotransmitter that is responsible for the fast inhibitory synaptic transmission in the central nervous system. A major post-translational mechanism that can rapidly regulate GABAAR function is receptor phosphorylation. This study was designed to test the effect of endogenous calcium and calmodulin-dependent kinase II (CaM kinase II) activation on both allosteric modulator binding and GABAA receptor subunit phosphorylation. Endogenous CaM kinase II activity was stimulated, and GABAA receptors were subsequently analyzed for bothallosteric modulator binding properties and immunoprecipitated and analyzed for subunit phosphorylation levels. A significant increase in allosteric-modulator binding of the GABAAR was observed under conditions maximal for CaM kinase II activation. In addition, CaM kinase II activation resulted in a direct increase in phosphorylation of the GABAA receptor alpha1 subunit. The data suggest that the CaM kinase II-dependent phosphorylation of the GABAA receptor alpha1 subunit modulated allosteric modulator binding to the GABAA receptor.     

Vitronectin Induces Phosphorylation of Ezrin/Radixin/Moesin Actin-binding Proteins through Binding to Its Novel Neuronal Receptor Telencephalin

Vitronectin (VN) is an extracellular matrix protein abundantly present in blood and a wide variety of tissues and plays important roles in a number of biological phenomena mainly through its binding to αV integrins. However, its definite function in the brain remains largely unknown. Here we report the identification of telencephalin (TLCN/ICAM-5) as a novel VN receptor on neuronal dendrites. VN strongly binds to TLCN, a unique neuronal member of the ICAM family, which is specifically expressed on dendrites of spiny neurons in the mammalian telencephalon. VN-coated microbeads induce the formation of phagocytic cup-like plasma membrane protrusions on dendrites of cultured hippocampal neurons and trigger the activation of TLCN-dependent intracellular signaling cascade including the phosphorylation of ezrin/radixin/moesin actin-binding proteins and recruitment of F-actin and phosphatidylinositol 4,5-bisphosphate for morphological transformation of the dendritic protrusions. These results suggest that the extracellular matrix molecule VN and its neuronal receptor TLCN play a pivotal role in the phosphorylation of ezrin/radixin/moesin proteins and the formation of phagocytic cup-like structures on neuronal dendrites.     

Extrasynaptic GABAA receptors in the crosshairs of hormones and ethanol

Gamma-aminobutyric acid (GABA) is the main chemical inhibitory neurotransmitter in the brain. In the central nervous system (CNS) it acts on two distinct types of receptor: an ion channel, i.e., an "ionotropic" receptor permeable to Cl- and HCO3- (GABAA receptors) and a G-protein coupled "metabotropic" receptor that is linked to various effector mechanisms (GABAB receptors). This review will summarize novel developments in the physiology and pharmacology of GABAA receptors (GABAARs), specifically those found outside synapses. The focus will be on a particular combination of GABAAR subunits sensitive to ovarian and adrenal cortical steroid hormone metabolites that are synthesized in the brain (neurosteroids) and to sobriety impairing concentrations of ethanol. These receptors may be the final common pathway for interactions between ethanol and ovarian and stress-related neurosteroids.     

Identification of alpha 2-adrenergic receptor sites in human retinoblastoma (Y-79) and neuroblastoma (SH-SY5Y) cells

The existence of specific alpha 2-adrenergic receptor sites has been shown in human retinoblastoma (Y-79) and neuroblastoma (SH-SH5Y) cells using direct radioligand binding. [3H]Rauwolscine, a selective alpha 2-adrenergic receptor antagonist, exhibited high affinity, saturable binding to both Y-79 and SH-SY5Y cell membranes. The binding of alpha 1 specific antagonist, [3H]Prazocine, was not detectable in either cell type. Competition studies with antagonists yielded pharmacological characteristics typical of alpha 2-adrenergic receptors: rauwolscine greater than yohimbine greater than phentolamine greater than prazocine. Based on the affinity constants of prazocine and oxymetazoline, it appears that Y-79 cells contain alpha 2A receptor, whereas SH-SY5Y cells probably represent a mixture of alpha 2A and alpha 2B receptors. alpha 2-agonists clonidine and (-)epinephrine inhibition curves yielded high and low affinity states of the receptor in SH-SY5Y cells. Gpp(NH)p and sodium ions reduced the proportion of high affinity sites of alpha 2 receptors. These two neuronal cell lines of human origin would prove useful in elucidating the action and regulation of human alpha 2-adrenergic receptors and their interaction with other receptor systems.     

Using an α-Bungarotoxin Binding Site Tag to Study GABA A Receptor Membrane Localization and Trafficking

It is increasingly evident that neurotransmitter receptors, including ionotropic GABA A receptors (GABAAR), exhibit highly dynamic trafficking and cell surface mobility^1-7. To study receptor cell surface localization and endocytosis, the technique described here combines the use of fluorescent α-bungarotoxin with cells expressing constructs containing an α-bungarotoxin (Bgt) binding site (BBS). The BBS (WRYYESSLEPYPD) is based on the α subunit of the muscle nicotinic acetylcholine receptor, which binds Bgt with high affinity^8,9. Incorporation of the BBS site allows surface localization and measurements of receptor insertion or removal with application of exogenous fluorescent Bgt, as previously described in the tracking of GABAA and metabotropic GABAB receptors^2,10. In addition to the BBS site, we inserted a pH-sensitive GFP (pHGFP¹¹) between amino acids 4 and 5 of the mature GABAAR subunit by standard molecular biology and PCR cloning strategies (see Figure 1)¹². The BBS is 3'' of the pH-sensitive GFP reporter, separated by a 13-amino acid alanine/proline linker. For trafficking studies described in this publication that are based on fixed samples, the pHGFP serves as a reporter of total tagged GABAAR subunit protein levels, allowing normalization of the Bgt labeled receptor population to total receptor population. This minimizes cell to cell Bgt staining signal variability resulting from higher or lower baseline expression of the tagged GABAAR subunits. Furthermore the pHGFP tag enables easy identification of construct expressing cells for live or fixed imaging experiments.     

Three complement-like repeats compose the complete alpha2-macroglobulin binding site in the second ligand binding cluster of the low density lipoprotein receptor-related protein

Given the importance of the low density lipoprotein receptor-related protein (LRP) as an essential endocytosis and signaling receptor for many protein ligands, and of alpha2-macroglobulin (alpha2M)-proteinase complexes as one such set of ligands, an understanding of the specificity of their interaction with LRP is an important goal. A starting point is the known role of the 138-residue receptor binding domain (RBD) in binding to LRP. Previous studies have localized high affinity alpha2M binding to the eight complement repeat (CR)-containing cluster 2 of LRP. In the present study we have identified the minimum CR domains that constitute the full binding site for RBD and, hence, for alpha2M on LRP. We report on the ability of the triple construct of CR3-4-5 to bind RBD with an affinity (Kd = 130 nM) the same as for isolated RBD to intact LRP. This Kd is 30-fold smaller than for RBD to CR5-6-7, demonstrating the specificity of the interaction with CR3-4-5. Binding requires previously identified critical lysine residues but is almost pH-independent within the range of pH values encountered between extracellular and internal compartments, consistent with an earlier proposed model of intracellular ligand displacement by intramolecular YWTD domains. The present findings suggest a model to explain the ability of LRP to bind a wide range of structurally unrelated ligands in which a nonspecific ligand interaction with the acidic region present in most CR domains is augmented by interactions with other CR surface residues that are unique to a particular CR cluster.     

Structural determinants within residues 180-199 of the rodent alpha 5 nicotinic acetylcholine receptor subunit involved in alpha-bungarotoxin binding

Synthetic peptides corresponding to sequence segments of the nicotinic acetylcholine receptor (nAChR) alpha subunits have been used to identify regions that contribute to formation of the binding sites for cholinergic ligands. We have previously defined alpha-bungarotoxin (alpha-BTX) binding sequences between residues 180 and 199 of a putative rat neuronal nAChR alpha subunit, designated alpha 5 [McLane, K. E., Wu, X., & Conti-Tronconi, B. M. (1990) J. Biol. Chem. 265, 9816-9824], and between residues 181 and 200 of the chick neuronal alpha 7 and alpha 8 subunits [McLane, K. E., Wu, X., Schoepfer, R., Lindstrom, J., & Conti-Tronconi, B. M. (1991) J. Biol. Chem. (in press)]. These sequences are relatively divergent compared with the Torpedo and muscle nAChR alpha 1 alpha-BTX binding sites, which indicates a serious limitation of predicting functional domains of proteins based on homology in general. Given the highly divergent nature of the alpha 5 sequence, we were interested in determining the critical amino acid residues for alpha-BTX binding. In the present study, the effects of single amino acid substitutions of Gly or Ala for each residue of the rat alpha 5(180-199) sequence were tested, using a competition assay, in which peptides compete for 125I-alpha-BTX binding with native Torpedo nAChR.(ABSTRACT TRUNCATED AT 250 WORDS)     

Direct binding of neutral endopeptidase 24.11 to ezrin/radixin/moesin (ERM) proteins competes with the interaction of CD44 with ERM proteins

Neutral endopeptidase 24.11 (NEP) is a cell surface peptidase expressed by numerous tissues including prostatic epithelial cells. We reported that NEP inhibits prostate cancer cell proliferation and cell migration by enzymatic inactivation of neuropeptide substrates and through protein-protein interaction independent of catalytic function. The cytoplasmic domain of NEP contains a positively charged amino acid cluster, previously identified as a binding site for ezrin/radixin/moesin (ERM) proteins. We report here that NEP co-immunoprecipitates with ERM proteins in NEP-expressing LNCaP prostate cancer cells and MeWo melanoma cells. Co-immunoprecipitation showed that ERM proteins associate with wild-type NEP protein but not NEP protein containing a truncated cytoplasmic domain or point mutations replacing the positively charged amino acid cluster. In vitro binding assays showed that NEP binds directly to recombinant N terminus fragments of ERM proteins at the positively charged amino acid cluster within the NEP cytoplasmic domain. Binding of ERM proteins to NEP results in decreased binding of ERM proteins to the hyaluronan receptor CD44, a main binding partner of ERM proteins. Moreover, cells expressing wild-type NEP demonstrate decreased adhesion to hyaluronic acid and cell migration. These data suggest that NEP can affect cell adhesion and migration through direct binding to ERM proteins.     

Rabies virus binding to an acetylcholine receptor alpha-subunit peptide

The binding of 125I-labeled rabies virus to a synthetic peptide comprising residues 173-204 of the alpha 1-subunit of the nicotinic acetylcholine receptor was investigated. Binding of rabies virus to the receptor peptide was dependent on pH, could be competed with by unlabeled homologous virus particles, and was saturable. Synthetic peptides of snake venom, curaremimetic neurotoxins and of the structurally similar segment of the rabies virus glycoprotein, were effective in competing with labeled virus binding to the receptor peptide at micromolar concentrations. Similarly, synthetic peptides of the binding domain on the acetylcholine receptor competed for binding. These findings suggest that both rabies virus and neurotoxins bind to residues 173-204 of the alpha 1-subunit of the acetylcholine receptor. Competition studies with shorter alpha-subunit peptides within this region indicate that the highest affinity virus binding determinants are located within residues 179-192. A rat nerve alpha 3-subunit peptide, that does not bind alpha-bungarotoxin, inhibited binding of virus to the alpha 1 peptide, suggesting that rabies binds to neuronal nicotinic acetylcholine receptors. These studies indicate that synthetic peptides of the glycoprotein binding domain and of the receptor binding domain may represent useful antiviral agents by targeting the recognition event between the viral attachment protein and the host cell receptor, and inhibiting attachment of virus to the receptor.     

Layilin, a cell surface hyaluronan receptor, interacts with merlin and radixin

Layilin is a widely expressed integral membrane hyaluronan receptor, originally identified as a binding partner of talin located in membrane ruffles. We have identified merlin, the neurofibromatosis type 2 tumor suppressor protein and radixin, as other interactors with the carboxy-terminal domain of layilin. We show that the carboxy-terminal domain of layilin is capable of binding to the amino-terminal domain of radixin. An interdomain interaction between the amino- and the carboxy-terminal domains of radixin inhibits its ability to bind to layilin. In the presence of acidic phospholipids, the interdomain interaction of radixin is inhibited and layilin can bind to full-length radixin. In contrast, layilin binds both full-length and amino-terminal merlin-GST fusion proteins without a requirement for phospholipids. Furthermore, layilin antibody can immunoprecipitate merlin, confirming association in vivo between these two proteins, which also display similar subcellular localizations in ruffling membranes. No interaction was observed between layilin and ezrin or layilin and moesin. These findings expand the known binding partners of layilin to include other members of the talin/band 4.1/ERM (ezrin, radixin, and moesin) family of cytoskeletal-membrane linker molecules. This in turn suggests that layilin may mediate signals from extracellular matrix to the cell cytoskeleton via interaction with different intracellular binding partners and thereby be involved in the modulation of cortical structures in the cell.     

Trafficking and synaptic anchoring of ionotropic inhibitory neurotransmitter receptors

Neurotransmitter receptors are subject to microtubule-based transport between intracellular organelles and the neuronal plasma membrane. Receptors that arrive at plasma membrane compartments diffuse laterally within the plane of the cellular surface. To achieve immobilization at their sites of action, cytoplasmic receptor residues bind to submembrane proteins, which are coupled to the underlying cytoskeleton by multiprotein scaffolds. GABA(A)Rs (gamma-aminobutyric type A receptors) and GlyRs (glycine receptors) are the major inhibitory receptors in the central nervous system. At inhibitory postsynaptic sites, all GlyRs and the majority of GABA(A)Rs directly or indirectly couple to gephyrin, a multimeric PSD (postsynaptic density) component. In addition to cluster formations at axo-dendritic contacts, individual GABA(A)R subtypes also anchor and concentrate at extrasynaptic positions, either through association with gephyrin or direct interaction with the ERM (ezrin/radixin/moesin) family protein radixin. In addition to their role in diffusion trapping of surface receptors, scaffold components also undergo rapid exchange to/from and between postsynaptic specializations, leading to a dynamic equilibrium of receptor-scaffold complexes. Moreover, scaffold components serve as adaptor proteins that mediate specificity in intracellular transport complexes. In the present review, we discuss the dynamic delivery, stabilization and removal of inhibitory receptors at synaptic sites.     

Identification of calcium binding sites that regulate potentiation of a neuronal nicotinic acetylcholine receptor.

The divalent cation calcium potentiates the physiological response of neuronal nicotinic receptors to agonists by enhancing ionic current amplitudes, apparent agonist affinity and cooperativity. Here we show that mutations in several consensus Ca2+ binding sequences from the N-terminal domain of the neuronal alpha 7 nicotinic acetylcholine receptor alter Ca2+ potentiation of the alpha 7-V201-5HT3 chimera. Mutations E18Q or E44Q abolish calcium-enhanced agonist affinity but preserve the calcium increase of plateau current amplitudes and cooperativity. On the other hand, mutations of amino acids belonging to the 12 amino acid canonical domain (alpha 7 161-172) alter all features of potentiation by enhancing (D163, S169), reducing (E161, S165, Y167) or abolishing (E172) calcium effects on ionic current amplitudes and agonist affinity. Introduction of the alpha 7 161-172 domain in the calcium insensitive 5-hydroxytryptamine (5HT3) serotoninergic receptor results in a receptor activated by 5HT and potentiated by calcium. In vitro terbium fluorescence studies with an alpha 7 160-174 peptide further show that mutation E172Q also alters in vitro calcium binding. Data are consistent with the occurrence of distinct categories of regulatory calcium binding sites, among which the highly conserved (alpha 7 161-172) domain may simultaneously contribute to calcium and agonist binding.