17-beta estradiol and progesterone-induced alterations in human type-1/type-2 cytokine balance and the role of costimulatory and apoptotic mechanisms

Evidence suggests that sex-based differences in immune function may predispose women to numerous hypersensitivity conditions such as Systemic lupus erythematosus (SLE), Hashimoto's thyroiditis and asthma. To date, the exact mechanisms of sexual dimorphism in immunity are not fully characterized but sex hormones such as 17-β estradiol (E2) and progesterone (PR) are believed to be involved. Since E2 and PR may modulate the production of critical regulatory cytokines, we sought to characterize their effects on the in vitro human type-1/type-2 cytokine balance. We hypothesized that E2 and/or PR vary cytokine production and influence costimulatory molecule expression and apoptosis. We first described the effect of E2 and/or PR on type-1 (IFN-γ and IL-12) and type-2 (IL-4 and IL-10) cytokine production by human peripheral blood mononuclear cells (PBMC) treated with various T-lymphocyte and monocyte stimuli. E2 and/or PR were each used at concentrations similar to those found at the maternal-fetal interface during pregnancy. At this dose, E2 increased IFN-γ and IL-12 production and PR decreased IFN-γ production and tended to increase IL-4 production. Furthermore, the combination of E2+PR decreased IL-12 production. This suggests that E2 shifts the type-1/type-2 cytokine balance towards a type-1 response and that PR and E2+PR shift the balance towards a type-2 response. Next, we used intracellular cytokine detection to demonstrate that E2 and/or PR are capable of altering cytokine production of CD3+ T-cells and the CD3+CD4+ and CD3+CD8+ subsets. In addition, we used the H9 T-lymphocyte cell line and the THP-1 monocyte cell line to show that E2 and/or PR can induce cytokine effects in both T-cells and monocytes independent of their interaction. Lastly, we determined the effect of E2 and/or PR on costimulatory molecule expression and apoptosis as potential mechanisms for the cytokine-induced alterations. E2 increased and PR decreased CD80 expression on THP-1 cells and PR and E2+PR decreased CD28 expression in PBMC and Jurkat cells. Furthermore, E2, PR and E2+PR increased Fas-mediated apoptosis in Jurkat cells and E2 increased FasL expression on THP-1 cells. Thus, E2 and/or PR may alter the cytokine balance by modulating the CD28/CD80 costimulatory pathway and apoptosis. ^

Hypoxia-induced transcriptional regulation through chromatin modifications and NC2 function

In response to tumor hypoxia, specific genes that promote angiogenesis, proliferation, and survival are induced. Globally, I find that hypoxia induces a mixed pattern of histone modifications that are typically associated with either transcriptional activation or repression. Furthermore, I find that selective activation of hypoxia-inducible genes occurs simultaneously with widespread repression of transcription. I analyzed histone modifications at the core promoters of hypoxia-repressed and -activated genes and find that distinct patterns of histone modifications correlate with transcriptional activity. Additionally, I discovered that trimethylated H3-K4, a modification generally associated with transcriptional activation, is induced at both hypoxia-activated and repressed genes, suggesting a novel pattern of histone modifications induced during hypoxia. ^ In order to determine the mechanism of hypoxia-induced widespread repression of transcription, I focused my studies on negative cofactor 2 (NC2). Previously, we found that hypoxia-induced repression of the alpha-fetoprotein (AFP) gene occurs during preinitiation complex (PIC) assembly, and I find that NC2, an inhibitor of PIC assembly, is induced during hypoxia. Moreover, I find that the beta subunit of NC2 is essential for hypoxia-mediated repression of AFP, as well as the widespread repression of transcription observed during hypoxia. Previous data in Drosophila and S. cerevisiae indicate that NC2 functions as either an activator or a repressor of transcription. The mechanism of NC2-mediated activation remains unclear; although, Drosophila NC2 function correlates with specific core promoter elements. I tested if NC2 activates transcription in mammalian cells using this core promoter-specific model as a guide. Utilizing site-specific mutagenesis, I find that NC2 function in mammalian cells is not dependent upon specific core promoter elements; however, I do find that mammalian NC2 does function in a gene-specific manner as either an activator or repressor of transcription during hypoxia. Furthermore, I find that binding of the alpha subunit of NC2 specifically correlates with NC2-mediated transcriptional activation. NC2α and NC2β are both required for NC2-mediated transcriptional activation; whereas, NC2β alone is required for hypoxia-induced transcriptional repression. Together, these data indicate that hypoxia mediates changes in gene expression through both chromatin modifications and NC2 function. ^

The molecular basis for beta-lactamase gene expression in B. anthracis, B. cereus and B. thuringiensis

The susceptibility of most Bacillus anthracis strains to β-lactam antibiotics is intriguing considering that the B. anthracis genome harbors two β-lactamase genes, bla1 and bla2, and closely-related species, Bacillus cereus and Bacillus thuringiensis, typically produce β-lactamases. This work demonstrates that B. anthracis bla expression is affected by two genes, sigP and rsp, predicted to encode an extracytoplasmic function sigma factor and an antisigma factor, respectively. Deletion of the sigP/rsp locus abolished bla expression in a penicillin-resistant clinical isolate and had no effect on bla expression in a prototypical penicillin-susceptible strain. Complementation with sigP/rsp from the penicillin-resistant strain, but not the penicillin-susceptible strain, conferred β-lactamase activity upon both mutants. These results are attributed to a nucleotide deletion near the 5' end of rsp in the penicillin-resistant strain that is predicted to result in a nonfunctional protein. B. cereus and B. thuringiensis sigP and rsp homologues are required for inducible penicillin resistance in those species. Expression of the B. cereus or B. thuringiensis sigP and rsp genes in a B. anthracis sigP/rsp-null mutant confers resistance to β-lactam antibiotics, suggesting that while B. anthracis contains the genes necessary for sensing β-lactam antibiotics, the B. anthracis sigP/rsp gene products are insufficient for bla induction. ^ Because alternative sigma factors recognize unique promoter sequence, direct targets can be elucidated by comparing transcriptional profiling results with an in silico search using the sigma factor binding sequence. Potential σP -10 and -35 promoter elements were identified upstream from bla1 bla2 and sigP. Results obtained from searching the B. anthracis genome with the conserved sequences were evaluated against transcriptional profiling results comparing B. anthracis 32 and an isogenic sigP/rsp -null strain. Results from these analyses indicate that while the absence of the sigP gene significantly affects the transcript levels of 16 genes, only bla1, bla2 and sigP are directly regulated by σP. The genomes of B. cereus and B. thuringiensis strains were also analyzed for the potential σP binding elements. The sequence was located upstream from the sigP and bla genes, and previously unidentified genes predicted to encode a penicillin-binding protein (PBP) and a D-alanyl-D-alanine carboxypeptidase, indicating that the σ P regulon in these species responds to cell-wall stress caused by β-lactam antibiotics. ^ β-lactam antibiotics prevent attachment of new peptidoglycan to the cell wall by blocking the active site of PBPs. A B. cereus and B. thuringiensis pbp-encoding gene located near bla1 contains a potential σP recognition sequence upstream from the annotated translational start. Deletion of this gene abolished β-lactam resistance in both strains. Mutations in the active site of the PBP were detrimental to β-lactam resistance in B. cereus, but not B. thuringiensis, indicating that the transpeptidase activity is only important in B. cereus. I also found that transcript levels of the PBP-encoding gene are not significantly affected by the presence of β-lactam antibiotic. Based on these data I hypothesize that the gene product acts a sensor of β-lactam antibiotic. ^

The role of beta-arrestin 2 in lysophosphatidic acid-induced NF-kappaB activation

Lysophosphatidic acid (LPA) is a bioactive phospholipid and binds to its receptors, a family of G protein-coupled receptors (GPCR), which initiates multiple signaling cascades and leads to activation of several transcription factors, including NF-κB. NF-κB critically regulates numerous gene expressions, and is persistently active in many diseases. In our previous studies, we have demonstrated that LPA-induced NF-κB activation is dependent on a novel scaffold protein, CARMA3. However, how CARMA3 is recruited to receptor remains unknown. β-Arrestins are a family of proteins involved in desensitization of GPCR signaling. Additionally, β-arrestins function as signaling adaptor proteins, and mediate multiple signaling pathways. Therefore, we have hypothesized that β-arrestins may link CARMA3 to LPA receptors, and facilitate LPA-induced NF-κB activation. ^ Using β-arrestin-deficient MEFs, we found that β-arrestin 2, but not β-arrestin 1, was required for LPA-induced NF-κB activation. Also, we showed that the expression of NF-κB-dependent cytokines, such as interlukin-6, was impaired in β-arrestin 2-deficient MEFs. Mechanistically, we demonstrated the inducible association of endogenous β-arrestin 2 and CARMA3, and we found the CARD domain of CARMA3 interacted with 60-320 residues of β-arrestin 2. To understand why β-arrestin 2, but not β-arrestin 1, mediated NF-κB activation, we generated β-arrestin mutants. However, some mutants degraded quickly, and the rest did not rescue NF-κB activation in β-arrestin-deficient MEFs, though they had similar binding affinities with CARMA3. Therefore, it indicates that slight changes in residues may determine the different functions of β-arrestins. Moreover, we found β-arrestin 2 deficiency impaired LPA-induced IKK kinase activity, while it did not affect LPA-induced IKKα/β phosphorylation. ^ In summary, our results provide the genetic evidence that β-arrestin 2 serves as a positive regulator in NF-κB signaling pathway by connecting CARMA3 to LPA receptors. Additionally, we demonstrate that β-arrestin 2 is required for IKKα/β activation, but not for the inducible phosphorylation of IKKα/β. Because the signaling pathways around the membrane-proximal region of LPA receptors and GPCRs are quite conserved, our results also suggest a possible link between other GPCRs and CARMA3-mediated NF-κB activation. To fully define the role of β-arrestins in LPA-induced NF-κB signaling pathways will help to identify new drug targets for clinical therapeutics.^

14-3-3 ZETA OVEREXPRESSION SERVES AS A NOVEL MOLECULAR SWITCH TURNING TGF-BETA FROM TUMOR SUPPRESSOR TO TUMOR PROMOTER

TGF-β plays an important role in differentiation and tissue morphogenesis as well as cancer progression. However, the role of TGF-β in cancer is complicate. TGF-β has primarily been recognized as tumor suppressor, because it can directly inhibit cell proliferation of normal and premalignant epithelial cell. However, in the last stage of tumor progression, TGF-β functions as tumor promoter to enhance tumor cells metastatic dissemination and expands metastatic colonies. Currently, the mechanism of how TGF-β switches its role from tumor suppressor to promoter still remains elusive. Here we identify that overexpression of 14-3-3ζ inhibits TGF-β’s cell cytostatic program through destabilizing p53 in non-transformed human mammary epithelial cells. Mechanistically, we found that 14-3-3ζ overexpression leads to 14-3-3σ downregulation, thereby activates PI3K/Akt signaling pathway and degrades p53, and further inhibits TGF-β induced p21 expression and cell cytostatic function. In addition, we found that overexpression of 14-3-3ζ promotes TGF-β induced breast cancer cells bone metastatic colonization through stabilizing Gli2, which is an important co-transcriptional factor for p-smad2 to activate PTHrP expression and bone osteolytic effect. Taken together, we reveal a novel mechanism that 14-3-3ζ dictates the tumor suppressor or metastases promoter activities of TGF-β signaling pathway through switching p-smad2 binding partner from p53 to Gli2. The expected results will not only provide us the better understanding of the important role of 14-3-3ζ in the early stage of breast cancer development, but also deeply impact our knowledge of signaling mechanisms underlying the complex roles of TGF-β in cancer, which will give us a more accurate strategy to determine when and how anti-TGF-β targeted therapy might be effective.

Heregulin -beta 1 directly associates with RhoA via the immunoglobulin -like domain thereby modulating breast cancer metastasis-related properties

Heregulins constitute a family of growth factors belonging to the epidermal growth factor (EGF) family. Breast cancers that overexpress specific members of the EGF receptor family (EGFR, ErbB2, ErbB3, ErbB4) have increased metastatic potential, and Heregulin-β1 (HRGβ1), a ligand for ErbB3 and ErbB4, has also been shown to induce metastasis-related properties in breast cancer cells in vitro. The secreted form of the HRGβ1 is composed of five distinct structural domains, including the N-terminal domain, an immunoglobulin-like domain (IgG-like), a glycosylation domain, an EGF-like domain, and a β1-specific domain. Of these, the EGF-like domain is well characterized for its function in metastasis-related properties as well as its structure. However, the contributions of the other HRGβ1 domains in breast cancer metastasis remains unclear. ^ To investigate this, HRGβ1 proteins with targeted domain deletions were purified and subjected to assays for metastasis-related properties, including aggregation, invasion, activation of EGFR family members, and motility of breast cancer cells. These assays showed that retaining the EGF-like domain of HRGβ1 is important for activation of EGFRs. Interestingly, the HRGβ1 protein lacking the IgG-like domain (NGEB) led to a decrease in breast cancer cell motility, indicating the IgG-like domain modulates cell motility, an important step in cancer metastasis. ^ To understand the underlying mechanisms, I performed protein sequence and structural analysis of HRGβ1 and identified that the IgG-like domain of HRGβ1 shares sequence homology and three-dimensional structural similarity with the IgG-like domain of TRIO. TRIO is a cytoplasmic protein that directly associates with RhoA, a GTPase involved in cell reorganization and cell motility. Therefore, I hypothesized that HRGβ1 may translocate inside the breast cancer cells through receptor mediated endocytosis and bind to RhoA via its IgG-like domain. I show wild type HRGβ1 but not NGEB binds RhoA in vitro and in vivo, leading to RhoA activation. Inhibition of HRG-β1 internalization via endocytosis disrupted HRGβ1 binding to RhoA. Additionally, breast cancer cell motility induced by HRG-β1 is reduced after treatment with inhibitors to both endocytosis and RhoA function, similar to levels seen with NGEB treatment. ^ Thus, in addition to the well-known role of HRGβ1 as an extracellular stimulator of the EGFR family members, HRGβ1 also functions within the cell as a binding partner and activator of RhoA to modulate cancer cell motility. ^

The Function of Fish Cytokines

Peer reviewed

Identification of bdm-1, a gene involved in G protein β-subunit function and α-subunit accumulation

Targeted disruption of Gα and Gβ genes has established the requirement of an intact G protein signaling pathway for optimal execution of several important physiological processes, including pathogenesis, in the chestnut blight fungus Cryphonectria parasitica. We now report the identification of a G protein signal transduction component, beta disruption mimic factor-1, BDM-1. Disruption of the corresponding gene, bdm-1, resulted in a phenotype indistinguishable from that previously observed after disruption of the Gβ subunit gene, cpgb-1. The BDM-1 deduced amino acid sequence contained several significant clusters of identity with mammalian phosducin, including a domain corresponding to a highly conserved 11-amino acid stretch that has been implicated in binding to the Gβγ dimer and two regions of defined Gβ/phosducin contact points. Unlike the negative regulatory function proposed for mammalian phosducin, the genetic data presented in this report suggest that BDM-1 is required for or facilitates Gβ function. Moreover, disruption of either bdm-1 or cpgb-1 resulted in a significant, posttranscriptional reduction in the accumulation of CPG-1, a key Gα subunit required for a range of vital physiological processes.

Absence of fetal liver hematopoiesis in mice deficient in transcriptional coactivator core binding factor beta.

Core binding factor beta (CBF beta) is considered to be a transcriptional coactivator that dimerizes with transcription factors core binding factor alpha 1 (CBFA1), -2, and -3, and enhances DNA binding capacity of these transcription factors. CBF beta and CBFA2, which is also called acute myeloid leukemia 1 gene, are frequently involved in chromosomal translocations in human leukemia. To elucidate the function of CBF beta, mice carrying a mutation in the Cbfb locus were generated. Homozygous mutant embryos died between embryonic days 11.5-13.5 due to hemorrhage in the central nervous system. Mutant embryos had primitive erythropoiesis in yolk sac but lacked definitive hematopoiesis in fetal liver. In the yolk sac of mutant embryos, no erythroid or myeloid progenitors of definitive hematopoietic origin were detected, and the expression of flk-2/flt-3, the marker gene for early precursor cells of definitive hematopoiesis, was absent. These data suggest that Cbfb is essential for definitive hematopoiesis in liver, especially for the commitment to early hematopoietic precursor cells.

Identification of a functionally important sequence in the cytoplasmic tail of integrin beta 3 by using cell-permeable peptide analogs.

Integrins are major two-way signaling receptors responsible for the attachment of cells to the extracellular matrix and for cell-cell interactions that underlie immune responses, tumor metastasis, and progression of atherosclerosis and thrombosis. We report the structure-function analysis of the cytoplasmic tail of integrin beta 3 (glycoprotein IIla) based on the cellular import of synthetic peptide analogs of this region. Among the four overlapping cell-permeable peptides, only the peptide carrying residues 747-762 of the carboxyl-terminal segment of integrin beta 3 inhibited adhesion of human erythroleukemia (HEL) cells and of human endothelial cells (ECV) 304 to immobilized fibrinogen mediated by integrin beta 3 heterodimers, alpha IIb beta 3, and alpha v beta 3, respectively. Inhibition of adhesion was integrin-specific because the cell-permeable beta 3 peptide (residues 747-762) did not inhibit adhesion of human fibroblasts mediated by integrin beta 1 heterodimers. Conversely, a cell-permeable peptide representing homologous portion of the integrin beta 1 cytoplasmic tail (residues 788-803) inhibited adhesion of human fibroblasts, whereas it was without effect on adhesion of HEL or ECV 304 cells. The cell-permeable integrin beta 3 peptide (residues 747-762) carrying a known loss-of-function mutation (Ser752Pro) responsible for the genetic disorder Glanzmann thrombasthenia Paris I did not inhibit cell adhesion of HEL or ECV 304 cells, whereas the beta 3 peptide carrying a Ser752Ala mutation was inhibitory. Although Ser752 is not essential, Tyr747 and Tyr759 form a functionally active tandem because conservative mutations Tyr747Phe or Tyr759Phe resulted in a nonfunctional cell permeable integrin beta 3 peptide. We propose that the carboxyl-terminal segment of the integrin beta 3 cytoplasmic tail spanning residues 747-762 constitutes a major intracellular cell adhesion regulatory domain (CARD) that modulates the interaction of integrin beta 3-expressing cells with immobilized fibrinogen. Import of cell-permeable peptides carrying this domain results in inhibition "from within" of the adhesive function of these integrins.

In vivo tracking of platelets: circulating degranulated platelets rapidly lose surface P-selectin but continue to circulate and function.

To examine the hypothesis that surface P-selectin-positive (degranulated) platelets are rapidly cleared from the circulation, we developed novel methods for tracking of platelets and measurement of platelet function in vivo. Washed platelets prepared from nonhuman primates (baboons) were labeled with PKH2 (a lipophilic fluorescent dye), thrombin-activated, washed, and reinfused into the same baboons. Three-color whole blood flow cytometry was used to simultaneously (i) identify platelets with a mAb directed against glycoprotein (GP)IIb-IIIa (integrin alpha 11b beta 3), (ii) distinguish infused platelets by their PKH2 fluorescence, and (iii) analyze platelet function with mAbs. Two hours after infusion of autologous thrombin-activated platelets (P-selectin-positive, PKH2-labeled), 95 +/- 1% (mean +/- SEM, n = 5) of the circulating PKH2-labeled platelets had become P-selectin-negative. Compared with platelets not activated with thrombin preinfusion, the recovery of these circulating PKH2-labeled, P-selectin-negative platelets was similar 24 h after infusion and only slightly less 48 h after infusion. The loss of platelet surface P-selectin was fully accounted for by a 67.1 +/- 16.7 ng/ml increase in the plasma concentration of soluble P-selectin. The circulating PKH2-labeled, P-selectin-negative platelets were still able to function in vivo, as determined by their (i) participation in platelet aggregates emerging from a bleeding time wound, (ii) binding to Dacron in an arteriovenous shunt, (iii) binding of mAb PAC1 (directed against the fibrinogen binding site on GPIIb-IIIa), and (iv) generation of procoagulant platelet-derived microparticles. In summary, (i) circulating degranulated platelets rapidly lose surface P-selectin to the plasma pool, but continue to circulate and function; and (ii) we have developed novel three-color whole blood flow cytometric methods for tracking of platelets and measurement of platelet function in vivo.

Regulation of the myeloperoxidase enhancer binding proteins Pu1, C-EBP alpha, -beta, and -delta during granulocyte-lineage specification.

We have compared the molecular architecture and function of the myeloperoxidase upstream enhancer in multipotential versus granulocyte-committed hematopoietic progenitor cells. We show that the enhancer is accessible in multipotential cell chromatin but functionally incompetent before granulocyte commitment. Multipotential cells contain both Pu1 and C-EBP alpha as enhancer-binding activities. Pu1 is unphosphorylated in both multipotential and granulocyte-committed cells but is phosphorylated in B lymphocytes, raising the possibility that differential phosphorylation may play a role in specifying its lymphoid versus myeloid functions. C-EBP alpha exists as multiple phosphorylated forms in the nucleus of both multipotential and granulocyte-committed cells. C-EBP beta is unphosphorylated and cytoplasmically localized in multipotential cells but exists as a phosphorylated nuclear enhancer-binding activity in granulocyte-committed cells. Granulocyte colony-stimulating factor-induced granulocytic differentiation of multipotential progenitor cells results in activation of C-EBP delta expression and functional recruitment of C-EBP delta and C-EBP beta to the nucleus. Our results implicate Pu1 and the C-EBP family as critical regulators of myeloperoxidase gene expression and are consistent with a model in which a temporal exchange of C-EBP isoforms at the myeloperoxidase enhancer mediates the transition from a primed state in multipotential cells to a transcriptionally active configuration in promyelocytes.

Functional analysis of retinoid Z receptor beta, a brain-specific nuclear orphan receptor.

The retinoid Z receptor beta (RZR beta), an orphan receptor, is a member of the retinoic acid receptor (RAR)/thyroid hormone receptor (TR) subfamily of nuclear receptors. RZR beta exhibits a highly restricted brain-specific expression pattern. So far, no natural RZR beta target gene has been identified and the physiological role of the receptor in transcriptional regulation remains to be elucidated. Electrophoretic mobility shift assays reveal binding of RZR beta to monomeric response elements containing the sequence AnnTAGGTCA, but RZR beta-mediated transactivation of reporter genes is only achieved with two property spaced binding sites. We present evidence that RZR beta can function as a cell-type-specific transactivator. In neuronal cells, GaI-RZR beta fusion proteins function as potent transcriptional activators, whereas no transactivation can be observed in nonneuronal cells. Mutational analyses demonstrate that the activation domain (AF-2) of RZR beta and RAR alpha are functionally interchangeable. However, in contrast to RAR and TR, the RZR beta AF-2 cannot function autonomously as a transactivation domain. Furthermore, our data define a novel repressor function for the C-terminal part of the putative ligand binding domain. We propose that the transcriptional activity of RZR beta is regulated by an interplay of different receptor domains with coactivators and corepressors.

Long-lasting reduction of inhibitory function and gamma-aminobutyric acid type A receptor subunit mRNA expression in a model of temporal lobe epilepsy.

This study evaluated hippocampal inhibitory function and the level of expression of gamma-aminobutyric acid type A (GABAA) receptor mRNA in an in vivo model of epilepsy. Chronic recurrent limbic seizures were induced in rats using injections of pilocarpine. Electrophysiological studies performed on hippocampal slices prepared from control and epileptic animals 1 to 2 months after pilocarpine injections demonstrated a significant hyperexcitability in the epileptic animals. Reduced levels of mRNA expression for the alpha 2 and alpha 5 subunits of the GABAA receptors were evident in the CA1, CA2, and CA3 regions of the hippocampus of epileptic animals. No decrease in mRNA encoding alpha 1, beta 2, or gamma 2 GABAA receptor subunits was observed. In addition, no change in the mRNA levels of alpha CaM kinase II was seen. Selective decreases in mRNA expression did not correlate with neuronal cell loss. The results indicate that selective, long-lasting reduction of GABAA subunit mRNA expression and increased excitability, possibly reflecting loss of GABAergic inhibition, occur in an in vivo model of partial complex epilepsy.

Subcellular localization of gamma-aminobutyric acid type A receptors is determined by receptor beta subunits.

gamma-aminobutyric acid type A (GABAA) receptors are the major sites of fast synaptic inhibition in the brain. They are constructed from four subunit classes with multiple members: alpha (1-6), beta (1-4), gamma (1-4), and delta (1). The contribution of subunit diversity in determining receptor subcellular targeting was examined in polarized Madin-Darby canine kidney (MDCK) cells. Significant detection of cell surface homomeric receptor expression by a combination of both immunological and electrophysiological methodologies was only found for the beta 3 subunit. Expression of alpha/beta binary combinations resulted in a nonpolarized distribution for alpha 1 beta 1 complexes, but specific basolateral targeting of both alpha 1 beta 2 and alpha 1 beta 3 complexes. The polarized distribution of these alpha/beta complexes was unaffected by the presence of the gamma 2S subunit. Interestingly, delivery of receptors containing the beta 3 subunit to the basolateral domain occurs via the apical surface. These results show that beta subunits can selectively target GABAA receptors to distinct cellular locations. Changes in the spatial and temporal expression of beta-subunit isoforms may therefore provide a mechanism for relocating GABAA receptor function between distinct neuronal domains. Given the critical role of these receptors in mediating synaptic inhibition, the contribution of different beta subunits in GABAA receptor function, may have implications in neuronal development and for receptor localization/clustering.