BARX2 and estrogen receptor-alpha (ESR1) coordinately regulate the production of alternatively spliced ESR1 isoforms and control breast cancer cell growth and invasion.

The estrogen receptor-alpha gene (ESR1) was previously identified as a direct target of the homeobox transcription factor BARX2 in MCF7 cells. Here, we show that BARX2 and ESR1 proteins bind to different ESR1 gene promoters and regulate the expression of alternatively spliced mRNAs that encode 66 and 46 kDa ESR1 protein isoforms. BARX2 increases the expression of both ESR1 isoforms; however, it has a greater effect on the 46 kDa isoform, leading to an increased ratio between the 46 and 66 kDa proteins. BARX2 also influences estrogen-dependent processes such as anchorage-independent growth and modulates the expression of the estrogen-responsive genes SOX5, RBM15, Dynein and Mortalin. In addition, BARX2 expression promotes cellular invasion and increases the expression of active matrix metalloproteinase-9 (MMP9). BARX2 also increases the expression of the tissue inhibitor of metalloproteinase (TIMP) genes, TIMP1 and TIMP3, in cooperation with estrogen signaling. Overall, these data indicate that BARX2 and ESR1 may coordinately regulate cell growth, survival and invasion pathways that are critical to breast cancer progression.

Effects of trypsin on large-conductance Ca(2+)-activated K(+) channels of guinea-pig outer hair cells.

High-conductance Ca(2+)-activated K(+) (BK(Ca)) channels from isolated adult guinea-pig outer hair cells were studied in inside-out membrane patches. They had a 300 pS unitary conductance and were inhibited by tetraethyl ammonium (1 mM), iberiotoxin (33 nM) and charybdotoxin (50 nM). In symmetrical 144 mM KCl their K(+) permeability (P(K)) was 5.4 x 10(-13) cm(3)/s; this was reduced to around 4.5 x 10(-13) cm(3)/s with 160 mM Na(+) in place of K(+) on either internal or external membrane surface. BK(Ca) channels from trypsin-isolated hair cells had a high open probability, that depended on both membrane voltage (16 mV/e-fold change) and the concentration of calcium ions at their intracellular surface ([Ca(2+)](i)). The Hill coefficient was 3-4. About 50% of BK(Ca) channels from mechanically isolated outer hair cells had similar characteristics; the remainder had the same high conductance but a low open probability. Trypsin (<0.5 mg/ml) applied to the intracellular face of these 'inactive' channels markedly increased their open probability. It is possible that exposure to trypsin during cell isolation removes an inactivating beta subunit. This would account for the absence of 'inactive' BK(Ca) channels in trypsin-isolated cells.

Central circuitry in the jellyfish Aglantha digitale IV. Pathways coordinating feeding behaviour.

The hydromedusan jellyfish Aglantha digitale feeds on small planktonic organisms carried to the margin by tentacle flexions. During feeding, the manubrium bends across ("points") and seizes the prey with flared lips. In immobilized preparations, pointing to a source of electrical stimulation was accurate, 70% of the time, to within 15 degrees. Cutting experiments showed that the conduction pathways concerned with pointing and lip flaring are located in eight radial strands consisting of a radial canal, a giant nerve axon and a bundle of small axons with FMRFamide-like immunoreactivity. Application of food juices to sites on the margin and tentacles evoked trains of impulses in the axon bundles (F events; conduction velocity 15.5+/-3.7 cm s(-1)) and in the epithelium lining the radial canals (E events; conduction velocity 28.5+/-3.5 cm s(-1)). Impulses were conducted circularly in the outer nerve ring (F events) or in the ring canal (E events). Unilateral flexions of the manubrium during pointing arise from preferential excitation of one or more of eight longitudinal "muscle bands" in the wall of the manubrium and peduncle. Lip flaring represents symmetrical contraction of all eight bands. Cutting experiments revealed that F events mediate pointing; E events mediate lip flaring. Thus the endodermal radial canals, which in other hydromedusae mediate protective 'crumpling', provide the conduction pathway for manubrial lip flaring. Aglantha's alternative protective response--escape swimming--makes crumpling unnecessary, releasing the pathway for use in feeding. Trains of E events, generated in the manubrium during ingestion, propagate to the margin and inhibit rhythmic (slow) swimming with a duration that depended on their number and frequency. Inhibition of swimming appeared to facilitate transfer of food from the margin to the mouth, but how it comes about is unclear.

Prx1 controls vascular smooth muscle cell proliferation and tenascin-C expression and is upregulated with Prx2 in pulmonary vascular disease.

Prx1 and Prx2 are homeobox transcription factors expressed during vasculogenesis. To begin to elucidate how Prx1 and Prx2 are regulated and function in the adult vasculature, in situ hybridization studies were performed. Prx1 and Prx2 mRNAs were not detected in normal adult rat pulmonary arteries; however, both genes were induced with vascular disease, colocalizing to sites of tenascin-C (TN-C) expression. Because catabolism of the extracellular matrix (ECM) is a critical step in the development of vascular disease, we investigated whether changes in vascular smooth muscle cell (SMC)-ECM interactions regulate Prx1 and Prx2. A10 SMCs cultured on native type I collagen showed low levels of Prx1 and Prx2 mRNA expression, whereas cells cultured on denatured collagen showed higher levels of expression of both genes. At a functional level, transfection of SMCs with a Prx1 expression plasmid significantly increased their growth. Because TN-C also promotes SMC growth and its expression is also upregulated by denatured collagen, we tested and thereafter showed that Prx1 expression significantly enhances TN-C gene promoter activity 20-fold. Similar experiments conducted with truncated Prx1 proteins showed that the N-terminal portion and the homeodomain of Prx1 were necessary to induce the bulk of TN-C promoter activity. These findings support the hypothesis that Prx genes are regulated by changes in SMC adhesion and play key morphoregulatory roles during the development and progression of pulmonary vascular disease in adults.

Potassium inhibition of sodium-activated potassium (K(Na)) channels in guinea-pig ventricular myocytes.

N(a+)-activated potassium channels (K(Na) channels) were studied in inside-out patches from guinea-pig ventricular myocytes at potentials between -100 and +80 mV. External K(+) (K(+)(o)) was set to 140 mM. For inwardly directed currents with 105 mM internal K(+) (K(+)(1)), the unitary current-voltage relationship was fitted by the constant field equation with a potassium permeability coefficient, P(K), of 3.72 x 10(-13) cm(3) s(-1). The slope conductance (-100 to -10 mV) was 194 +/- 4.5 pS (mean +/- s.d., n = 4) with 105 mM K(+)(i) (35 mM Na(+)(i)) but it decreased to 181 +/- 5.6 pS (n = 5) in 70 mM K(+)(i) (70 mM Na(+)(i)). K(Na) channels were activated by internal Na(+) in a concentration-dependent fashion. With 4 mM K(+)(i), maximal activation was recorded with 100 mM Na(+)(i) (open probability, P(o), about 0.78); half-maximal activation required about 35 mM Na(+)(i). When K(+)(i) was increased to 70 mM, half-maximal activation shifted to about 70 mM Na(+)(i). With Na(+)(i) set to 105 mM, channel activity was markedly inhibited when K(+)(i) was increased from 35 to 105 mM. Channel openings were abolished with 210 mM K(+)(i). The inhibitory effect of internal K(+) was also observed at more physiological conditions of osmolarity, ionic strength and chloride concentration. With 35 mM Na(+)(i) and 4 mM K(+)(i), P(o) was 0.48 +/- 0.10 (n = 6); when K(+)(i)was increased to 35 mM, P(o) was reduced to 0.04 +/- 0.05 (n = 7, P < 0.001). The relationship between P(o) and Na(+)(i) concentration at different levels of K(+)(i) is well described by a modified Michaelis-Menten equation for competitive inhibition; the Hill coefficients were 4 for the P(o)-Na(+)(i) relationship and 1.2 for the P(o)-K(+)(i) relationship. It is suggested that Na(+) and K(+) compete for a superficial site on the channel's permeation pathway. K(Na) channels would be most likely to be activated in vivo when an increase in Na(+)(i) is accompanied by a decrease of K(+)(i).

Central circuitry in the jellyfish Aglantha digitale. III. The rootlet and pacemaker systems.

Tactile stimulation of the subumbrella of Aglantha digitale was found to evoke an escape swimming response similar to that evoked by stimulation of the outer surfaces of the margin but that does not involve the ring giant axon. Evidence is presented that conduction around the margin takes place via an interconnected system of rootlet interneurones. Confocal microscopy of carboxyfluorescein-filled axons showed that the rootlet neurones run out from the bases of the motor giant axons within the inner nerve ring and come into close contact with those of the neighbouring motor giant axons on either side. Transmission between the rootlet neurones has the properties of chemical synaptic transmission. A distinct type of fast excitatory postsynaptic potential (rootlet PSP) was recorded in motor giant axons following stimulation of nearby axons in 3-5 mmol l(-)(1) Mn(2+), which lowered the PSP below spike threshold. Immune labelling with anti-syntaxin 1 showed structures tentatively identified as synapses in the inner nerve ring, including some on the rootlet neurones. Neuromuscular junctions were not labelled. A secondary consequence of stimulating motor giant axons was the triggering of events in the pacemaker system. Triggering was blocked in 105 mmol l(-)(1) Mg(2+), indicating a synaptic link. Activity in the pacemaker system led indirectly to tentacle contractions (as described in earlier papers in this series), but the contractions were not as sudden or as violent as those seen when escape swimming was mediated by the ring giant axon. Events triggered in the pacemaker system fed back into the motor giants, producing postsynaptic potentials that appeared as humps in the spike after-potential. The conduction velocity of events propagating in the relay system was increased when the rootlet pathway was simultaneously excited (piggyback effect). With the addition of the rootlet pathway, the number of identified systems concerned with locomotion, feeding and tentacle contractions comes to fourteen, and the list is probably nearly complete.

The homeodomain protein Barx2 contains activator and repressor domains and interacts with members of the CREB family.

Barx1 and Barx2 are homeodomain proteins originally identified using regulatory elements of genes encoding certain cell adhesion molecules (CAMs). In the present study, we characterize regions of Barx2 that bind to regulatory elements of genes encoding three CAMs, L1, neuron-glia CAM (Ng-CAM), and neural CAM (N-CAM), and identify domains of Barx2 that regulate N-CAM transcription. The homeodomain of Barx2 was sufficient for binding to homeodomain binding sites (HBS) from all three CAM genes. The presence of a 17-amino acid Barx basic region resulted in a 2-fold decrease in binding to HBS sequences from the Ng-CAM and L1 genes, whereas it led to a 6.5-fold increase in binding to the HBS from the N-CAM promoter. Thus, the Barx basic region influences the strength and specificity of Barx2 binding to DNA. In co-transfection experiments, Barx2 repressed N-CAM promoter activity. A 24-residue N-terminal region of Barx2 was essential for repression. When this region was absent, Barx2 activated the N-CAM promoter. A 63-residue C-terminal domain was required for this activation. In GST pull-down experiments, Barx2 bound to proteins of the CREB family, CREB1 and ATF2. Overall, these findings provide a framework for understanding developmental and physiological contexts that influence repressor or activator functions of Barx2.

Synthetic promoter elements obtained by nucleotide sequence variation and selection for activity.

Eukaryotic transcriptional regulation in different cells involves large numbers and arrangements of cis and trans elements. To survey the number of cis regulatory elements that are active in different contexts, we have devised a high-throughput selection procedure permitting synthesis of active cis motifs that enhance the activity of a minimal promoter. This synthetic promoter construction method (SPCM) was used to identify >100 DNA sequences that showed increased promoter activity in the neuroblastoma cell line Neuro2A. After determining DNA sequences of selected synthetic promoters, database searches for known elements revealed a predominance of eight motifs: AP2, CEBP, GRE, Ebox, ETS, CREB, AP1, and SP1/MAZ. The most active of the selected synthetic promoters contain composites of a number of these motifs. Assays of DNA binding and promoter activity of three exemplary motifs (ETS, CREB, and SP1/MAZ) were used to prove the effectiveness of SPCM in uncovering active sequences. Up to 10% of 133 selected active sequences had no match in currently available databases, raising the possibility that new motifs and transcriptional regulatory proteins to which they bind may be revealed by SPCM. The method may find uses in constructing databases of active cis motifs, in diagnostics, and in gene therapy.

Evidence that the product of the human X-linked CGD gene, gp91-phox, is a voltage-gated H(+) pathway.

Expression of gp91-phox in Chinese hamster ovary (CHO91) cells is correlated with the presence of a voltage-gated H(+) conductance. As one component of NADPH oxidase in neutrophils, gp91-phox is responsible for catalyzing the production of superoxide (O(2).(2)). Suspensions of CHO91 cells exhibit arachidonate-activatable H(+) fluxes (Henderson, L.M., G. Banting, and J.B. Chappell. 1995. J. Biol. Chem. 270:5909-5916) and we now characterize the electrical properties of the pathway. Voltage-gated currents were recorded from CHO91 cells using the whole-cell configuration of the patch-clamp technique under conditions designed to exclude a contribution from ions other than H(+). As in other voltage-gated proton currents (Byerly, L., R. Meech, and W. Moody. 1984. J. Physiol. 351:199-216; DeCoursey, T.E., and V.V. Cherny. 1993. Biophys. J. 65:1590-1598), a lowered external pH (pH(o)) shifted activation to more positive voltages and caused the tail current reversal potential to shift in the manner predicted by the Nernst equation. The outward currents were also reversibly inhibited by 200 microM zinc. Voltage-gated currents were not present immediately upon perforating the cell membrane, but showed a progressive increase over the first 10-20 min of the recording period. This time course was consistent with a gradual shift in activation to more negative potentials as the pipette solution, pH 6.5, equilibrated with the cell contents (reported by Lucifer yellow included in the patch pipette). Use of the pH-sensitive dye 2'7' bis-(2-carboxyethyl)-5(and 6) carboxyfluorescein (BCECF) suggested that the final intracellular pH (pH(i)) was approximately 6.9, as though pH(i) was largely determined by endogenous cellular regulation. Arachidonate (20 microM) increased the amplitude of the currents by shifting activation to more negative voltages and by increasing the maximally available conductance. Changes in external Cl(-) concentration had no effect on either the time scale or the appearance of the currents. Examination of whole cell currents from cells expressing mutated versions of gp91-phox suggest that: (a) voltage as well as arachidonate sensitivity was retained by cells with only the NH(2)-terminal 230 amino acids, (b) histidine residues at positions 111, 115, and 119 on a putative membrane-spanning helical region of the protein contribute to H(+) permeation, (c) histidine residues at positions 111 and 119 may contribute to voltage gating, (d) the histidine residue at position 115 is functionally important for H(+) selectivity. Mechanisms of H(+) permeation through gp91-phox include the possible protonation/deprotonation of His-115 as it is exposed alternatively to the interior and exterior faces of the cell membrane (see Starace, D.M., E. Stefani, and F. Bezanilla. 1997. Neuron. 19:1319-1327) and the transfer of protons across an "H-X-X-X-H-X-X-X-H" motif lining a conducting pore.

D-Methionine protects against cisplatin damage to the stria vascularis.

D-Methionine (D-met) protects against cisplatin (CDDP)-induced hearing loss and outer hair cell loss (Campbell et al., 1996). However, D-met's protective effects on the stria vascularis has not been previously investigated. The purpose of this study was to examine, using semi-quantitative analysis, whether D-met also protects the stria vascularis. We removed a basal turn section of the stria vascularis from five groups of five male Wistar rats each: (1) a CDDP-treated control group receiving a 30 min i.p. infusion of 16 mg/kg CDDP, (2) a saline-injected control group receiving an equivalent volume of saline, and (3) three groups injected with either 75, 150, or 300 mg/kg D-methionine (D-met) i.p. 30 min prior to receiving the 16 mg/kg CDDP dosing. Using transmission electron microscopy and light microscopy, we analyzed strial volume (i.e. edema), marginal cell damage classification (bulging and/or compression), and relative optical density (ROD) ratios (i.e. depletion of marginal cell cytoplasmic organelles). All three levels of D-met provided complete protection against marginal cell bulging and/or compression but only partial protection against strial edema. At 300 mg/kg, D-met significantly reduced ROD ratio degradation in the spiral prominence and middle stria vascularis regions. In Reissner's membrane region, values from the D-met pretreated group were not significantly different from either the treated or untreated control groups suggesting only partial protection for that area. Protection of marginal cell cytoplasmic organelles was also noted. In summary, D-met partially or fully protects the stria vascularis from several types of CDDP-induced damage.

Knockout of REST/NRSF shows that the protein is a potent repressor of neuronally expressed genes in non-neural tissues.

The protein repressor element 1 silencing transcription factor/neuron restrictive silencer factor (REST/NRSF) is a negative regulator of neuronal genes that contain a particular DNA sequence, the neuron restrictive silencer element (NRSE). REST is expressed ubiquitously in non-neural tissues but is down-regulated in neural precursors and turned off in postmitotic neurons, suggesting that it can act both to prevent extraneural expression of certain genes and to delay the differentiation of neuronal subtypes. In a recent paper, Chen et al.(1) describe the production of a null mutant for REST in mice and the mosaic inactivation of REST function in chicken embryos. Knockout of REST led to malformations in several non-neural tissues, as well as apoptosis and embryonic lethality in mice. In addition, the expression of several REST target genes was derepressed in non-neural tissues and in neural progenitors in both mouse and chicken embryos. These studies clearly demonstrate that active repression of tissue-specific genes is required for proper tissue differentiation during embryonic development.

Impulse conduction in a sponge.

All-or-none propagated electrical impulses were recorded from the hexactinellid sponge Rhabdocalyptus dawsoni using suction electrodes attached to lumps of aggregated sponge tissue grafted onto the surface of pieces of the same sponge. Impulses were normally evoked by means of externally applied electrical shocks. Recorded externally using an a.c.-coupled amplifier, the electrical event was triphasic and lasted approximately 30 s; integration gave a diphasic waveform. A further integration to give the form of the membrane action potential produced a monophasic signal. Impulses propagated at 0.27+/-0.1 cm s-1 with an absolute refractory period of 29 s and a relative refractory period of approximately 150 s. Concurrent thermistor flow meter recordings confirmed that water flow through the sponge was arrested following the passage of an impulse, presumably as result of the cessation of beating of the flagella in the flagellated chambers. Tactile stimuli also evoked impulses, as did addition of particulate material to the incoming water stream. Impulses continued to propagate through the sponge during arrests, indicating that the conduction and effector systems were independent. Sponges lack nerves, and a variety of evidence indicates that the conducting tissues are the syncytial trabecular reticulum and pinacoderm layers. Na+-deficient solutions had little effect on the action potential, but propagation was blocked by 10 mmol l-1 Co2+, 1 mmol l-1 Mn2+ or 24 micromol l-1 nimodipine. Tetraethylammonium ions at 1-5 mmol l-1 also blocked propagation without prolonging the action potential. Impulse conduction in the sponge is discussed in relation to excitability and conduction in the protozoa and in plants and to non-nervous conduction in more advanced animals.

A binding site for homeodomain and Pax proteins is necessary for L1 cell adhesion molecule gene expression by Pax-6 and bone morphogenetic proteins.

The cell adhesion molecule L1 regulates axonal guidance and fasciculation during development. We previously identified the regulatory region of the L1 gene and showed that it was sufficient for establishing the neural pattern of L1 expression in transgenic mice. In the present study, we characterize a DNA element within this region called the HPD that contains binding motifs for both homeodomain and Pax proteins and responds to signals from bone morphogenetic proteins (BMPs). An ATTA sequence within the core of the HPD was required for binding to the homeodomain protein Barx2 while a separate paired domain recognition motif was necessary for binding to Pax-6. In cellular transfection experiments, L1-luciferase reporter constructs containing the HPD were activated an average of 4-fold by Pax-6 in N2A cells and 5-fold by BMP-2 and BMP-4 in Ng108 cells. Both of these responses were eliminated on deletion of the HPD from L1 constructs. In transgenic mice, deletion of the HPD from an L1-lacZ reporter resulted in a loss of beta-galactosidase expression in the telencephalon and mesencephalon. Collectively, our experiments indicate that the HPD regulates L1 expression in neural tissues via homeodomain and Pax proteins and is likely to be a target of BMP signaling during development.

A semiquantitative analysis of the effects of cisplatin on the rat stria vascularis.

Cisplatin (CDDP) is a very effective chemotherapeutic agent but is highly ototoxic. Most studies have focused on the effects of CDDP on the outer hair cells. The purpose of this study was to examine changes in the stria vascularis in cisplatin treated male Wistar rats and to provide semiquantitative analysis of the results. We removed a section of the stria vascularis from the basal turn of five control and five CDDP (16 mg/kg) treated rats. Using transmission electron microscopy (TEM) we analyzed: (1) changes to the strial tissue as a whole; and (2) intracellular changes in the marginal cells. We also subjected the samples to semiquantitative analysis using the MCID, focusing on three aspects of strial profile abnormalities; the number of abnormal marginal cells in CDDP treated tissue, intracellular strial edema and densitometry. Controls appeared normal, but many pathologic changes were apparent in the experimental group. Results from the semiquantitative analysis indicate cisplatin has a deleterious effect on the stria vascularis including strial edema; bulging, rupture and/or compression of the marginal cells and depletion of the cytoplasmic organelles.

Chickenpox immunisation in New Zealand.

PREVENTION: The appropriate use of varicella vaccine, effective in the prevention of chickenpox, has been considered by a Ministry of Health Working Party in 1996 and 1997, including discussion at a workshop held in Wellington, 26-27 June 1996. The introduction of varicella vaccine into the routine childhood immunisation schedule was not supported at this stage. The use of the only varicella vaccine for which the Minister of Health has given consent for distribution in New Zealand, Varilrix (SmithKline Beecham Limited), in healthy children aged nine months to 13 years inclusive, was supported. Consent has not been given for the use of Varilrix in immunocompromised people or in adults. This report discusses other groups that could be candidates for vaccination, such as children with deteriorating renal function and susceptible health care workers who regularly come into contact with especially vulnerable patients. In these cases, the vaccine would need to be administered on a named patient basis. The use of Varilrix in immunocompromised people was not supported. SURVEILLANCE: Enhanced surveillance of chickenpox and zoster are required in New Zealand. Adverse reactions to Varilrix should be carefully monitored. OUTBREAK CONTROL: There are insufficient data at present to support the use of Varilrix in outbreak control. The frequency, cost and current management of nosocomial outbreaks should be ascertained. This information may also assist in the decision whether to incorporate a varicella vaccine into the routine childhood immunisation schedule in the future.

Determinants of UDP glucuronosyltransferase membrane association and residency in the endoplasmic reticulum.

The UDP glucuronosyltransferases (UGT)2 are a family of enzymes which detoxify small hydrophobic compounds in mammalian cells. It is believed that UGTs are type I endoplasmic reticulum (ER) resident membrane proteins with a single membrane spanning domain near the carboxyl-terminus. The determinants of endoplasmic reticulum subcellular localization and membrane association for the UDP glucuronosyltransferase, UGT2B1, were examined. The construction and analysis of truncated and chimeric forms of UGT2B1 demonstrated that the protein contains regions of membrane interaction in the amino-terminal half of the lumenal domain in addition to the carboxyl-terminal transmembrane domain. UGT2B1 also remained resident in the ER in the absence of the cytosolic tail and transmembrane domain. Construction and analysis of an active, truncated form of UGT2B1 indicated that the cytosolically located dilysine motif, which is a putative ER membrane targeting signal, may be redundant for residency of UGT in the ER.

The effect of polyamines on KATP channels in guinea-pig ventricular myocytes.

1. The effect of natural polyamines on KATP channels was studied using inside-out patches from guinea-pig ventricular myocytes. 2. At a holding potential of +40 mV, spermine at the intracellular membrane surface reduced the KATP channel open probability (Popen) in a dose-dependent manner. Half-maximal inhibition occurred at 29 microM with a Hill coefficient of 1.2. 3. The effect of spermine on Popen was not greatly influenced by the membrane potential but there appeared to be a small reduction in unitary current amplitude during strong depolarizations. 4. Analysis of KATP single channel kinetics showed that spermine inhibited the channel by decreasing the mean open time and introducing transitions to a long closed state. 5. Spermidine (0.1 mM) was found to have a similar effect to spermine. Putrescine (10 mM) was found to block more effectively at positive membrane potentials. Up to 20 mM arginine had no significant effect on KATP channels. 6. Our results indicate that natural polyamines influence native KATP channel gating in cardiac myocytes.

Structure and function of uridine diphosphate glucuronosyltransferases.

1. The uridine diphosphate (UDP)-glucuronosyltransferases (UGT) are a family of enzymes that catalyse the covalent addition of glucuronic acid to a wide range of lipophilic chemicals. They play a major role in the detoxification of many exogenous and endogenous compounds by generating products that are more polar and, thus, more readily excreted in bile or urine. 2. Inherited deficiencies in UGT forms are deleterious, as exemplified by the debilitating effects of hyperbilirubinaemia and neurotoxicity in subjects with mutations in the enzyme that converts bilirubin to its more polar glucuronide. 3. The UGT protein can be conceptually divided into two domains with the amino-terminal half of the protein demonstrating greater sequence divergence between isoforms. This region apparently determines aglycone specificity. The aglycone binding site is presumed to be a 'loose' fit, as many structurally diverse substrates can be bound by the same UGT isoform. The carboxyl-terminal half, which is more conserved in sequence between different isoforms, is believed to contain a binding site for the cosubstrate UDP glucuronic acid (UDPGA). 4. Uridine diphosphate glucuronosyltransferase is localized to the endoplasmic reticulum (ER) and spans the membrane with a type I topology. The putative transmembrane domain is located near the carboxyl terminus of the protein such that only a small portion of the protein resides in the cytosol. This cytosolic tail is believed to contain an ER-targeting signal. The major portion of the protein is located in the ER lumen, including the proposed substrate-binding domains and the catalytic site. 5. The microsomal membrane impedes the access of UDPGA to the active site, resulting in latency of UGT activity in intact ER-derived microsomes. Active transport of UDPGA is believed to occur in hepatocytes, but the transport system has not been fully characterized. Uridine diphosphate glucuronosyltransferase activity is also highly lipid dependent and the enzyme may contain regions of membrane association in addition to the transmembrane domain.

UDP-glucuronosyltransferase, the role of the amino terminus in dimerization.

UDP-glucuronosyltransferases (UGTs) comprise an important enzyme system in mammals that is involved in detoxification of a variety of small hydrophobic compounds of both endogenous and exogenous origin. Some evidence suggests that these enzymes may function as oligomers; however, little is known about the domain of interaction or the mechanism of oligomerization. In this work, evidence for a functional dimerization between UGTs is provided by studies on mutated forms of UGT2B1. When two inactive forms of UGT2B1 were co-expressed in cell culture, catalytic activity was restored, indicating that UGT2B1 forms functional dimers. To delineate the dimerization domain, inactive fusion proteins containing the amino- or carboxyl-terminal domains of UGT2B1 were generated and expressed with active UGT2B1. Expression of a fusion protein containing only the amino-terminal half of UGT2B1 with active UGT2B1 caused a reduction in UGT2B1 catalytic activity. This reduction in activity was not observed when UGT2B1 was co-expressed with a fusion protein containing only the carboxyl-terminal half of UGT2B1, strongly suggesting that the amino-terminal domain is involved in dimerization. Truncation of the immediate amino terminus of UGT2B1 abolished UGT2B1 activity and dimer formation. Activity was also abolished by an L4R substitution in this region of the mature protein, which is highly conserved in the UGT family. These results indicate that UGTs can interact through their amino-terminal domains to form catalytically active dimers. Possible mechanisms resulting in the formation and stabilization of the UGT2B1 dimer are discussed.