Presence Of Agonist Research Articles

Molecular Dissection of Purinergic P2X Receptor Channels

The P2X receptors (P2XRs) are a family of ATP-gated channels expressed in the plasma membrane of numerous excitable and nonexcitable cells and play important roles in control of cellular functions, such as neurotransmission, hormone secretion, transcriptional regulation, and protein synthesis. P2XRs are homomeric or heteromeric proteins, formed by assembly of at least three of seven subunits named P2X(1)-P2X(7). All subunits possess intracellular N- and C-termini, two transmembrane domains, and a relatively large extracellular ligand-binding loop. ATP binds to still an unidentified extracellular domain, leading to a sequence of conformational transitions between closed, open, and desensitized states. Removal of extracellular ATP leads to deactivation and resensitization of receptors. Activated P2XRs generate inward currents caused by Na(+) and Ca(2+) influx through the pore of channels, and thus mediate membrane depolarization and facilitation of voltage-gated calcium entry in excitable cells. No crystal structures are available for P2XRs and these receptors have no obvious similarity to other ion channels or ATP binding proteins, which limits the progress in understanding the relationship between molecular structure and conformational transitions of receptor in the presence of agonist and after its washout. We summarize here the alternative approaches in studies on molecular properties of P2XRs, including heteromerization, chimerization, mutagenesis, and biochemical studies.

Solution X-ray Scattering Evidence for Agonist- and Antagonist-induced Modulation of Cleft Closure in a Glutamate Receptor Ligand-binding Domain

Agonist-induced conformational changes in the ligand-binding domains (LBD) of glutamate receptor ion channels provide the driving force for molecular rearrangements that mediate channel opening and subsequent desensitization. The resulting regulated transmembrane ion fluxes form the basis for most excitatory neuronal signaling in the brain. Crystallographic analysis of the GluR2 LBD core has revealed a ligand-binding cleft located between two lobes. Channel antagonists stabilize an open cleft, whereas agonists stabilize a closed cleft. The crystal structure of the apo form is similar to the antagonist-bound, open state. To understand the conformational behavior of the LBD in the absence of crystal lattice constraints, and thus better to appreciate the thermodynamic constraints on ligand binding, we have undertaken a solution x-ray scattering study using two different constructs encoding either the core or an extended LBD. In agreement with the GluR2 crystal structures, the LBD is more compact in the presence of agonist than it is in the presence of antagonist. However, the time-averaged conformation of the ligand-free core in solution is intermediate between the open, antagonist-bound state and the closed, agonist-bound state, suggesting a conformational equilibrium. Addition of peptide moieties that connect the core domain to the other functional domains in each channel subunit appears to constrain the conformational equilibrium in favor of the open state.

Ethanol Modulates the VR-1 Variant Amiloride-insensitive Salt Taste Receptor. II. Effect on Chorda Tympani Salt Responses

The effect of ethanol on the amiloride- and benzamil (Bz)-insensitive salt taste receptor was investigated by direct measurement of intracellular Na+ activity ([Na+]i) using fluorescence imaging in polarized fungiform taste receptor cells (TRCs) and by chorda tympani (CT) taste nerve recordings. CT responses to KCl and NaCl were recorded in Sprague-Dawley rats, and in wild-type (WT) and vanilloid receptor-1 (VR-1) knockout mice (KO). CT responses were monitored in the presence of Bz, a specific blocker of the epithelial Na+ channel (ENaC). CT responses were also recorded in the presence of agonists (resiniferatoxin and elevated temperature) and antagonists (capsazepine and SB-366791) of VR-1 that similarly modulate the Bz-insensitive VR-1 variant salt taste receptor. In the absence of mineral salts, ethanol induced a transient decrease in TRC volume and elicited only transient phasic CT responses. In the presence of mineral salts, ethanol increased the apical cation flux in TRCs without a change in volume, increased transepithelial electrical resistance across the tongue, and elicited CT responses that were similar to salt responses, consisting of both a phasic component and a sustained tonic component. At concentrations <50%, ethanol enhanced responses to KCl and NaCl, while at ethanol concentrations >50%, those CT responses were inhibited. Resiniferatoxin and elevated temperature increased the sensitivity of the CT response to ethanol in salt-containing media, and SB-366791 inhibited the effect of ethanol, resiniferatoxin, and elevated temperature on the CT responses to mineral salts. VR-1 KO mice demonstrated no Bz-insensitive CT response to NaCl and no sensitivity to ethanol. We conclude that ethanol increases salt taste sensitivity by its direct action on the Bz-insensitive VR-1 variant salt taste receptor.

Hsp90 and environmental impacts on epigenetic states: a model for the trans-generational effects of diethylstibesterol on uterine development and cancer

Hsp90 is a chaperone for over 100 'client proteins' in the cell, most of which are involved in signaling pathways. For example, Hsp90 maintains several nuclear hormone receptors, such as the estrogen receptor (ER), as agonist-receptive monomers in the cytoplasm. In the presence of agonist, Hsp90 dissociates and the receptors dimerize, enter the nucleus and ultimately activate transcription of the target genes. Increasing evidence suggests that Hsp90 also has a role in modifying the chromatin conformation of many genes. For example, Hsp90 has recently been shown to increase the activity of the histone H3 lysine-4 methyltransferase SMYD3, which activates the chromatin of target genes. Further evidence for chromatin-remodeling functions is that Hsp90 acts as a capacitor for morphological evolution by masking epigenetic variation. Release of the capacitor function of Hsp90, such as by environmental stress or by drugs that inhibit the ATP-binding activity of Hsp90, exposes previously hidden morphological phenotypes in the next generation and for several generations thereafter. The chromatin-modifying phenotypes of Hsp90 have striking similarities to the trans-generational effects of the ER agonist diethylstilbesterol (DES). Prenatal and perinatal exposure to DES increases the predisposition to uterine developmental abnormalities and cancer in the daughters and granddaughters of exposed pregnant mice. In this review, we propose that trans-generational epigenetic phenomena involving Hsp90 and DES are related and that chromatin-mediated WNT signaling modifications are required. This model suggests that inhibitors of Hsp90, WNT signaling and chromatin-remodeling enzymes might function as anticancer agents by interfering with epigenetic reprogramming and canalization in cancer stem cells.

G-Protein–Coupled Receptor Mas Is a Physiological Antagonist of the Angiotensin II Type 1 Receptor

We previously identified the G-protein-coupled receptor Mas, encoded by the Mas proto-oncogene, as an endogenous receptor for the heptapeptide angiotensin-(1-7); however, the receptor is also suggested to be involved in actions of angiotensin II. We therefore tested whether this could be mediated indirectly through an interaction with the angiotensin II type 1 receptor, AT1. In transfected mammalian cells, Mas was not activated by angiotensin II; however, AT1 receptor-mediated, angiotensin II-induced production of inositol phosphates and mobilization of intracellular Ca2+ was diminished by 50% after coexpression of Mas, despite a concomitant increase in angiotensin II binding capacity. Mas and the AT1 receptor formed a constitutive hetero-oligomeric complex that was unaffected by the presence of agonists or antagonists of the 2 receptors. In vivo, Mas acts as an antagonist of the AT1 receptor; mice lacking the Mas gene show enhanced angiotensin II-mediated vasoconstriction in mesenteric microvessels. These results demonstrate that Mas can hetero-oligomerize with the AT1 receptor and by so doing inhibit the actions of angiotensin II. This is a novel demonstration that a G-protein-coupled receptor acts as a physiological antagonist of a previously characterized receptor. Consequently, the AT1-Mas complex could be of great importance as a target for pharmacological intervention in cardiovascular diseases.

G protein‐coupled receptors: a count of 1001 conformations

G protein-coupled receptors (GPCRs) were initially regarded to adopt an inactive and an active conformation and to activate a single type of G protein. Studies with recombinant cell systems have led to a more complex picture. First, GPCRs can activate distinct G protein species. Second, GPCR multistate models have been invoked to explain their complex behaviour in the presence of agonists, antagonists and other binding partners. The occurrence of intermediate receptor conformational states during GPCR activation and antagonist binding is suggested by fluorescence measurements and studies with constitutively active receptor mutants and insurmountable antagonists. Different agonists may trigger distinct effector pathways through a single receptor by dictating its preference for certain G proteins (i.e. 'agonist trafficking'). Structural modification and exogenous and endogenous (e.g. other cellular proteins, lipids) allosteric modulators also affect ligand-GPCR interaction and receptor activation. These new developments in GPCR research could lead to the development of more selective therapeutic drugs.

Neuronal nitric oxide modulates morphine antinociceptive tolerance by enhancing constitutive activity of the μ-opioid receptor

NO is a key mediator of morphine antinociceptive tolerance. This work was conducted to evaluate the specific effects of NO on μ-opioid receptor activity. To investigate the effects of morphine- and l-arginine (the NO precursor)-induced increases in NO, five groups of rats were treated with saline, l-arginine (100-, 300-, or 500-mg/kg/h), or morphine 3-mg/kg/h for 8 h on Day 1; brain tissue was collected on Day 2. To evaluate the effects of additional increases in NO on morphine-induced alterations of the μ-opioid receptor, six groups of rats were treated with 8-h intravenous infusions for two consecutive days as per the following scheme (Day 1:Day 2): saline:saline (control); saline:morphine 3-mg/kg/h (tolerant); l-arginine 500-mg/kg/h:saline (NO control); l-arginine 100-mg/kg/h:morphine 3-mg/kg/h; l-arginine 300-mg/kg/h:morphine 3-mg/kg/h; and l-arginine 500-mg/kg/h:morphine 3-mg/kg/h (supertolerant). Brain tissue was collected at the end of Day 2. The time course of effects on morphine-induced receptor alterations due to increased NO also was evaluated. Brain tissue was analyzed for changes in radioligand (agonist and antagonist) binding and [ 35S]GTPγS binding (agonist and antagonist). In the absence of agonist exposure, NO produced an alteration in the μ-opioid receptor that increased receptor activity. In the presence of agonist, NO increased constitutive activation of the μ-opioid receptor and reduced the ability of a selective μ-opioid agonist to activate the μ-opioid G-protein-coupled receptor; these molecular effects occurred in a time course consistent with the development of antinociceptive tolerance. This work establishes important NO-induced alterations in μ-opioid receptor functionality, which directly lead to the development of opioid antinociceptive tolerance.

Protons trap NR1/NR2B NMDA receptors in a nonconducting state.

NMDA receptors are highly expressed in the CNS and are involved in excitatory synaptic transmission, as well as synaptic plasticity. Given that overstimulation of NMDA receptors can cause cell death, it is not surprising that these channels are under tight control by a series of inhibitory extracellular ions, including zinc, magnesium, and H+. We studied the inhibition by extracellular protons of recombinant NMDA receptor NR1/NR2B single-channel and macroscopic responses in transiently transfected human embryonic kidney HEK 293 cells using patch-clamp techniques. We report that proton inhibition proceeds identically in the absence or presence of agonist, which rules out the possibility that protonation inhibits receptors by altering coagonist binding. The response of macroscopic currents in excised patches to rapid jumps in pH was used to estimate the microscopic association and dissociation rates for protons, which were 1.4 x 10(9) m(-1) sec(-1) and 110-196 sec(-1), respectively (K(d) corresponds to pH 7.2). Protons reduce the open probability without altering the time course of desensitization or deactivation. Protons appear to slow at least one time constant describing the intra-activation shut-time histogram and modestly reduce channel open time, which we interpret to reflect a reduction in the overall channel activation rate and possible proton-induced termination of openings. This is consistent with a modest proton-dependent slowing of the macroscopic response rise time. From these data, we propose a physical model of proton inhibition that can describe macroscopic and single-channel properties of NMDA receptor function over a range of pH values.

Expression and regulation of cyclooxygenase‐2 in normal and neoplastic canine keratinocytes

Squamous cell carcinoma (SCC) is one of the most common cancers in dogs, yet relatively little is known about the molecular events involved in its development. Increasing evidence implicates cyclooxygenase-2 (COX-2) in the pathogenesis of various cancers in humans and animals. COX-2 overexpression has recently been demonstrated in canine SCCs. The objective of our study was to characterize the expression and regulation of COX-2 in normal and neoplastic canine keratinocytes (CKs) to provide an in vitro system to investigate the implication of COX-2 in SCC oncogenesis in dogs. Cell lines derived from normal CKs and neoplastic CKs (SCCs) were cultured in the absence or presence of agonists, and immunoblots, immunocytochemistry, radioimmunoassays and a cell proliferation assay were used to characterize COX-2 expression and action. Results showed that neoplastic keratinocytes had a higher basal COX-2 expression than normal keratinocytes. In both cell lines, stimulation with the tumour promoter phorbol 12-myristate 13-acetate induced a time-dependent increase in COX-2 protein, with COX-2 induction being stronger in cancerous SCC than in normal CK cells. Moreover, SCC cells produced significantly more PGE(2) than CK cells, under both baseline and stimulated conditions (P < 0.05). NS-398, a selective COX-2 inhibitor, inhibited prostaglandin (PG)E(2) synthesis and decreased proliferation of CK and SCC cells (P < 0.05). Collectively, our results indicate that the canine neoplastic keratinocyte SCC cell line expresses more COX-2 and produces more PGE(2) than the normal keratinocyte CK cell line, thus providing an in vitro system to study the molecular basis of elevated COX-2 expression in SCCs in dogs.

Fluorescence Resonance Energy Transfer Analysis Reveals the Existence of Endothelin-A and Endothelin-B Receptor Homodimers

Endothelin-1 is a potent vasoactive peptide that exerts its action via endothelin-A (ETA) and endothelin-B (ETB) receptors. Both types of receptor belong to the rhodopsin-like (class A) G protein-coupled receptors. Since there is an increasing body of evidence that G protein-coupled receptors function as homo- and heterodimers, we sought to prove whether endothelin receptor subtypes also exist as homodimers. To this end we generated plasmids encoding fusion proteins of the endothelin-A and endothelin-B receptor with cyan (ETA.CFP, ETB.CFP) or yellow fluorescent protein (ETA.YFP, ETB.YFP), suitable for fluorescence resonance energy transfer experiments. HEK293 cells transiently co-expressing either ETA.CFP and ETA.YFP or ETB.CFP and ETB.YFP showed significant fluorescence resonance energy transfer. The calculated efficiencies of fluorescence resonance energy transfer were between 12 and 18% for both endothelin-A and endothelin-B receptor homodimers. When cells co-expressing ETA.CFP and ETA.YFP or ETB.CFP and ETB.YFP were treated with endothelin-1 for 30 minutes at 37degreesC, no change in fluorescence resonance energy transfer efficiencies was found. Similarly, the endothelin-A receptor-selective antagonist, BQ123, and the endothelin-B receptor-selective antagonist, BQ788, did not change the fluorescence resonance energy transfer efficiencies of ETA.CFP/ ETA.YFP and ETB.CFP/ETB.YFP homodimers, respectively. The data demonstrate that endothelin-A and endothelin-B receptors exist as constitutive homodimers, which are not altered in the presence of agonists or antagonists.

Channel Gating of the Glycine Receptor Changes Accessibility to Residues Implicated in Receptor Potentiation by Alcohols and Anesthetics

The glycine receptor is a target for both alcohols and anesthetics, and certain amino acids in the alpha1 subunit transmembrane segments (TM) are critical for drug effects. Introducing larger amino acids at these positions increases the potency of glycine, suggesting that introducing larger residues, or drug molecules, into the drug-binding cavity facilitates channel opening. A possible mechanism for these actions is that the volume of the cavity expands and contracts during channel opening and closing. To investigate this hypothesis, mutations for amino acids in TM1 (I229C) and TM2 (G256C, T259C, V260C, M263C, T264C, S267C, S270C) and TM3 (A288C) were individually expressed in Xenopus laevis oocytes. The ability of sulfhydryl-specific alkyl methanethiosulfonate (MTS) compounds of different lengths to covalently react with introduced cysteines in both the closed and open states of the receptor was determined. S267C was accessible to short chain (C3-C8) MTS in both open and closed states, but was only accessible to longer chain (C10-C16) MTS compounds in the open state. Reaction with S267C was faster in the open state. I229C and A288C showed state-dependent reaction with MTS only in the presence of agonist. M263C and S270C were also accessible to MTS labeling. Mutated residues more intracellular than M263C did not react, indicating a floor of the cavity. These data demonstrate that the conformational changes accompanying channel gating increase accessibility to amino acids critical for drug action in TM1, TM2, and TM3, which may provide a mechanism by which alcohols and anesthetics can act on glycine (and likely other) receptors.

High-affinity interactions between human alpha1A-adrenoceptor C-terminal splice variants produce homo- and heterodimers but do not generate the alpha1L-adrenoceptor.

Using combinations of bioluminescence resonance energy transfer, time-resolved fluorescence resonance energy transfer and the functional complementation of pairs of inactive receptor-G protein fusion proteins, the human alpha(1A-1)-adrenoceptor was shown to form homodimeric/oligomeric complexes when expressed in human embryonic kidney (HEK) 293 cells. Saturation bioluminescence resonance energy transfer studies indicated the alpha(1A-1)-adrenoceptor homodimer interactions to be high affinity and some 75 times greater than interactions between the alpha(1A-1)-adrenoceptor and the delta opioid peptide receptor. Only a fraction of the alpha(1A-1)-adrenoceptors was at the plasma membrane of HEK293 cells at steady state. However, dimers of alpha(1A-1)-adrenoceptors were also present in intracellular membranes, and the dimer status of those delivered to the cell surface was unaffected by the presence of agonist. Splice variation can generate at least three forms of the human alpha(1A-1)-adrenoceptor with differences limited to the C-terminal tail. Each of the alpha(1A-1), alpha(1A-2a), and alpha(1A-3a)-adrenoceptor splice variants formed homodimers/oligomers, and all combinations of these splice variants were able to generate heterodimeric/oligomeric interactions. Despite the coexpression of these splice variants in human tissues that possess the pharmacologically defined alpha(1L)-adrenoceptor binding site, coexpression of any pair in HEK293 cells failed to generate ligand binding characteristic of the alpha(1L)-adrenoceptor.

Effects of partial agonists and Mg2+ ions on the interaction of M2 muscarinic acetylcholine receptor and G protein Galpha i1 subunit in the M2-Galpha i1 fusion protein.

We have expressed a M(2)-Galpha(i1) fusion protein in insect cells, in which the G protein alpha(i1) subunit was fused with a mutant of the muscarinic receptor M(2) subtype without glycosylation sites and the central part of the third intracellular loop. The M(2)-Galpha(i1) fusion protein showed GTP-sensitive, high-affinity agonist binding. Displacement curves by GDP of [(35)S]GTPgammaS binding shifted to the right in the presence of muscarinic agonists. The extent of the shift was greater for full agonists (120-150 fold) than for partial agonists (25-35 fold), and virtually no shift was observed for antagonists. The affinity for GDP decreased with increasing MgCl(2) concentration in the presence of an agonist but was not affected by MgCl(2) in the presence of an antagonist. These results indicate that the apparent affinity for GDP of the M(2)-Galpha(i1) fusion protein bound to a ligand represents the efficacy of the given ligand, and that Mg(2+) is required for the agonist-bound M(2) to interact with Galpha(i1), reducing its affinity for GDP. We propose that the agonist-M(2)-Galpha(i1) complex represents the transition state for the GDP-GTP exchange reaction catalyzed by agonist-bound receptors, and that the complex has different affinities for GDP depending on the species of the ligand bound to M(2) receptors.

The Receptor-G Fusion Protein as a Tool for Ligand Screening: a Model Study Using a Nociceptin Receptor-G i2 Fusion Protein

As a model system to screen endogenous ligands for G(i)-coupled receptors, we have prepared and characterized a fusion protein of nociceptin receptor and alpha subunit of G(i2). We detected nociceptin binding to the fusion protein by measuring stimulation of [(35)S]GTPgammaS binding with an EC(50) of 2.0 nM and a gain of approximately five times. The stimulation by nociceptin of [(35)S]GTPgammaS binding to the fusion protein was clearly observed in the presence of an appropriate concentration of GDP, because the affinity for GDP was decreased in the presence of agonist. Full and partial agonists differed in their effects on apparent the affinity of the fusion protein for GDP: the IC(50) values for GDP to displace 100 pM [(35)S]GTPgammaS were estimated to be 2 micro M, 0.4 micro M, and 0.05 micro M in the presence of full agonist (nociceptin), partial agonist (F/G-NC), and antagonist (NBZH), respectively. We also detected the activity to stimulate [(35)S]GTPgammaS binding to the fusion protein in the brain extract derived from 2-3 g wet weight tissue without false-positive results. The active component was identified as endogenous nociceptin itself. These results indicate that the fusion protein of GPCR and Galpha(i) is useful for screening of endogenous ligands.

Evidence that the TM1-TM2 Loop Contributes to the ρ1 GABA Receptor Pore

Considerable evidence indicates the second transmembrane domain (TM2) of the gamma-aminobutyric acid (GABA) receptor lines the integral ion pore. To further delineate the structures that constitute the ion pore and selectivity filter of the rho1 GABA receptor, we used the substituted cysteine accessibility method with charged reagents to identify anion- and cation-accessible surfaces. Twenty-one consecutive residues were mutated to cysteine, one at a time, in the presumed intracellular end of the first transmembrane domain (TM1; Ala(271)-Met(276)), the entire linker connecting TM1 to TM2 (Leu(277)-Arg(287)), and the presumed intracellular end of TM2 (Ala(288)-Ala(291)). Positively (MTSEA(+)) and negatively (pCMBS(-)) charged sulfhydryl reagents, as well as Cd(2+), were added extracellularly to test accessibility of the engineered cysteines. Four of the mutants, all at the intracellular end of TM2 (R287C, V289C, P290C, A291C), were accessible to positively charged reagents, whereas seven mutants (A271C, T272C, L277C, W279C, V280C, P290C, A291C) were functionally modified by negatively charged pCMBS(-). These seven modified residues were at the intracellular end of TM2, in the TM1-TM2 linker, and at the intracellular end of TM1. In nearly all cases (excluding P290C), the rate and the degree of modification were state-dependent, with greater accessibility in the presence of agonist. Select cysteine mutants were combined with a point mutation (A291E) that converted the pore from chloride- to non-selective. In this case, positively charged reagents could modify residues in the TM1-TM2 linker (Leu(277) and Val(280)), supporting the notion that the modifying reagents were reaching their target through the pore. Taken together, our results suggest that, up to its intracellular end, the TM2 domain is not charge selective. In addition, we propose that the TM1-TM2 linker and the intracellular end of TM1 are along the pathway of the permeating ion. These findings may lend new insights into the structure of the GABA receptor pore.

Characterization of Four Autonomous Repression Domains in the Corepressor Receptor Interacting Protein 140

Receptor interacting protein (RIP) 140 is a corepressor that can be recruited to nuclear receptors by means of LXXLL motifs. We have characterized four distinct autonomous repression domains in RIP140, termed RD1-4, that are highly conserved in mammals and birds. RD1 at the N terminus represses transcription in the presence of trichostatin A, suggesting that it functions by a histone deacetylase (HDAC)-independent mechanism. The repressive activity of RD2 is dependent upon carboxyl-terminal binding protein recruitment to two specific binding sites. Use of specific inhibitors indicates that RD2, RD3, and RD4 are capable of functioning by HDAC-dependent and HDAC-independent mechanisms, depending upon cell type.

Effects of prolonged cholecystokinin administration on rat pituitary-adrenocortical axis: Role of the CCK receptor subtypes 1 and 2

Many lines of evidence indicate that cholecystokinin (CCK) and its receptors, named CCK1-R and CCK2-R, are expressed in the hypothalamo-pituitary-adrenal (HPA) axis, the function of which they acutely stimulate. However, the role of endogenous CCK system in the regulation of HPA axis is still unknown. To address this issue we investigated the effect of the prolonged (6-day) administration of CCK, CCK-R antagonists (CCK-RAs) and pentagastrin (PG), a CCK2-R agonist, on adult rat HPA axis. Semiquantitative reverse transcription-polymerase chain reaction showed that CCK treatment lowered the expression of CCK1-R and CCK2-R mRNAs in the pituitary, but not adrenal gland. ACTH plasma concentration was not affected by any treatment. Neither CCK nor PG administration induced significant changes in the blood levels of aldosterone and corticosterone. CCK1-RA, although being per se ineffective, in the presence of CCK raised plasma levels of aldosterone and corticosterone; conversely, CCK2-RA, either alone or in the presence of agonists, lowered the blood concentrations of the two hormones. CCK, but not PG, treatment decreased relative adrenal weight, and morphometry showed that CCK-induced adrenal atrophy was coupled to decreases in the volume of adrenocortical zones, which in turn mainly depended on the lowering in the volume and number of adrenocortical cells. PG administration raised and CCK2-RA per se decreased the volume and number of adrenocortical cells. Taken together, these findings allow us to draw the following main conclusions: i) the prolonged exposure to elevated CCK concentrations down-regulates CCK-R expression in the pituitary gland, which accounts for the lack of effect of CCK on ACTH secretion; ii) adrenal CCK1-Rs and CCK2-Rs inhibit and stimulate, respectively, corticosteroid secretion; and iii) endogenous CCK system plays a minor role in the physiological regulation of rat HPA axis, its main effect being the CCK2-R-mediated maintenance of adrenocortical-cell number.

Angiotensin-(1–7) stimulates water transport in rat inner medullary collecting duct: evidence for involvement of vasopressin V2 receptors

The peptide angiotensin-(1-7) [Ang-(1-7)] is known to enhance water transport in rat inner medullary collecting duct (IMCD). The aim of this study was to determine the mechanism of the Ang-(1-7) effect on osmotic water permeability (Pf). Pf was measured in the normal rat IMCD perfused in vitro in presence of agonists [Ang-(1-7), arginine vasopressin (AVP) and Ang-(3-8)], and antagonists of the angiotensin and the vasopressin cascade. Ang-(1-7), but not Ang-(3-8), increased Pf significantly. The effect of Ang-(1-7) on Pf was abolished by its selective antagonist, A-779, added before or after Ang-(1-7). Prostaglandin E2 and the protein kinase A inhibitor H8 also blocked the Ang-(1-7) effect. Blockade of vasopressin V1 receptors by antagonists did not change the Ang-(1-7) effect, but pre-treatment with a V2 antagonist abolished the effect of Ang-(1-7) on Pf. Similarly, pre-treatment with A-779 inhibited AVP's effect on Pf. Forskolin-stimulated Pf was blocked both by A-779 and by the V2 antagonist. Finally, Ang-(1-7) increased cAMP levels in fresh IMCD cell suspensions whilst the forskolin-stimulated cAMP synthesis was decreased by A-779 and the V2 antagonist. These data provide evidence that Ang-(1-7) interacts via its receptor with the AVP V2 system through a mechanism involving adenylate-cyclase activation.

Repressors of Androgen and Progesterone Receptor Action

Androgen and progesterone receptors (AR and PR) are two determining factors in gonadal differentiation that are highly expressed in developing and mature gonads. Loss of AR results in XY sex reversal and mutations causing reduced AR activity lead to varying degrees of defects in masculinization. Female PR knockout mice are infertile due to ovarian defects. While much has been discovered about positive regulation of these receptors by coactivators little is known about repression of the transcriptional activity of AR and PR in the presence of agonists. In this study we assessed the effect of SMRT and DAX-1 on AR and PR activity in the presence of both agonists and partial antagonists. We show that SMRT and DAX-1 repress agonist-dependent activity of both receptors, and the mechanism of repression includes disruption of the receptor dimer interactions rather than recruitment of histone deacetylases. We demonstrate that endogenous agonist-bound PR and DAX-1 in T47D breast cancer cells and endogenous AR and DAX-1 in LNCaP prostate cancer cells can be coimmunoprecipitated suggesting that the interaction is physiological. Surprisingly, although DAX-1 represses partial antagonist activity of AR, it was ineffective in repressing partial antagonist induced activity of PR. In contrast to most reported repressors, the expression of DAX-1 is restricted. We found that although DAX-1 is expressed in normal human prostate, its expression is strongly reduced in benign prostatic hyperplasia suggesting that DAX-1 plays a role in limiting AR activity in prostate.

ERbeta Binds N-CoR in the Presence of Estrogens via an LXXLL-like Motif in the N-CoR C-terminus.

Nuclear receptors (NRs) usually bind the corepressors N-CoR and SMRT in the absence of ligand or in the presence of antagonists. Agonist binding leads to corepressor release and recruitment of coactivators. Here, we report that estrogen receptor β (ERβ) binds N-CoR and SMRT in the presence of agonists, but not antagonists, in vitro and in vivo. This ligand preference differs from that of ERα interactions with corepressors, which are inhibited by estradiol, and resembles that of ERβ interactions with coactivators. ERβ /N-CoR interactions involve ERβ AF-2, which also mediates coactivator recognition. Moreover, ERβ recognizes a sequence (PLTIRML) in the N-CoR C-terminus that resembles coactivator LXXLL motifs. Inhibition of histone deacetylase activity specifically potentiates ERβ LBD activity, suggesting that corepressors restrict the activity of AF-2. We conclude that the ER isoforms show completely distinct modes of interaction with a physiologically important corepressor and discuss our results in terms of ER isoform specificity in vivo.