Effect of the glucocorticoid receptor antagonist Org 34850 on basal and stress-induced corticosterone secretion.

The activity of the hypothalamic-pituitary-adrenal (HPA) axis is characterised both by an ultradian pulsatile pattern of glucocorticoid secretion and an endogenous diurnal rhythm. Glucocorticoid feedback plays a major role in regulating HPA axis activity and this mechanism occurs via two different receptors: mineralocorticoid (MR) and glucocorticoid receptors (GR). In the present study, the effects of both acute and subchronic treatment with the GR antagonist Org 34850 on basal and stress-induced HPA axis activity in male rats were evaluated. To investigate the effect of Org 34850 on basal diurnal corticosterone rhythm over the 24-h cycle, an automated blood sampling system collected samples every 10 min. Acute injection of Org 34850 (10 mg/kg, s.c.) did not affect basal or stress-induced corticosterone secretion, but was able to antagonise the inhibitory effect of the glucocorticoid agonist methylprednisolone on stress-induced corticosterone secretion. However, 5 days of treatment with Org 34850 (10 mg/kg, s.c., two times a day), compared to rats treated with vehicle (5% mulgofen in 0.9% saline, 1 ml/kg, s.c.), increased corticosterone secretion over the 24-h cycle and resulted in changes in the pulsatile pattern of hormone release, but had no significant effect on adrenocorticotrophic hormone secretion or on stress-induced corticosterone secretion. Subchronic treatment with Org 34850 did not alter GR mRNA expression in the hippocampus, paraventricular nucleus of the hypothalamus or anterior-pituitary, or MR mRNA expression in the hippocampus. Our data suggest that a prolonged blockade of GRs is required to increase basal HPA axis activity. The changes observed here with ORG 34850 are consistent with inhibition of GR-mediated negative feedback of the HPA axis. In light of the evidence showing an involvement of dysfunctional HPA axis in the pathophysiology of depression, Org 34850 could be a potential treatment for mood disorders.

How do G-proteins stay at the plasma membrane?

G-protein alpha-subunits are modified by combinations of fatty acyl groups. G-protein gamma-subunits are isoprenylated. These modifications play central roles in membrane association of the G-proteins. Attachment of long-chain fatty acyl chains to G-protein alpha-subunits via a thioester linkage allows the possibility of dynamic regulation of membrane attachment and function.

Sex-specific association between bipolar affective disorder in women and GPR50, an X-linked orphan G protein-coupled receptor.

GPR50 is an orphan G protein-coupled receptor (GPCR) located on Xq28, a region previously implicated in multiple genetic studies of bipolar affective disorder (BPAD). Allele frequencies of three polymorphisms in GPR50 were compared in case-control studies between subjects with BPAD (264), major depressive disorder (MDD) (226), or schizophrenia (SCZ) (263) and ethnically matched controls (562). Significant associations were found between an insertion/deletion polymorphism in exon 2 and both BPAD (P=0.0070), and MDD (P=0.011) with increased risk associated with the deletion variant (GPR50(Delta502-505)). When the analysis was restricted to female subjects, the associations with BPAD and MDD increased in significance (P=0.00023 and P=0.0064, respectively). Two other single-nucleotide polymorphisms (SNPs) tested within this gene showed associations between: the female MDD group and an SNP in exon 2 (P=0.0096); and female SCZ and an intronic SNP (P=0.0014). No association was detected in males with either MDD, BPAD or SCZ. These results suggest that GPR50(Delta502-505), or a variant in tight linkage disequilibrium with this polymorphism, is a sex-specific risk factor for susceptibility to bipolar disorder, and that other variants in the gene may be sex-specific risk factors in the development of schizophrenia.

Analysis of the relative interactions between the alpha 2C10 adrenoceptor and the guanine-nucleotide-binding proteins G(o)1 alpha and Gi 2 alpha following co-expression of these polypeptides in rat 1 fibroblasts.

Rat 1 fibroblasts which had been transfected to express the human alpha 2C10 adrenoceptor (clone 1C) were further co-transfected with a plasmid containing the hygromycin-B-resistance gene and a plasmid containing a cDNA encoding the alpha-subunit of the rat pertussis-toxin-sensitive G-protein G(o)1. In clone 3 the receptor was expressed at some 2.2 pmol/mg of membrane protein, and G(o)1 alpha at approx. 100 pmol/mg of membrane protein. The interaction of these two polypeptides and that between the receptor and Gi2 alpha (endogenously expressed at some 50 pmol/mg of membrane protein) were studied. Agonist activation of G(o)1 alpha was observed in membranes of the alpha 2C10-adrenoceptor(+)-G(o)1 alpha+ cells (clone 3), but not in alpha 2C10-adrenoceptor(+)-G(o)alpha-cells (clone 1C), whereas similar agonist-dependent activation of Gi2 alpha was observed in both cell types. alpha 2C10-adrenoceptor activation of G(o)1 alpha and Gi2 alpha in clone-3 membranes was produced with similar agonist-dose-effect curves. These observations indicate that the receptor interacts with equivalent affinity with each of these G-proteins. Agonist-dependent cholera-toxin-catalysed [32P]ADP-ribosylation of G(o)1 alpha was terminated when the alpha 2-adrenoceptor antagonist yohimbine was added subsequent to agonist-induced initiation of the reaction and release of GDP, demonstrating the conformational requirement for this reaction to be the ternary complex of agonist-occupied receptor and guanine-nucleotide-denuded G-protein.

Stimulation of high-affinity GTPase activity and cholera toxin-catalysed [32P]ADP-ribosylation of Gi by lysophosphatidic acid (LPA) in wild-type and alpha 2C10 adrenoceptor-transfected Rat 1 fibroblasts.

Lysophosphatidic acid (LPA) stimulated high-affinity GTPase activity in membranes of Rat 1 fibroblasts. This effect was dose-dependent, with maximal effects at 10 microM LPA, and was attenuated by pertussis toxin but not by cholera toxin pretreatment of the cells, indicating that the effect was likely to be produced by a Gi-like G-protein. LPA stimulation of high-affinity GTPase was also observed in a clone of Rat 1 fibroblasts that had been transfected to express the human alpha 2C10 adrenoceptor. The alpha 2 adrenoceptor agonist UK14304 also stimulated high-affinity GTPase activity in membranes of these cells, but not in parental Rat 1 cells. LPA was also able to promote cholera toxin-catalysed [32P]ADP-ribosylation of Gi. This effect of LPA was also prevented by pretreatment of the cells with pertussis toxin but not cholera toxin. LPA-stimulated cholera toxin-catalysed [32P]ADP-ribosylation of Gi in membranes of the alpha 2C10 adrenoceptor-expressing clone was additive with that produced by UK14304. Dose-response curves for LPA in the two assays of G-protein activation were coincident. The results presented herein demonstrate conclusively that the pertussis toxin-sensitive effects of LPA in Rat 1 fibroblasts and a clone of these cells expressing the alpha 2C10 adrenoceptor are produced directly by the activation of Gi.

The characteristic which makes the cell-coded HSV-inducible U90 distinctive in transformed cells is its greatly increased half-life.

A set of polypeptides detected in transformed cells of M(r) values 90,000 (a doublet) 40,000 and 32,000 that are recognised by immunoprecipitation of L-[35S]methionine-labelled tumour cell lysates, with sera from tumour-bearing rats and with antisera raised against herpes simplex virus type 2 (HSV-2)-infected cells, were previously reported (Macnab et al., 1985, 1992; Hewitt et al., 1991). These proteins are cell-coded and cannot be precipitated from similarly radiolabelled control cells. Two of these polypeptides are significantly induced by HSV-2 infection (Hewitt et al., 1991; Macnab et al., 1992). This paper further characterises one of these polypeptides, U90, and addresses which properties distinguish the behaviour of U90 in tumour cells, whether there is an equivalent in control cells and whether U90 can be induced without HSV replication. U90 can be immunoprecipitated from radiolabelled human cells which are capable of extended passage in culture, as well as from human tumour cells, rodent tumour cells and cells of different lineages, e.g. epithelial, fibroblastic and lymphoid. Purification of U90 and the subsequent production of monospecific antibodies revealed, by western blotting, a homologue of 90 kDa in control cells. Western blotting shows that HSV can increase the amount of the U90 homologue in these normal cells. The absence of an immunoprecipitate of the U90 homologue from control cells is a result of the very short half-life (i.e. high turnover) of the protein in these cells (32.9 minutes) as opposed to tumour cells (about 13 hours).(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemical approaches to examine the specificity of interactions between receptors and guanine nuclotide binding proteins.

It is now appreciated both that G-protein-linked receptors and signal transducing heterotrimeric G-proteins consist of large multi-member superfamilies and that regulation of a signal transduction cascade can be produced by a variety of means following activation of a G-protein by a receptor. To begin to unravel the complexities of this regulation it is clearly important to be able to define the molecular identity of the G-protein or G-proteins activated by a receptor and to assess the quantitative importance of such interactions for the integration of signals produced by a receptor agonist. Substantial progress has been made towards these goals in recent years and the purpose of this short review will be to discuss the use and potential limitations of some of the currently most widely used approaches.

A cysteine-3 to serine mutation of the G-protein Gi1 alpha abrogates functional activation by the alpha 2A-adrenoceptor but not interactions with the beta gamma complex.

Pertussis toxin-resistant (C351G) and also palmitoylation-negative (C3S/C351G), myristoylation-negative (G2A/C351G) and combined acylation-negative (G2A/C3S/C351G) forms of the G-protein Gi1 alpha were expressed in COS-7 cells along with the porcine alpha 2A-adrenoceptor. G2A/C3S/C351G Gi1 alpha and G2A/C351G Gi1 alpha were largely cytosolic and failed to interact with the agonist-occupied alpha 2A-adrenoceptor in membrane preparations. In contrast, C351G Gi1 alpha was almost entirely particulate and the alpha 2-adrenoceptor agonist UK14304 caused a marked stimulation of its GTPase activity and binding of [35S]GTP gamma S which was not prevented by pertussis toxin treatment of the cells. C3S/C351G Gi1 alpha was present in both the particulate and cytosolic fractions but the GTPase activity of the membrane bound fraction was only slightly activated by the alpha 2A-adrenoceptor. Coexpression of C3S/C351G Gi1 alpha and the alpha 2A-adrenoceptor along with beta 1 and gamma 2 subunits increased the P2 membrane complement of the alpha subunit and increased substantially the ratio of membrane bound to cytosolic protein. However, this also failed to allow marked stimulation of high-affinity GTPase activity by the alpha 2A-adrenoceptor despite the increased proportion of G-protein in the P2 membrane fraction. Despite the low fractional activation of C3S/C351G Gi1 alpha by the alpha 2A-adrenoceptor compared to C351G Gi1 alpha, the palmitoylation-resistant G-protein caused a marked reduction in pertussis toxin-resistant, agonist (UK14304)-mediated stimulation of adenylyl cyclase activity. UK14304 caused the same degree of effect on adenylyl cyclase activity in pertussis toxin-treated cells following transfection of the same amounts of C351G Gi1 alpha and C3S/C351G Gi1 alpha, as both appear to act to sequester beta gamma subunits. By contrast, neither G2A/C351G Gi1 alpha nor G2A/C3S/C351G Gi1 alpha resulted in effective regulation of adenylyl cyclase activity.

Thyrotropin-releasing hormone-induced subcellular redistribution and down-regulation of G11alpha: analysis of agonist regulation of coexpressed G11alpha species variants.

Human embryonic kidney 293 cells that had been transfected to express the long isoform of the rat thyrotropin-releasing hormone (TRH) receptor (clone E2) were further transfected with a cDNA encoding the murine version of G11alpha. A clone was isolated (clone E2M11) that stably expressed murine as well as the endogenous human G11alpha. Subcellular fractionation demonstrated identical cellular distribution of the two species variants of this G protein. Sustained exposure of clone E2M11 cells to TRH resulted in substantial cellular redistribution and reduction in total cellular levels of G11alpha immunoreactivity. Fractions of both the exogenously introduced murine and endogenously expressed human isoforms of G11alpha were transferred from plasma membranes to low density membranes (detected as a shift from middle to low density regions on sucrose density gradients) and cytosol fractions. The plasma membrane redistribution to low density membrane was accompanied by a parallel redistribution of G protein beta subunits; however, there was no increase in beta subunits in the cytosol. The total cellular amount of G11alpha subunits was decreased to 21% and 59% for human and murine isoforms, respectively, and beta subunits were decreased to 68% after sustained treatment with TRH compared with controls (100%). Such data are consistent with the notion that the agonist-occupied long isoform of the rat TRH receptor may be able to partially differentiate between the endogenous (human) and exogenous (murine) G11alpha. This was not a reflection that the murine G protein was expressed but incorrectly folded as both species variants of G11alpha were solubilized equally from E2M11 membranes by sodium cholate. Using this system, we demonstrate both agonist-induced subcellular redistribution and down-regulation of G11alpha and beta subunit proteins in response to activation of a phospholipase C coupled receptor.

The palmitoylation status of the G-protein G(o)1 alpha regulates its activity of interaction with the plasma membrane.

Plasmids containing cDNAs encoding either the wild-type guanine-nucleotide-binding protein G(o)1 alpha or the palmitoylation-negative cysteine-3-to-serine (C3S) mutant of G(o)1 alpha were transfected into Rat 1 cells, and clones stably expressing immunoreactivity corresponding to these polypeptides were isolated. Clones C5B (expressing wild-type G(o)1 alpha) and D3 (expressing the mutant form) were selected for detailed study. Immunoprecipitation of whole cell lysates of each clone labelled with either [3H]palmitate or [3H]myristate demonstrated incorporation of [3H]myristate into both wild-type and the C3S mutant of G(o)1 alpha, but that incorporation of hydroxylamine-sensitive [3H]palmitate was restricted to the wild type. When membrane and cytoplasmic fractions were prepared from cells of either the C5B or D3 clones, although immunodetection of wild-type G(o)1 alpha was observed only in the membrane fraction, the C3S mutant was present in both membrane and cytoplasmic fractions. Furthermore, a significant proportion of the C3S G(o)1 alpha immunoreactivity was also detected in the cytoplasmic fraction if immunoprecipitation of recently synthesized G(o)1 alpha was performed from fractions derived from cells pulse-labelled with [35S]Trans label. Pretreatment of cells of both clones C5B and D3 with pertussis toxin led to complete ADP-ribosylation of the cellular population of G(o)1 alpha in both cell types, irrespective of whether the polypeptide was subsequently found in the membrane or cytoplasmic fraction following cellular disruption. By contrast, separation of membrane and cytoplasmic fractions before pertussis-toxin-catalysed [32P]ADP-ribosylation allowed modification only of the membrane-associated G(o)1 alpha (whether wild-type or the C3S mutant). This labelling was decreased substantially by incubation of the membranes with guanosine 5'-[beta gamma-imido]triphosphate. No cytoplasmic G-protein beta subunit was detected immunologically, and the non-membrane-associated C3S G(o)1 alpha from D3 cells migrated as an apparently monomeric 40 kDa protein on a Superose 12 gel-filtration column. Membrane-associated wild-type and C3S G(o)1 alpha appeared to interact with guanine nucleotides with similar affinity, as no alteration in the dose-response curves for guanine-nucleotide-induced maintenance of a stable 37 kDa tryptic fragment was noted for the two forms of G(o)1 alpha. Chemical depalmitoylation of membranes of clone C5B with neutral 1 M hydroxylamine caused a release of some 25-30% of each of G(o)1 alpha, Gi2 alpha and Gq alpha/G11 alpha from the membranes. Equivalent treatment of D3 cells caused an equivalent release of Gi2 alpha and Gq alpha/G11 alpha, but was unable to cause any appreciable release of the CS3 form of G(o)1 alpha, which was membrane-bound.

The role of palmitoylation of the guanine nucleotide binding protein G11 alpha in defining interaction with the plasma membrane.

Mutations of Cys-9 to serine, Cys-10 to serine and a combination of both alterations were produced in a cDNA encoding murine G11 alpha to potentially interfere with the ability of the expressed polypeptides to act as substrates for post-translational palmitoylation. Each of these mutants and the wild-type protein were expressed in simian COS-1 cells. Mutation of either cysteine-9 or cysteine-10 decreased the degree of palmitoylation of the protein by some 80% compared with the wild-type, while the double mutant totally failed to incorporate [3H]palmitate. By contrast, in all transfections the endogenously expressed simian G11 alpha incorporated [3H]palmitate to similar levels. Particulate and cytoplasmic fractions from these cells were subjected to SDS/PAGE under conditions which allow resolution of primate and rodent forms of G11 alpha. Immunoblotting of these fractions demonstrated that in all cases the endogenously expressed simian G11 alpha was exclusively associated with the particulate fraction, as was the transfected and expressed wild-type murine G11 alpha. By contrast, each of the mutated forms of murine G11 alpha displayed a distribution in which approx. 70% of the expressed protein was present in the particulate fraction and 30% in the supernatant. To examine the conformation of the particulate expressed forms of murine G11 alpha, these fractions were treated with various concentrations of sodium cholate and immunoblots were subsequently performed on the solubilized and remaining particulate proteins. Whereas essentially all of the endogenous simian G11 alpha was solubilized by treatment with 1% (w/v) sodium cholate and some 50% with 0.32% cholate, expressed wild-type murine G11 alpha was more recalcitrant to solubilization. However, that fraction of wild-type murine G11 alpha which was solubilized behaved identically to the endogenous simian G11 alpha on Superose-12 gel-exclusion chromatography. The particulate fraction of the C9S/C10S double mutant of murine G11 alpha was highly resistant to solubilization by sodium cholate, whereas the particulate fractions of the two single cysteine to serine mutants were intermediate to the wild-type and double mutant in their ability to be solubilized by this detergent. These data demonstrate that the palmitoylation status of the cysteine residues at positions 9 and 10 in murine G11 alpha plays a central role in defining membrane association of this G-protein and indicate that much of the particulate fraction of the expressed palmitoylation-resistant mutants is likely to represent non-functional rather than correctly folded protein.(ABSTRACT TRUNCATED AT 400 WORDS)

A transformation-specific polypeptide distinct from heat shock proteins is induced by herpes simplex virus type 2 infection.

A tumour-specific polypeptide designated U90 is one of a set of polypeptides which are encoded by the host cell and are specific for the transformed cell state, being immunoprecipitated by the sera of tumour-bearing animals. The interest in these tumour-specific polypeptides centres on the finding that they are also recognized by antisera raised against herpes simplex virus type 2 (HSV-2)-infected cells, implying some role for HSV-2 in tumorigenesis. The peptide map of HSV-2-induced U90 is indistinguishable from that of U90 present in uninfected tumour cells, including mouse cells transformed by human papillomavirus type 16. In tumour cells, U90 is located principally in the plasma membrane fraction and cannot be induced by heat shock, glucose starvation, or treatment with tunicamycin or calcium ionophore. U90 is not related to either the heat shock protein of Mr 90,000 (HSP90) or the glucose-related polypeptide of Mr 94,000 (GRP94) as determined by peptide mapping and the use of monospecific, monoclonal and antipeptide antibodies. This suggests that U90 is a novel transformation-specific protein which can be induced by infection with HSV-2.