Biomedical subjects

Paul R Albert

Publications and source records attributed to Paul R Albert.

At least 19 recordsLinked to original sources

PubMed2006

RGS17/RGSZ2 and the RZ/A family of regulators of G-protein signaling.

Regulators of G-protein signaling (RGS proteins) comprise over 20 different proteins that have been classified into subfamilies on the basis of structural homology. The RZ/A family includes RGSZ2/RGS17 (the most recently discovered member of this family), GAIP/RGS19, RGSZ1/RGS20, and the RGSZ1 variant Ret-RGS. The RGS proteins are GTPase activating proteins (GAPs) that turn off G-proteins and thus negatively regulate the signaling of G-protein coupled receptors (GPCRs). In addition, some RZ/A family RGS proteins are able to modify signaling through interactions with adapter proteins (such as GIPC and GIPN). The RZ/A proteins have a simple structure that includes a conserved amino-terminal cysteine string motif, RGS box and short carboxyl-terminal, which confer GAP activity (RGS box) and the ability to undergo covalent modification and interact with other proteins (amino-terminal). This review focuses on RGS17 and its RZ/A sibling proteins and discusses the similarities and differences among these proteins in terms of their palmitoylation, phosphorylation, intracellular localization and interactions with GPCRs and adapter proteins. The specificity of these RGS protein for different Galpha proteins and receptors, and the consequences for signaling are discussed. The tissue and brain distribution, and the evolving understanding of the roles of this family of RGS proteins in receptor signaling and brain function are highlighted.

Animals↗

PubMed2006

Cell-specific repressor or enhancer activities of Deaf-1 at a serotonin 1A receptor gene polymorphism.

The serotonin-1A (5-HT1A) receptor is the primary somatodendritic autoreceptor that inhibits the activity of serotonergic raphe neurons and is also expressed in nonserotonergic cortical and limbic neurons. Alterations in 5-HT1A receptor levels are implicated in mood disorders, and a functional C(-1019)G 5-HT1A promoter polymorphism has been associated with depression, suicide, and panic disorder. We examined the cell-specific activity of identified transcription factors, human nuclear deformed epidermal autoregulatory factor-1 (DEAF-1)-related (NUDR)/Deaf-1 and Hes5, at the 5-HT1A C(-1019) site. In serotonergic raphe RN46A cells, Deaf-1 and Hes5 repressed the 5-HT1A receptor gene at the C(-1019)-allele but not the G(-1019)-allele. However, in nonserotonergic cells that express 5-HT1A receptors (septal SN48, neuroblastoma SKN-SH, and neuroblastoma/glioma NG108-15 cells), Deaf-1 enhanced 5-HT1A promoter activity at the C(-1019)-allele but not the G-allele, whereas Hes5 repressed in all cell types. The enhancer activity of Deaf-1 was orientation independent and competed out Hes5 repression. To test whether Deaf-1 activity is intrinsic, the activity of a Gal4DBD (DNA binding domain)-Deaf-1 fusion protein at a heterologous Gal4 DNA element was examined. Gal4DBD-Deaf-1 repressed transcription in RN46A cells but enhanced transcription in SN48 cells, indicating that these opposite activities are intrinsic to Deaf-1. Repressor or enhancer activities of Deaf-1 or Gal4DBD-Deaf-1 were blocked by histone deacetylase inhibitor trichostatin A. Thus, the intrinsic activity of Deaf-1 at the 5-HT1A promoter is opposite in presynaptic versus postsynaptic neuronal cells and requires deacetylation. Cell-specific regulation by Deaf-1 could underlie region-specific alterations in 5-HT1A receptor expression in different mood disorders.

Cell Line, Tumor↗

PubMed2006

Molecular determinants in the second intracellular loop of the 5-hydroxytryptamine-1A receptor for G-protein coupling.

This study provides the first comprehensive evidence that the second intracellular loop C-terminal domain (Ci2) is critical for receptor-G protein coupling to multiple responses. Although Ci2 is weakly conserved, its role in 5-hydroxytryptamine-1A (5-HT1A) receptor function was suggested by the selective loss of Gbetagamma-mediated signaling in the T149A-5-HT1A receptor mutant. More than 60 point mutant 5-HT1A receptors in the alpha-helical Ci2 sequence (143DYVNKRTPRR152) were generated. Most mutants retained agonist binding and were tested for Gbetagamma signaling to adenylyl cyclase II or phospholipase C and Galphai coupling to detect constitutive and agonist-induced Gi/Go coupling. Remarkably, most point mutations markedly attenuated 5-HT1A signaling, indicating that the entire Ci2 domain is critical for receptor G-protein coupling. Six signaling phenotypes were observed: wild-type-like, Galphai-coupled/weak Gbetagamma-coupled, Gbetagamma-uncoupled, Gbetagamma-selective coupled, uncoupled, and inverse coupling. Our data elucidate specific roles of Ci2 residues consistent with predictions based on rhodopsin crystal structure. The absolute coupling requirement for lysine, arginine, and proline residues is consistent with a predicted amphipathic alpha-helical Ci2 domain that is kinked at Pro150. Polar residues (Thr149, Asn146) located in the externally oriented positively charged face were required for Gbetagamma but not Galphai coupling, suggesting a direct interface with Gbetagamma subunits. The hydrophobic face includes the critical Tyr144 that directs the specificity of coupling to both Gbetagamma and Galphai pathways. The key coupling residues Tyr144/Lys147 (Ci2) are predicted to orient internally, forming hydrogen and ionic bonds with Asp133/Arg134 (Ni2 DRY motif) and Glu340 (Ci3) to stabilize the Gprotein coupling domain. Thus, the 5-HT1A receptor Ci2 domain determines Gbetagamma specificity and stabilizes Galphai-mediated signaling.

Amino Acid Sequence↗

PubMed2005

Differential roles of nuclear and cytoplasmic cyclin-dependent kinase 5 in apoptotic and excitotoxic neuronal death.

Cyclin-dependent kinase 5 (cdk5) is a member of the cyclin-dependent kinase family whose activity is localized mainly to postmitotic neurons attributable to the selective expression of its activating partners p35 and p39. Deregulation of cdk5, as a result of calpain cleavage of p35 to a smaller p25 form, has been suggested to be a central component of neuronal death underlying numerous neurodegenerative diseases. However, the relevance of cdk5 in apoptotic death that relies on the mitochondrial pathway is unknown. Furthermore, evidence that cdk5 can also promote neuronal survival has necessitated a more complex understanding of cdk5 in the control of neuronal fate. Here we explore each of these issues using apoptotic and excitotoxic death models. We find that apoptotic death induced by the DNA-damaging agent camptothecin is associated with early transcription-mediated loss of p35 and with late production of p25 that is dependent on Bax, Apaf1, and caspases. In contrast, during excitotoxic death induced by glutamate, neurons rapidly produce p25 independent of the mitochondrial pathway. Analysis of the localization of p35 and p25 revealed that p35 is mainly cytoplasmic, whereas p25 accumulates selectively in the nucleus. By targeting a dominant-negative cdk5 to either the cytoplasm or nucleus, we show that cdk5 has a death-promoting activity within the nucleus and that this activity is required in excitotoxic death but not apoptotic death. Moreover, we also find that cdk5 contributes to pro-survival signaling selectively within the cytoplasm, and manipulation of this signal can modify death induced by both excitotoxicity and DNA damage.

Animals↗

PubMed2005

Coupling of 5-HT1A autoreceptors to inhibition of mitogen-activated protein kinase activation via G beta gamma subunit signaling.

The 5-HT1A receptor is expressed presynaptically as the primary somatodendritic autoreceptor on serotonergic raphe neurons, and postsynaptically in several brain regions. Signaling of the 5-HT1A autoreceptor was studied in RN46A cells, a model of serotonergic raphe neurons that express endogenous 5-HT1A receptors. In undifferentiated RN46A cells stably transfected with the wild-type 5-HT1A receptor, 5-HT1A receptor activation inhibited forskolin-induced cyclic adenosine monophosphate (cAMP) formation (by 50%), increased [Ca2+]i, and induced a novel inhibition (up to 60%) of phospho-p42/p44-mitogen-activated protein kinase (MAPK). Upon differentiation of non-transfected or 5-HT1A-transfected RN46A cells, agonist-mediated inhibition of MAPK was enhanced. These actions were blocked by pretreatment with pertussis toxin indicating mediation via Gi/Go proteins and the calcium response was blocked by preactivation of protein kinase C (PKC). In cells overexpressing the G beta gamma scavenger carboxyl-terminal domain of G protein receptor kinase 2 (GRK-CT), 5-HT1A receptor activation inhibited cAMP formation, but coupling to calcium mobilization and inhibition of MAPK was abolished. The activity of 5-HT1A receptors containing mutations of PKC sites in the second (i2: T149A) or third intracellular loop (i3: T229A/S253G/T343A) was tested. At comparable levels of receptor expression, the signaling of the 5-HT1A i3 mutant was similar to the 5-HT1A wild-type receptor, while the i2 and quadruple (i2/i3) mutants failed to couple to G beta gamma-mediated increase in [Ca2+]i or inhibition of MAPK, but did couple to G alpha i-mediated inhibition of cAMP. Thus, the i2-domain of the 5-HT1A autoreceptor is crucial for coupling to G beta gamma subunits and their subsequent responses (e.g. calcium mobilization and inhibition of MAPK activity).

8-Hydroxy-2-(di-n-propylamino)tetralin↗

PubMed2004

Association of the C(-1019)G 5-HT1A functional promoter polymorphism with antidepressant response.

Antidepressants, such as serotonin or noradrenaline reuptake inhibitors (e.g. fluoxetine, nefadozone) or 5-HT1A agonists (flibanserin), desensitize the 5-HT1A autoreceptor, which may contribute to their clinical efficacy. The 5-HT1A receptor gene is repressed by NUDR/DEAF-1 in raphe cells at the C-, but not at the G-allele of the C(-1019)G polymorphism that is associated with major depression and suicide. Depressed patients (n=118) were treated with antidepressants including fluoxetine or nefadozone combined with pindolol or flibanserin alone. The severity of depression was assesssed using the Hamilton Rating Scale for Depression. Although patients had similar severity initially, those with the homozygous G(-1019) genotype responded significantly less to flibanserin (p=0.039) and in pooled antidepressant treatment groups (p=0.0497) and were approximately twice as likely to be non-responders as those with the C(-1019)C genotype. These results implicate the C(-1019)G 5-HT1A gene polymorphism as a potential marker for antidepressant response, suggesting a role for repression of the 5-HT1A gene.

Adrenergic beta-Antagonists↗

PubMed2004

The proapoptotic gene SIVA is a direct transcriptional target for the tumor suppressors p53 and E2F1.

The p53 tumor suppressor gene is believed to play an important role in neuronal cell death in acute neurological disease and in neurodegeneration. The p53 signaling cascade is complex, and the mechanism by which p53 induces apoptosis is cell type-dependent. Using DNA microarray analysis, we have found a striking induction of the proapoptotic gene, SIVA. SIVA is a proapoptotic protein containing a death domain and interacts with members of the tumor necrosis factor receptor family as well as anti-apoptotic Bcl-2 family proteins. SIVA is induced following direct p53 gene delivery, treatment with a DNA-damaging agent camptothecin, and stroke injury in vivo. SIVA up-regulation is sufficient to initiate the apoptotic cascade in neurons. Through isolation and analysis of the SIVA promoter, we have identified response elements for both p53 and E2F1. Like p53, E2F1 is another tumor suppressor gene involved in the regulation of apoptosis, including neuronal injury models. We have identified E2F consensus sites in the promoter region, whereas p53 recognition sequences were found in intron1. Sequence analysis has shown that these consensus sites are also conserved between mouse and human SIVA genes. Electrophoretic mobility shift assays reveal that both transcription factors are capable of binding to putative consensus sites, and luciferase reporter assays reveal that E2F1 and p53 can activate transcription from the SIVA promoter. Here, we report that the proapoptotic gene, SIVA, which functions in a broad spectrum of cell types, is a direct transcriptional target for both tumor suppressors, p53 and E2F1.

Adenoviridae↗

PubMed2004

RGS17/RGSZ2, a novel regulator of Gi/o, Gz, and Gq signaling.

To identify novel regulators of Galpha(o), the most abundant G-protein in brain, we used yeast two-hybrid screening with constitutively active Galpha(o) as bait and identified a new regulator of G-protein signaling (RGS) protein, RGS17 (RGSZ2), as a novel human member of the RZ (or A) subfamily of RGS proteins. RGS17 contains an amino-terminal cysteine-rich motif and a carboxyl-terminal RGS domain with highest homology to hRGSZ1- and hRGS-Galpha-interacting protein. RGS17 RNA was strongly expressed as multiple species in cerebellum and other brain regions. The interactions between hRGS17 and active forms of Galpha(i1-3), Galpha(o), Galpha(z), or Galpha(q) but not Galpha(s) were detected by yeast two-hybrid assay, in vitro pull-down assay, and co-immunoprecipitation studies. Recombinant RGS17 acted as a GTPase-activating protein (GAP) on free Galpha(i2) and Galpha(o) under pre-steady-state conditions, and on M2-muscarinic receptor-activated Galpha(i1), Galpha(i2), Galpha(i3), Galpha(z), and Galpha(o) in steady-state GTPase assays in vitro. Unlike RGSZ1, which is highly selective for G(z), RGS17 exhibited limited selectivity for G(o) among G(i)/G(o) proteins. All RZ family members reduced dopamine-D2/Galpha(i)-mediated inhibition of cAMP formation and abolished thyrotropin-releasing hormone receptor/Galpha(q)-mediated calcium mobilization. RGS17 is a new RZ member that preferentially inhibits receptor signaling via G(i/o), G(z), and G(q) over G(s) to enhance cAMP-dependent signaling and inhibit calcium signaling. Differences observed between in vitro GAP assays and whole-cell signaling suggest additional determinants of the G-protein specificity of RGS GAP effects that could include receptors and effectors.

Amino Acid Motifs↗

PubMed2004

Expression of adenylyl cyclase-4 (AC-4) in Y1 and forskolin-resistant adrenal cells.

Forskolin-resistant mutants of a mouse adrenocortical cell line present a complex phenotype in which adenylyl cyclase (AC) is resistant to activation by forskolin and by ACTH. ACTH-resistance results from a defect affecting transcription of the ACTH receptor and can be overcome by transfecting mutant cells with expression vectors encoding G beta/gamma. Forskolin-resistance results from an AC-4 deficiency. We now demonstrate that the AC-4 deficiency in forskolin-resistant mutants results from a transcription defect affecting the promoter activity of the AC-4 gene. Furthermore, the underlying defect leading to AC-4 deficiency and forskolin-resistance can be overcome by transfection of mutant clones with expression vectors encoding G beta/gamma. These data support our hypothesis that AC-4 is a preferred target of forskolin action in Y1 cells, demonstrate novel roles for G beta/gamma in gene expression and indicate that a common underlying defect, suppressible by G beta/gamma, accounts for both the resistance to ACTH and to forskolin.

Adenylyl Cyclases↗

PubMed2004

Forskolin-resistant Y1 adrenal cell mutants are deficient in adenylyl cyclase type 4.

Four mutant clones independently derived from the Y1 mouse adrenocortical tumor cell line have adenylyl cyclase (AC) activities that are resistant to forskolin, a direct activator of AC. In this study the AC isoform composition of the forskolin-resistant mutants was examined in order to explore the underlying basis for the resistance to forskolin. As determined by Western blot and RT-PCR analysis, the four forskolin-resistant mutants all were deficient in AC-4; the levels of other AC isoforms (AC-1, AC-3 and AC-5/6) were comparable to the levels in parent Y1 cells. Transfection of one of the mutant clones with an AC-4 expression vector increased forskolin-stimulated cAMP signaling, and restored forskolin-induced changes in cell morphology and growth. Taken together, these observations indicate that AC-4 deficiency is a hallmark of the forskolin-resistant phenotype of these mutants and suggest that AC-4 is an important target of forskolin action in the Y1 adrenal cell line.

Adenylyl Cyclases↗

PubMed2004

5-HT1A-mediated promotion of mitogen-activated T and B cell survival and proliferation is associated with increased translocation of NF-kappaB to the nucleus.

Mitogenic activation of T and B lymphocytes induces expression of the 5-HT(1A) receptor through an NF-kappaB-dependent signaling pathway. In the present study, it is shown that serotonin (5-HT), as well as the selective 5-HT(1A) receptor agonist R-DPAT, increase cell survival and S phase transition in mouse splenocytes stimulated by T or B cell mitogens. Further examination of the mechanisms underlying increased cell survival revealed that 5-HT and R-DPAT inhibited apoptotic cell death, assessed both by soluble DNA content, internucleosomal DNA cleavage, and hypodiploid DNA content. Additionally, 5-HT and R-DPAT treatment increased intranuclear levels of the p50 and p65 subunits of NF-kappaB. Potentiation by 5-HT and R-DPAT of mitogen-activated cell survival, S phase transition, and nuclear localization of NF-kappaB, as well as inhibition of apoptosis, were all reversed by the selective 5-HT(1A) receptor antagonist WAY-100635. These results indicate that 5-HT(1A)-mediated promotion of cell survival and proliferation of mitogen-activated T and B lymphocytes is associated with increased translocation of NF-kappaB in the nucleus.

8-Hydroxy-2-(di-n-propylamino)tetralin↗

PubMed2004

Cell type-dependent recruitment of trichostatin A-sensitive repression of the human 5-HT1A receptor gene.

Regulation of serotonin (5-HT)1A receptor expression in brain is implicated in mood disorders such as depression and anxiety. Transcriptional activity of the human 5-HT1A receptor gene was strongly repressed by a negative regulatory region containing a consensus repressor element-1 (RE-1) and two copies of the dual repressor element (DRE) identified in the rat 5-HT1A receptor gene. REST/NRSF, a silencer of neuronal genes, bound the 5-HT1A RE-1 and repressed the 5-HT1A promoter. Inactivation of RE-1 completely abolished REST-mediated repression, but resulted in only partial (15-50%) de-repression of basal 5-HT1A promoter activity. The human 5-HT1A DRE sequences bound specifically to the novel repressor Freud-1 (5'repressor element under dual repression binding protein-1) and conferred repressor activity at 5-HT1A or SV40 promoters. In 5-HT1A-negative cells [L6, human embryonic kidney (HEK) 293], the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) abolished repression mediated by both RE-1/REST and DRE/Freud-1, and induced almost complete de-repression of the 5-HT1A gene. By contrast, in 5-HT1A-expressing neuronal cells (RN46A, SN-48) TSA blocked RE-1/REST repression, but did not affect DRE/Freud-1-mediated repression. Thus in contrast to REST, Freud-1 mediates HDAC-independent repression of the 5-HT1A receptor promoter in neuronal 5-HT1A-positive cells, suggesting that HDAC recruitment might influence neuron-specific gene expression by further silencing expression in non-neuronal tissue.

Animals↗

PubMed2004

5-HT1A receptors, gene repression, and depression: guilt by association.

The serotonin system is implicated in major depression and suicide and is negatively regulated by somatodendritic 5-HT1A autoreceptors. Desensitization of 5-HT1A autoreceptors is implicated in the 2- to 3-week latency for antidepressant treatments. Alterations in 5-HT1A receptor levels are reported in depression and suicide, and gene knockout of the 5-HT1A receptor results in an anxiety phenotype, suggesting that abnormal transcriptional regulation of this receptor gene may underlie these disorders. The 5-HT1A receptor gene is negatively regulated in neurons by repressors including REST/NRSF, Freud-1, NUDR/Deaf-1, and Hes5. The association with major depression, suicide, and panic disorder of a new functional 5-HT1A polymorphism at C(-1019)G that selectively blocks repression of the 5-HT1A autoreceptor by NUDR further suggests a causative role for altered regulation of this receptor in predisposition to mental illness. The authors review evidence that altered transcription of the 5-HT1A receptor can affect the serotonin system and limbic and cortical areas, leading to predisposition to depression.

Animals↗

PubMed2003

Impaired repression at a 5-hydroxytryptamine 1A receptor gene polymorphism associated with major depression and suicide.

Inhibition of serotonergic raphe neurons is mediated by somatodendritic 5-HT1A autoreceptors, which may be increased in depressed patients. We report an association of the C(-1019)G 5-HT1A promoter polymorphism with major depression and suicide in separate cohorts. In depressed patients, the homozygous G(-1019) allele was enriched twofold versus controls (p = 0.0017 and 0.0006 for G/G genotype and G allele distribution, respectively), and in completed suicide cases the G(-1019) allele was enriched fourfold (p = 0.002 and 0.00008 for G/G genotype and G allele distribution, respectively). The C(-1019) allele was part of a 26 bp imperfect palindrome that bound transcription factors nuclear NUDR [nuclear deformed epidermal autoregulatory factor (DEAF-1)]/suppressin and Hairy/Enhancer-of-split-5 (Drosophila) (Hes5) to repress 5-HT1A or heterologous promoters, whereas the G(-1019) allele abolished repression by NUDR, but only partially impaired Hes5-mediated repression. Recombinant NUDR bound specifically to the 26 bp palindrome, and endogenous NUDR was present in the major protein-DNA complex from raphe nuclear extracts. Stable expression of NUDR in raphe cells reduced levels of endogenous 5-HT1A protein and binding. NUDR protein was colocalized with 5-HT1A receptors in serotonergic raphe cells, hippocampal and cortical neurons, and adult brain regions including raphe nuclei, indicating a role in regulating 5-HT1A autoreceptor expression. Our data indicate that NUDR is a repressor of the 5-HT1A receptor in raphe cells the function of which is abrogated by a promoter polymorphism. We suggest a novel transcriptional model in which the G(-1019) allele derepresses 5-HT1A autoreceptor expression to reduce serotonergic neurotransmission, predisposing to depression and suicide.

Adult↗

PubMed2003

Freud-1: A neuronal calcium-regulated repressor of the 5-HT1A receptor gene.

Altered regulation of 5-HT1A receptors is implicated in mood disorders such as anxiety and major depression. To provide insight into its transcriptional regulation, we previously identified a novel DNA element [14 bp 5'-repressor element (FRE)] of the 5-HT1A receptor gene that mediates repression in neuronal and non-neuronal cells (Ou et al., 2000). We have now cloned a novel DNA binding protein [five' repressor element under dual repression binding protein-1 (Freud-1)] that binds to FRE to mediate repression of the 5-HT1A receptor or heterologous promoters. Freud-1 is evolutionarily conserved and contains two DM-14 basic repeats, a predicted helix-loop-helix DNA binding domain, and a protein kinase C conserved region 2 (C2)/calcium-dependent lipid binding (CalB) calcium/phospholipid binding domain. An intact CalB domain was required for Freud-1-mediated repression. In serotonergic raphe cells, overexpression of Freud-1 repressed the 5-HT1A promoter and decreased 5-HT1A receptor protein levels, whereas transfection of antisense to Freud-1 derepressed the 5-HT1A gene and increased 5-HT1A receptor protein expression. Calcium-dependent signaling blocked Freud-1-FRE binding and derepressed the 5-HT1A promoter. Treatment with inhibitors of calmodulin or CAM-dependent protein kinase reversed calcium-mediated inhibition of Freud-1. Freud-1 RNA and protein were present in raphe nuclei, hippocampus, cortex, and hypothalamus, and Freud-1 protein was colocalized with 5-HT1A receptors, suggesting its importance in regulating 5-HT1A receptors in vivo. Thus, Freud-1 represents a novel calcium-regulated repressor that negatively regulates basal 5-HT1A receptor expression in neurons and may play a role in the altered regulation of 5-HT1A receptors associated with anxiety or major depression.

Adenosine Triphosphate↗

PubMed2002

Diacylglycerol and ceramide formation induced by dopamine D2S receptors via Gbeta gamma -subunits in Balb/c-3T3 cells.

Diacylglycerol (DAG) and ceramide are important second messengers affecting cell growth, differentiation, and apoptosis. Balb/c-3T3 fibroblast cells expressing dopamine-D2S (short) receptors (Balb-D2S cells) provide a model of G protein-mediated cell growth and transformation. In Balb-D2S cells, apomorphine (EC(50) = 10 nM) stimulated DAG and ceramide formation by 5.6- and 4.3-fold, respectively, maximal at 1 h and persisting over 6 h. These actions were blocked by pretreatment with pertussis toxin (PTX), implicating G(i)/G(o) proteins. To address which G proteins are involved, Balb-D2S clones expressing individual PTX-insensitive Galpha(i) proteins were treated with PTX and tested for apomorphine-induced responses. Neither PTX-insensitive Galpha(i2) nor Galpha(i3) rescued D2S-induced DAG or ceramide formation. Both D2S-induced DAG and ceramide signals required Gbetagamma-subunits and were blocked by inhibitors of phospholipase C [1-(6-[([17beta]-3-methoxyestra-1,2,3[10]-trien- 17yl)amino]hexyl)-1H-pyrrole-2,5-dione (U-73122) and partially by D609]. The similar G protein specificity of D2S-induced calcium mobilization, DAG, and ceramide formation indicates a common Gbetagamma-dependent phospholipase C-mediated pathway. Both D2 agonists and ceramide specifically induced mitogen-activated protein kinase (ERK1/2), suggesting that ceramide mediates a novel pathway of D2S-induced ERK1/2 activation, leading to cell growth.

3T3 Cells↗

PubMed2002

Growth hormone-induced diacylglycerol and ceramide formation via Galpha i3 and Gbeta gamma in GH4 pituitary cells. Potentiation by dopamine-D2 receptor activation.

Growth hormone (GH) secretion is regulated by indirect negative feedback mechanisms. To address whether GH has direct actions on pituitary cells, lipid signaling in GH(4)ZR(7) somatomammotroph cells was examined. GH (EC(50) = 5 nm) stimulated diacylglycerol (DAG) and ceramide formation in parallel by over 10-fold within 15 min and persisting for >3 h. GH-induced DAG/ceramide formation was blocked by pertussis toxin (PTX) implicating G(i)/G(o) proteins and was potentiated 1.5-fold by activation of G(i)/G(o)-coupled dopamine-D2S receptors, which had no effect alone. Following PTX pretreatment, only PTX-resistant Galpha(i)3, not Galpha(o) or Galpha(i)2, rescued GH-induced DAG/ceramide signaling. GH-induced DAG/ceramide formation was also blocked in cells expressing Gbetagamma blocker GRK-ct. In GH(4)ZR(7) cells, GH induced phosphorylation of JAK2 and STAT5, which was blocked by PTX and mimicked by ceramide analogue C2-ceramide or sphingomyelinase treatment to increase endogenous ceramide. We conclude that in GH(4) pituitary cells, GH induces formation of DAG/ceramide via a novel Galpha(i)3/Gbetagamma-dependent pathway. This novel pathway suggests a mechanism for autocrine feedback regulation by GH of pituitary function.

Apomorphine↗

PubMed2002

G protein specificity: traffic direction required.

This review focuses on the coupling specificity of the Galpha and Gbetagamma subunits of pertussis toxin (PTX)-sensitive G(i/o) proteins that mediate diverse signaling pathways, including regulation of ion channels and other effectors. Several lines of evidence indicate that specific combinations of G protein alpha, beta and gamma subunits are required for different receptors or receptor-effector networks, and that a higher degree of specificity for Galpha and Gbetagamma is observed in intact systems than reported in vitro. The structural determinants of receptor-G protein specificity remain incompletely understood, and involve receptor-G protein interaction domains, and perhaps other scaffolding processes. By identifying G protein specificity for individual receptor signaling pathways, ligands targeted to disrupt individual pathways of a given receptor could be developed.

Adenylyl Cyclases↗