Coupling of a cloned rat dopamine-D2 receptor to inhibition of adenylyl cyclase and prolactin secretion.

We have previously described a cDNA which encodes a binding site with the pharmacology of the D2-dopamine receptor (Bunzow, J. R., VanTol, H. H. M., Grandy, D. K., Albert, P., Salon, J., Christie, M., Machida, C., Neve, K. A., and Civelli, O. (1988) Nature 336, 783-787). We demonstrate here that this protein is a functional receptor, i.e. it couples to G-proteins to inhibit cAMP generation and hormone secretion. The cDNA was expressed in GH4C1 cells, a rat somatomammotrophic cell strain which lacks dopamine receptors. Stable transfectants were isolated and one clone, GH4ZR7, which had the highest levels of D2-dopamine receptor mRNA on Northern blot, was studied in detail. Binding of D2-dopamine antagonist [3H]spiperone to membranes isolated from GH4ZR7 cells was saturable, with KD = 96 pM, and Bmax = 2300 fmol/mg protein. Addition of GTP/NaCl increased the IC50 value for dopamine competition for [3H]spiperone binding by 2-fold, indicating that the D2-dopamine receptor interacts with one or more G-proteins. To assess the function of the dopamine-binding site, acute biological actions of dopamine were characterized in GH4ZR7 cells. Dopamine, at concentrations found in vivo, decreased resting intra- and extracellular cAMP levels (EC50 = 8 +/- 2 nM) by 50-70% and blocked completely vasoactive intestinal peptide (VIP) induced enhancement of cAMP levels (EC50 = 6 +/- 1 nM). Antagonism of dopamine-induced inhibition of VIP-enhanced cAMP levels by spiperone, (+)-butaclamol, (-)-sulpiride, and SCH23390 occurred at concentrations expected from KI values for these antagonists at the D2-receptor and was stereoselective. Dopamine (as well as several D2-selective agonists) inhibited forskolin-stimulated adenylate cyclase activity by 45 +/- 6%, with EC50 of 500-800 nM in GH4ZR7 membranes. Dopaminergic inhibition of cellular cAMP levels and of adenylyl cyclase activity in membrane preparations was abolished by pretreatment with pertussis toxin (50 ng/ml, 16 h). Dopamine (200 nM) abolished VIP- and thyrotropin-releasing hormone-induced acute prolactin release. These data show conclusively that the cDNA clone encodes a functional dopamine-D2 receptor which couples to G-proteins to inhibit adenylyl cyclase and both cAMP-dependent and cAMP-independent hormone secretion.     

Distinct roles for Galphai2, Galphai3, and Gbeta gamma in modulation offorskolin- or Gs-mediated cAMP accumulation and calcium mobilization by dopamine D2S receptors

Previous studies have shown that a single G protein-coupled receptor can regulate different effector systems by signaling through multiple subtypes of heterotrimeric G proteins. In LD2S fibroblast cells, the dopamine D2S receptor couples to pertussis toxin (PTX)-sensitive Gi/Go proteins to inhibit forskolin- or prostaglandin E1-stimulated cAMP production and to stimulate calcium mobilization. To analyze the role of distinct Galphai/o protein subtypes, LD2S cells were stably transfected with a series of PTX-insensitive Galphai/o protein Cys --> Ser point mutants and assayed for D2S receptor signaling after PTX treatment. The level of expression of the transfected Galpha mutant subunits was similar to the endogenous level of the most abundant Galphai/o proteins (Galphao, Galphai3). D2S receptor-mediated inhibition of forskolin-stimulated cAMP production was retained only in clones expressing mutant Galphai2. In contrast, the D2S receptor utilized Galphai3 to inhibit PGE1-induced (Gs-coupled) enhancement of cAMP production. Following stable or transient transfection, no single or pair set of mutant Galphai/o subtypes rescued the D2S-mediated calcium response following PTX pretreatment. On the other hand, in LD2S cells stably transfected with GRK-CT, a receptor kinase fragment that specifically antagonizes Gbeta gamma subunit activity, D2S receptor-mediated calcium mobilization was blocked. The observed specificity of Galphai2 and Galphai3 for different states of adenylyl cyclase activation suggests a higher level of specificity for interaction of Galphai subunits with forskolin- versus Gs-activated states of adenylyl cyclase than has been previously appreciated.     

Dopamine-D2S receptor inhibition of calcium influx,adenylyl cyclase,and mitogen-activated protein kinase in pituitary cells: distinct Galpha and Gbetagamma requirements

The G protein specificity of multiple signaling pathways of the dopamine-D2S (short form) receptor was investigated in GH4ZR7 lactotroph cells. Activation of the dopamine-D2S receptor inhibited forskolin-induced cAMP production, reduced BayK8644- activated calcium influx, and blocked TRH-mediated p42/p44 MAPK phosphorylation. These actions were blocked by pretreatment with pertussis toxin (PTX), indicating mediation by G(i/o) proteins. D2S stimulation also decreased TRH-induced MAPK/ERK kinase phosphorylation. TRH induced c-Raf but not B-Raf activation, and the D2S receptor inhibited both TRH-induced c-Raf and basal B-Raf kinase activity. After PTX treatment, D2S receptor signaling was rescued in cells stably transfected with individual PTX-insensitive Galpha mutants. Inhibition of adenylyl cyclase was partly rescued by Galpha(i)2 or Galpha(i)3, but Galpha(o) alone completely reconstituted D2S-mediated inhibition of BayK8644-induced L-type calcium channel activation. Galpha(o) and Galpha(i)3 were the main components involved in D2S-mediated p42/44 MAPK inhibition. In cells transfected with the carboxyl-terminal domain of G protein receptor kinase to inhibit Gbetagamma signaling, only D2S-mediated inhibition of calcium influx was blocked, but not inhibition of adenylyl cyclase or MAPK. These results indicate that the dopamine-D2S receptor couples to distinct G(i/o) proteins, depending on the pathway addressed, and suggest a novel Galpha(i)3/Galpha(o)-dependent inhibition of MAPK mediated by c-Raf and B-Raf-dependent inhibition of MAPK/ERK kinase.     

Stimulation of cAMP synthesis by Gi-coupled receptors upon ablation of distinct Galphai protein expression. Gi subtype specificity of the 5-HT1A receptor.

The three Galphai subunits were independently depleted from rat pituitary GH4C1 cells by stable transfection of each Galphai antisense rat cDNA construct. Depletion of any Galphai subunit eliminated receptor-induced inhibition of basal cAMP production, indicating that all Galphai subunits are required for this response. By contrast, receptor-mediated inhibition of vasoactive intestinal peptide (VIP)-stimulated cAMP production was blocked by selective depletions for responses induced by the transfected serotonin 1A (5-HT1A) (Galphai2 or Galphai3) or endogenous muscarinic-M4 (Galphai1 or Galphai2) receptors. Strikingly, receptor activation in Galphai1-depleted clones (for the 5-HT1A receptor) or Galphai3-depleted clones (for the muscarinic receptor) induced a pertussis toxin-sensitive increase in basal cAMP production, whereas the inhibitory action on VIP-stimulated cAMP synthesis remained. Finally, in Galphai2-depleted clones, activation of 5-HT1A receptors increased VIP-stimulated cAMP synthesis. Thus, 5-HT1A and muscarinic M4 receptor may couple dominantly to Galphai1 and Galphai3, respectively, to inhibit cAMP production. Upon removal of these Galphai subunits to reduce inhibitory coupling, stimulatory receptor coupling is revealed that may involve Gbetagamma-induced activation of adenylyl cyclase II, a Gi-stimulated cyclase that is predominantly expressed in GH4C1 cells. Thus Gi-coupled receptor activation involves integration of both inhibitory and stimulatory outputs that can be modulated by specific changes in alphai subunit expression level.     

Distinct roles for Galpha(i)2 and Gbetagamma in signaling to DNA synthesis and Galpha(i)3 in cellular transformation by dopamine D2S receptor activation in BALB/c 3T3 cells

Control of cell proliferation depends on intracellular mediators that determine the cellular response to external cues. In neuroendocrine cells, the dopamine D2 receptor short form (D2S receptor) inhibits cell proliferation, whereas in mesenchymal cells the same receptor enhances cell proliferation. Nontransformed BALB/c 3T3 fibroblast cells were stably transfected with the D2S receptor cDNA to study the G proteins that direct D2S signaling to stimulate cell proliferation. Pertussis toxin inactivates G(i) and G(o) proteins and blocks signaling of the D2S receptor in these cells. D2S receptor signaling was reconstituted by individually transfecting pertussis toxin-resistant Galpha(i/o) subunit mutants and measuring D2-induced responses in pertussis toxin-treated cells. This approach identified Galpha(i)2 and Galpha(i)3 as mediators of the D2S receptor-mediated inhibition of forskolin-stimulated adenylyl cyclase activity; Galpha(i)2-mediated D2S-induced stimulation of p42 and p44 mitogen-activated kinase (MAPK) and DNA synthesis, whereas Galpha(i)3 was required for formation of transformed foci. Transfection of toxin-resistant Galpha(i)1 cDNA induced abnormal cell growth independent of D2S receptor activation, while Galpha(o) inhibited dopamine-induced transformation. The role of Gbetagamma subunits was assessed by ectopic expression of the carboxyl-terminal domain of G protein receptor kinase to selectively antagonize Gbetagamma activity. Mobilization of Gbetagamma subunits was required for D2S-induced calcium mobilization, MAPK activation, and DNA synthesis. These findings reveal a remarkable and distinct G protein specificity for D2S receptor-mediated signaling to initiate DNA synthesis (Galpha(i)2 and Gbetagamma) and oncogenic transformation (Galpha(i)3), and they indicate that acute activation of MAPK correlates with enhanced DNA synthesis but not with transformation.     

G protein preferences for dopamine D2 inhibition of prolactin secretion and DNA synthesis in GH4 pituitary cells

Dopamine is the primary inhibitory regulator of lactotroph proliferation and prolactin (PRL) secretion in vivo, acting via dopamine D2 receptors (short D2S and long D2L forms). In GH4C1 pituitary cells transfected with D2S or D2L receptor cDNA, dopamine inhibits PRL secretion and DNA synthesis. These actions were blocked by pertussis toxin, implicating G(i)/G(o) proteins. To address roles of specific G(i)/G(o)4 proteins in these actions a series of GH4C1 cell lines specifically depleted of individual Galpha subunits was examined. D2S-mediated inhibition of BayK8644-stimulated PRL secretion was primarily dependent on G(o) over G(i), as observed for BayK8644-induced calcium influx. By contrast, inhibitory coupling of the D2S receptor to TRH-induced PRL secretion was partially impaired by depletion of any single G protein, but especially G(i)3. Inhibitory coupling of D2L receptors to PRL secretion required G(o), but not G(i)2, muscarinic receptor coupling was resistant to depletion of any G(i)/G(o) protein, whereas the 5-HT1A and somatostatin receptors required G(i)2 or G(i)3 for coupling. The various receptors also demonstrated distinct G protein requirements for inhibition of DNA synthesis: depletion of any G(i)/G(o) subunit completely uncoupled the D2S receptor, the D2L receptor was uncoupled by depletion of G(i)2, and muscarinic and somatostatin receptors were resistant to depletion of G(i)2 only. These results demonstrate distinct receptor-G protein preferences for inhibition of TRH-induced PRL secretion and DNA synthesis.     

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.     

Alpha-thrombin-mediated phosphatidylinositol 3-kinase activation through release of Gbetagamma dimers from Galphaq and Galphai2

Chinese hamster embryonic fibroblasts (IIC9 cells) express the Galpha subunits Galphas, Galphai2, Galphai3, Galphao, Galpha(q/11), and Galpha13. Consistent with reports in other cell types, alpha-thrombin stimulates a subset of the expressed G proteins in IIC9 cells, namely Gi2, G13, and Gq as measured by an in vitro membrane [35S]guanosine 5'-O-(3-thio)triphosphate binding assay. Using specific Galpha peptides, which block coupling of G-protein receptors to selective G proteins, as well as dominant negative xanthine nucleotide-binding Galpha mutants, we show that activation of the phosphatidylinositol 3-kinase/Akt pathway is dependent on Gq and Gi2. To examine the role of the two G proteins, we examined the events upstream of PI 3-kinase. The activation of the PI 3-kinase/Akt pathway by alpha-thrombin in IIC9 cells is blocked by the expression of dominant negative Ras and beta-arrestin1 (Phillips-Mason, P. J., Raben, D. M., and Baldassare, J. J. (2000) J. Biol. Chem. 275, 18046-18053, and Goel, R., Phillips-Mason, P. J., Raben, D. M., and Baldassare, J. J. (2002) J. Biol. Chem. 277, 18640-18648), indicating a role for Ras and beta-arrestin1. Interestingly, inhibition of Gi2 and Gq activation blocks Ras activation and beta-arrestin1 membrane translocation, respectively. Furthermore, expression of the Gbetagamma sequestrant, alpha-transducin, inhibits both Ras activation and membrane translocation of beta-arrestin1, suggesting that Gbetagamma dimers from Galphai2 and Galphaq activate different effectors to coordinately regulate the PI 3-kinase/Akt pathway.     

N-ethylmaleimide uncouples muscarinic receptors from acetylcholine- sensitive potassium channels in bullfrog atrium

The effect of N-ethylmaleimide (NEM), a sulphydryl alkylating agent, on the acetylcholine-activated K+ current, IK(ACh), has been studied in single cells from bullfrog atrium using a tight-seal, whole-cell voltage clamp technique. Addition of NEM (5 x 10(-5) M) produced a time-dependent complete block of IK(ACh). Dialysis of guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S, 5-10 x 10(-4) M), a nonhydrolyzable GTP analogue, into the myoplasm from the recording pipette gradually activated IK(ACh) even in the absence of acetylcholine. This effect is thought to be due to a GTP gamma S-induced dissociation of GTP-binding proteins (Gi and/or Go) into subunits that can directly activate these K+ channels. When NEM (5 x 10(-5) M) was applied after the GTP gamma S effect had fully developed, it failed to inhibit the GTP gamma S-induced K+ current, indicating that the NEM effect is unlikely to be on the dissociated subunits of the GTP-binding protein(s) or on the K+ channels. In contrast, pretreatment with NEM before GTP gamma S application markedly reduced the muscarinic K+ current, suggesting that NEM can block this K+ current by inhibition of the dissociation of the GTP-binding proteins into functional subunits. In NEM-treated cells the stimulatory effect of isoproterenol on ICa was present, but the inhibitory action of ACh on ICa was completely abolished. These results demonstrated that NEM can preferentially inhibit muscarinic receptor-effector interactions, probably by alkylating the GTP-binding proteins that are essential for these responses.     

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 cellsexpressing dopamine-D2S (short) receptors (Balb-D2S cells) provide amodel of G protein-mediated cell growth and transformation. In Balb-D2Scells, apomorphine (EC₅₀ = 10 nM) stimulated DAG and ceramide formation by 5.6- and 4.3-fold, respectively, maximal at1 h and persisting over 6 h. These actions were blocked by pretreatment with pertussis toxin (PTX), implicatingG_i/G_o proteins. To address which G proteins areinvolved, Balb-D2S clones expressing individual PTX-insensitiveG_i proteins were treated with PTX and tested forapomorphine-induced responses. Neither PTX-insensitive G_i2 nor G_i3 rescued D2S-induced DAG orceramide formation. Both D2S-induced DAG and ceramide signals requiredG-subunits and were blocked by inhibitors of phospholipaseC[1-(6-[([17]-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 ofD2S-induced calcium mobilization, DAG, and ceramide formation indicatesa common G-dependent phospholipase C-mediated pathway. Both D2agonists and ceramide specifically induced mitogen-activated proteinkinase (ERK1/2), suggesting that ceramide mediates a novel pathway ofD2S-induced ERK1/2 activation, leading to cell growth.

     

Guanosine 5'-O-(3-thiotriphosphate) and guanosine 5'-O-(2-thiodiphosphate) activate G proteins and potentiate fibroblast growth factor-induced DNA synthesis in hamster fibroblasts

Addition of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) to intact Chinese hamster lung fibroblasts (CCL39) depolarized by high K+ concentrations results in activation of phosphoinositide-specific phospholipase C (PLC) (at GTP gamma S concentrations greater than 0.1 mM), inhibition of adenylate cyclase (between 10 microM and 0.5 mM), and activation of adenylate cyclase (above 0.5 mM). Since GTP gamma S-induced activation of PLC is dramatically enhanced upon receptor-mediated stimulation of PLC by alpha-thrombin, we conclude that in depolarized CCL39 cells GTP gamma S directly activates various guanine nucleotide-binding regulatory proteins (G proteins) coupled to PLC (Gp(s)) and to adenylate cyclase (Gi and Gs). Pretreatment of cells with pertussis toxin strongly inhibits GTP gamma S-induced activation of PLC and inhibition of adenylate cyclase. GTP gamma S cannot be replaced by other nucleotides, except by guanosine 5'-O-(2-thiodiphosphate) (GDP beta S), which mimics after a lag period of 15-20 min all the effects of GTP gamma S, with the same concentration dependence and the same sensitivity to pertussis toxin. We suggest that GDP beta S is converted in cells into GTP beta S, which acts as GTP gamma S. Since cell viability is not affected by a transient depolarization, these observations provide a simple method to examine long-term effects of G protein activation on DNA synthesis. We show that a transient exposure of G0-arrested CCL39 cells to GTP gamma S or GDP beta S under depolarizing conditions is not sufficient by itself to induce a significant mitogenic response, but markedly potentiates the mitogenic action of fibroblast growth factor, a mitogen known to activate a receptor-tyrosine kinase. The potentiating effect is maximal after 60 min of pretreatment with 2 mM GTP gamma S. GDP beta S is equally efficient but only after a lag period of 15-20 min. Mitogenic effects of both guanine nucleotide analogs are suppressed by pertussis toxin. Since the activation of G proteins by GTP gamma S under these conditions vanishes after a few hours, we conclude that a transient activation of G proteins facilitates the transition G0----G1 in CCL39 cells, whereas tyrosine kinase-induced signals are sufficient to mediate the progression into S phase.     

Mediation of adenylyl cyclase sensitization by PTX-insensitive GalphaoA, Galphai1, Galphai2 or Galphai3

Chronic activation of mu-opioid receptors, which couple to pertussis toxin-sensitive Galphai/o proteins to inhibit adenylyl cyclase (AC), leads to a compensatory sensitization of AC. Pertussis toxin-insensitive mutations of Galphai/o subtypes, in which the pertussis toxin-sensitive cysteine is mutated to isoleucine (Galpha ), were used to determine whether each of the Galphai/o subtypes is able to mediate sensitization of AC. Galpha , G , G or G were individually transiently transfected into C6 glioma cells stably expressing the mu-opioid receptor, or transiently co-expressed with the mu-opioid receptor into human embryonic kidney (HEK)293T cells. Cells were treated with pertussis toxin to uncouple endogenous Galphai/o proteins, followed by acute or chronic treatment with the mu-opioid agonist, [D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin (DAMGO). Each Galphai/o subtype mediated acute DAMGO inhibition of AC and DAMGO-induced sensitization of AC. The potency for DAMGO to stimulate sensitization was independent of the Galphai/o subtype, but the level of sensitization was increased in clones expressing higher levels of Galphai/o subunits. Sensitization of AC mediated by a component of fetal bovine serum, which was also dependent on the level of functional Galphai/o subunits in the cell, was observed. This serum-mediated sensitization partially masked mu-opioid-mediated sensitization when expressed as percentage overshoot due to an apparent increase in AC activity.     

Differential effect of retinoic acid on growth regulation by phorbol ester in human cancer cell lines.

Phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) and all-trans-retinoic acid (trans-RA) are potent regulators of growth of cancer cells. In this study, we investigated the effect of TPA and trans-RA alone or their combination on proliferation of human breast cancer ZR75-1 and T47D and lung cancer H460 and H292 cell lines. trans-RA caused various degrees of growth inhibition of these cell lines. However, TPA showed inhibition of proliferation of H460 and H292 cells and induction of ZR75-1 cell growth. Although trans-RA did not significantly regulate the growth inhibitory effect of TPA, it completely prevented its growth stimulating function. The divergent effects of TPA were associated with specific disruption of cell cycle events, an induction of G(0)/G(1) arrest in H460 and H292 cells and inhibition of G(0)/G(1) arrest with increase of S phase in ZR75-1 cells. Induction of G(0)/G(1) arrest was accompanied by induction of p21(WAF1) and ERK activity, whereas inhibition of G(0)/G(1) arrest was associated with enhanced activity of JNK and AP-1 but not ERK. trans-RA did not affect TPA-induced p21(WAF1) expression. However, it inhibited TPA-induced AP-1 activity in ZR75-1 cells and the constitutive AP-1 activity in H460 and H292 cells. Thus, trans-RA modulates TPA activity through its interaction through TPA-induced JNK/AP-1 pathway but not TPA-induced ERK/p21(WAF1) pathway.     

Differential signaling of dopamine-D2S and -D2L receptors to inhibit ERK1/2 phosphorylation

Although they have distinct functions, the signaling of dopamine-D(2) receptor short and long isoforms (D(2)S and D(2)L) is virtually identical. We compared inhibitory regulation of extracellular signal-regulated kinases (ERK1/2) in GH4 pituitary cells separately transfected with these isoforms. Activation of rat or human dopamine-D(2)S, muscarinic or somatostatin receptors inhibited thyrotropin-releasing hormone-induced ERK1/2 phosphorylation, while the D(2)L receptor failed to inhibit this response. In order to address the structural basis for the differential signaling of D(2)S and D(2)L receptors, we examined the D(2)L-SS mutant, in which a protein kinase C (PKC) pseudosubstrate site that is present in the D(2)L but not D(2)S receptor was converted to a consensus PKC site. In transfected GH4 cells, the D(2)L-SS mutant inhibited thyrotropin-releasing hormone-induced ERK1/2 phosphorylation almost as strongly as the D(2)S receptor. A D(2)S-triple mutant that eliminates PKC sites involved in D(2)S receptor desensitization also inhibited ERK1/2 activation. Similarly, in striatal cultures, the D(2)-selective agonist quinpirole inhibited potassium-stimulated ERK1/2 phosphorylation, indicating the presence of this pathway in neurons. In conclusion, the D(2)S and D(2)L receptors differ in inhibitory signaling to ERK1/2 due to specific residues in the D(2)L receptor alternatively spliced domain, which may account for differences in their function in vivo.     

SKF83959 selectively regulates phosphatidylinositol-linked D1 dopamine receptors in rat brain

Previously a distinct D1-like dopamine receptor (DAR) that selectively couples to phospholipase C/phosphatidylinositol (PLC/PI) was proposed. However, lack of a selective agonist has limited efforts aimed at characterizing this receptor. We characterized the in vitro and in vivo effects of SKF83959 in regulating PI metabolism. SKF83959 stimulates (EC50, 8 micro m) phosphatidylinositol 4,5-biphosphate hydrolysis in membranes of frontal cortex (FC) but not in membranes from PC12 cells expressing classical D1A DARs. Stimulation of FC PI metabolism was attenuated by the D1 antagonist, SCH23390, indicating that SKF83959 activates a D1-like DAR. The PI-linked DAR is located in hippocampus, cerebellum, striatum and FC. Most significantly, administration of SKF83959 induced accumulations of IP3 in striatum and hippocampus. In contrast to other D1 DAR agonists, SKF83959 did not increase cAMP production in brain or in D1A DAR-expressing PC12 cell membranes. However, SKF83959 inhibited cAMP elevation elicited by the D1A DAR agonist, SKF81297, indicating that the compound is an antagonist of the classical D1A DAR. Lastly, we demonstrated that SKF83959 enhances [35S]guanosine 5'-O-(3-thiotriphosphate) binding to membrane Galphaq and Galphai proteins, suggesting that PI stimulation is mediated by activation of these guanine nucleotide-binding regulatory proteins. Results indicate that SKF83959 is a selective agonist for the PI-linked D1-like DAR, providing a unique tool for investigating the functions of this brain D1 DAR subtype.     

Recombinant MUC1 probe authentically reflects cell-specific O-glycosylation profiles of endogenous breast cancer mucin. High density and prevalent core 2-based glycosylation

Knowledge about the O-linked glycan chains of tumor-associated MUC1 is primarily based on enzymatic and immunochemical evidence. To obtain structural information and to overcome limitations by the scarcity of endogenous mucin, we expressed a recombinant glycosylation probe corresponding to six MUC1 tandem repeats in four breast cancer cell lines. Comparative analyses of the O-glycan profiles were performed after hydrazinolysis and normal phase chromatography of 2-aminobenzamide-labeled glycans. Except for a general reduction in the O-glycan chain lengths and a high density glycosylation, no common structural pattern was revealed. T47D fusion protein exhibits an almost complete shift from core 2 to core 1 expression with a preponderance of sialylated glycans. By contrast, MCF-7, MDA-MB231, and ZR75-1 cells glycosylate the MUC1 repeat peptide preferentially with core 2-based glycans terminating mostly with alpha 3-linked sialic acid (MDA-MB231, ZR75-1) or alpha 2/3-linked fucose (MCF-7). Endogenous MUC1 from T47D and MCF-7 cell supernatants revealed almost identical O-glycosylation profiles compared with the respective recombinant probes, indicating that the fusion proteins reflected the authentic O-glycan profiles of the cells. The structural patterns in the majority of cells under study are in conflict with biosynthetic models of MUC1 O-glycosylation in breast cancer, which claim that the truncation of normal core 2-based polylactosamine structures to short sialylated core 1-based glycans is due to the reduced activity of core 2-forming beta 6-N-acetylglucosaminyltransferases and/or to overexpression of competitive alpha 3- sialyltransferase.     

Differentiation between the somatostatin inhibition and the post-somatostatin rebound observed on growth hormone secretion in vitro

A rebound in growth hormone secretion following somatostatin treatment has been shown in several systems where somatostatin suppresses secretion of the hormone. We have developed an in vitro system in which isolated and cultured pituitary cells were perfused after mild trypsinization. After washing, these cells retained their sensitivity and secreted growth hormone (GH) in response to physiological activators (norepinephrine, dopamine, serotonin) or inhibitors (somatostatin) as well as pharmacological activators (PGE2). The variation in GH secretion occurred within a minute after commencement of the infusion and was as rapidly reversible and repeatable minutes later. During somatostatin infusion the GH secretion was not totally suppressed (residual secretion (mean +/- S.D.) 34 +/- 7%). After the infusion a rapid rebound in GH secretion occurred, reaching levels in excess of the pretreatment value of 138 +/- 13%. This rebound effect occurred at doses higher than (10(-10)M) but not at lower doses, even when significant inhibition was observed. The inhibitory effect is of greater magnitude than the rebound effect (rebound = inhibition X 57 +/- 7% (mean +/- S.D.)). Furthermore, rebound was not enhanced by prolongation of somatostatin infusion. These latter results indicate that the rebound in secretion cannot be explained on the sole basis of storage of intracellular GH during somatostatin infusion and in fact suggest the involvement of a process of GH degradation and/or an inhibition of GH synthesis.