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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

1. Unlike some interfaces between the blood and the nervous system (e.g., nerve perineurium), the brain endothelium forming the blood-brain barrier can be modulated by a range of inflammatory mediators. The mechanisms underlying this modulation are reviewed, and the implications for therapy of the brain discussed. 2. Methods for measuring blood-brain barrier permeability in situ include the use of radiolabeled tracers in parenchymal vessels and measurements of transendothelial resistance and rate of loss of fluorescent dye in single pial microvessels. In vitro studies on culture models provide details of the signal transduction mechanisms involved. 3. Routes for penetration of polar solutes across the brain endothelium include the paracellular tight junctional pathway (usually very tight) and vesicular mechanisms. Inflammatory mediators have been reported to influence both pathways, but the clearest evidence is for modulation of tight junctions. 4. In addition to the brain endothelium, cell types involved in inflammatory reactions include several closely associated cells including pericytes, astrocytes, smooth muscle, microglia, mast cells, and neurons. In situ it is often difficult to identify the site of action of a vasoactive agent. In vitro models of brain endothelium are experimentally simpler but may also lack important features generated in situ by cell:cell interaction (e.g. induction, signaling). 5. Many inflammatory agents increase both endothelial permeability and vessel diameter, together contributing to significant leak across the blood-brain barrier and cerebral edema. This review concentrates on changes in endothelial permeability by focusing on studies in which changes in vessel diameter are minimized. 6. Bradykinin (Bk) increases blood-brain barrier permeability by acting on B2 receptors. The downstream events reported include elevation of [Ca2+]i, activation of phospholipase A2, release of arachidonic acid, and production of free radicals, with evidence that IL-1 beta potentiates the actions of Bk in ischemia. 7. Serotonin (5HT) has been reported to increase blood-brain barrier permeability in some but not all studies. Where barrier opening was seen, there was evidence for activation of 5-HT2 receptors and a calcium-dependent permeability increase. 8. Histamine is one of the few central nervous system neurotransmitters found to cause consistent blood-brain barrier opening. The earlier literature was unclear, but studies of pial vessels and cultured endothelium reveal increased permeability mediated by H2 receptors and elevation of [Ca2+]i and an H1 receptor-mediated reduction in permeability coupled to an elevation of cAMP. 9. Brain endothelial cells express nucleotide receptors for ATP, UTP, and ADP, with activation causing increased blood-brain barrier permeability. The effects are mediated predominantly via a P2U (P2Y2) G-protein-coupled receptor causing an elevation of [Ca2+]i; a P2Y1 receptor acting via inhibition of adenyl cyclase has been reported in some in vitro preparations. 10. Arachidonic acid is elevated in some neural pathologies and causes gross opening of the blood-brain barrier to large molecules including proteins. There is evidence that arachidonic acid acts via generation of free radicals in the course of its metabolism by cyclooxygenase and lipoxygenase pathways. 11. The mechanisms described reveal a range of interrelated pathways by which influences from the brain side or the blood side can modulate blood-brain barrier permeability. Knowledge of the mechanisms is already being exploited for deliberate opening of the blood-brain barrier for drug delivery to the brain, and the pathways capable of reducing permeability hold promise for therapeutic treatment of inflammation and cerebral edema.
Cell Mol Neurobiol 2000 Apr
PMID:Inflammatory mediators and modulation of blood-brain barrier permeability. 1069 6

In this study, we have documented an essential role for ADP-ribosylation factor 6 (ARF6) in cell surface remodeling in response to physiological stimulus and in the down regulation of stress fiber formation. We demonstrate that the G-protein-coupled receptor agonist bombesin triggers the redistribution of ARF6- and Rac1-containing endosomal vesicles to the cell surface. This membrane redistribution was accompanied by cortical actin rearrangements and was inhibited by dominant negative ARF6, implying that bombesin is a physiological trigger of ARF6 activation. Furthermore, these studies provide a new model for bombesin-induced Rac1 activation that involves ARF6-regulated endosomal recycling. The bombesin-elicited translocation of vesicular ARF6 was mimicked by activated Galphaq and was partially inhibited by expression of RGS2, which down regulates Gq function. This suggests that Gq functions as an upstream regulator of ARF6 activation. The ARF6-induced peripheral cytoskeletal rearrangements were accompanied by a depletion of stress fibers. Moreover, cells expressing activated ARF6 resisted the formation of stress fibers induced by lysophosphatidic acid. We show that the ARF6-dependent inhibition of stress fiber formation was due to an inhibition of RhoA activation and was overcome by expression of a constitutively active RhoA mutant. The latter observations demonstrate that activation of ARF6 down regulates Rho signaling. Our findings underscore the potential roles of ARF6, Rac1, and RhoA in the coordinated regulation of cytoskeletal remodeling.
Mol Cell Biol 2000 May
PMID:ADP-ribosylation factor 6 regulates actin cytoskeleton remodeling in coordination with Rac1 and RhoA. 1077 58

In previous studies we showed that the wild-type histamine H(2) receptor stably expressed in Chinese hamster ovary cells is constitutively active. Because constitutive activity of the H(2) receptor is already found at low expression levels (300 fmol/mg protein) this receptor is a relatively unique member of the G-protein-coupled receptor (GPCR) family and a useful tool for studying GPCR activation. In this study the role of the highly conserved DRY motif in activation of the H(2) receptor was investigated. Mutation of the aspartate 115 residue in this motif resulted in H(2) receptors with high constitutive activity, increased agonist affinity, and increased signaling properties. In addition, the mutant receptors were shown to be highly structurally instable. Mutation of the arginine 116 residue in the DRY motif resulted also in a highly structurally instable receptor; expression of the receptor could only be detected after stabilization with either an agonist or inverse agonist. Moreover, the agonist affinity at the Arg-116 mutant receptors was increased, whereas the signal transduction properties of these receptors were decreased. We conclude that the Arg-116 mutant receptors can adopt an active conformation but have a decreased ability to couple to or activate the G(s)-protein. This study examines the pivotal role of the aspartate and arginine residues of the DRY motif in GPCR function. Disruption of receptor stabilizing constraints by mutation in the DRY motif leads to the formation of active GPCR conformations, but concomitantly to GPCR instability.
Mol Pharmacol 2000 May
PMID:The effect of mutations in the DRY motif on the constitutive activity and structural instability of the histamine H(2) receptor. 1077 71

Binding of the alpha-factor pheromone to its G-protein-coupled receptor (encoded by STE2) activates the mating pathway in MATa yeast cells. To investigate whether specific interactions between the receptor and the G protein occur prior to ligand binding, we analyzed dominant-negative mutant receptors that compete with wild-type receptors for G proteins, and we analyzed the ability of receptors to suppress the constitutive signaling activity of mutant Galpha subunits in an alpha-factor-independent manner. Although the amino acid substitution L236H in the third intracellular loop of the receptor impairs G-protein activation, this substitution had no influence on the ability of the dominant-negative receptors to sequester G proteins or on the ability of receptors to suppress the GPA1-A345T mutant Galpha subunit. In contrast, removal of the cytoplasmic C-terminal domain of the receptor eliminated both of these activities even though the C-terminal domain is unnecessary for G-protein activation. Moreover, the alpha-factor-independent signaling activity of ste2-P258L mutant receptors was inhibited by the coexpression of wild-type receptors but not by coexpression of truncated receptors lacking the C-terminal domain. Deletion analysis suggested that the distal half of the C-terminal domain is critical for sequestration of G proteins. The C-terminal domain was also found to influence the affinity of the receptor for alpha-factor in cells lacking G proteins. These results suggest that the C-terminal cytoplasmic domain of the alpha-factor receptor, in addition to its role in receptor downregulation, promotes the formation of receptor-G-protein preactivation complexes.
Mol Cell Biol 2000 Jul
PMID:The C terminus of the Saccharomyces cerevisiae alpha-factor receptor contributes to the formation of preactivation complexes with its cognate G protein. 1086 88

Recombinant receptor cell lines are widely used in G-protein-coupled receptor selectivity studies. To unequivocally interpret the results of such studies, it is essential that the host cell line does not endogenously express the receptor of interest and in addition is unresponsive to the receptor's natural ligand. Here we describe an approach to overcome such difficulties associated with orphan receptors or, as in the present case, receptors whose endogenous ligand ubiquitously affects mammalian cells. The functional heterologous assay system described is for the hEdg2 receptor, which uses lysophosphatidic acid as its endogenous ligand. Once activated, this receptor mediates its effects via multiple secondary messenger pathways, including a Gi-coupled pathway. We have transiently expressed a pertussis toxin-insensitive hEdg2 receptor-ratGialpha1 fusion protein into human embryonic kidney cells and have monitored the ability of compounds to stimulate [(35)S]GTPgammaS binding in membranes prepared from these cells after pretreatment with toxin. Because the assay conditions used favor Gi-mediated responses and because endogenous Gialpha subunits are rendered inactive, the response measured is, by definition, fusion protein-mediated. Consequently, we have developed an assay that monitors definitively Edg2 receptor-mediated responses in a mammalian cell line. A limited structure activity relationship study suggests that the lysophospholipid carbon chain has a role in receptor activation and in addition indicates that certain modifications to the phosphate group are tolerated.
Mol Pharmacol 2000 Aug
PMID:Edg2 receptor functionality: gialpha1 coexpression and fusion protein studies. 1090 9

The opposing effects on proliferation mediated by G-protein-coupled receptor isoforms differing in their COOH termini could be correlated with the abilities of the receptors to differentially activate p38, implicated in apoptotic events, or phosphatidylinositol 3-kinase (PI 3-K), which provides a source of survival signals. These contrasting growth responses of the somatostatin sst(2) receptor isoforms, which couple to identical Galpha subunit pools (Galpha(i3) > Galpha(i2) >> Galpha(0)), were both inhibited following betagamma sequestration. The sst(2(a)) receptor-mediated ATF-2 activation and inhibition of proliferation induced by basic fibroblast growth factor (bFGF) were dependent on prolonged phosphorylation of p38. In contrast, cell proliferation and the associated transient phosphorylation of Akt and p70(rsk) induced by sst(2(b)) receptors were blocked by the PI 3-K inhibitor LY 294002. Stimulation with bFGF alone had no effect on the activity of either p38 or Akt but markedly enhanced p38 phosphorylation mediated by sst(2(a)) receptors, suggesting that a complex interplay exists between the transduction cascades activated by these distinct receptor types. In addition, although all receptors mediated a sustained activation of extracellular signal-regulated kinases (ERK1 and ERK2), induction of the tumor suppressor p21(cip1) was detected only following amplification of ERK and p38 phosphorylation by concomitant bFGF and sst(2(a)) receptor activation. Expression of constitutively active Akt in the presence of a p38 inhibitor enabled a proliferative response to be detected in sst(2(a)) receptor-expressing cells. These findings demonstrate that the duration of activation and a critical balance between the mitogen-activated protein kinase and PI 3-K pathways are important for controlling cell proliferation and that the COOH termini of the sst(2) receptor isoforms may determine the selection of appropriate betagamma-pairings necessary for interaction with distinct kinase cascades.
Mol Cell Biol 2000 Aug
PMID:Receptor isoforms mediate opposing proliferative effects through gbetagamma-activated p38 or Akt pathways. 1091 80

Cannabinoids exert most of their effects through the CB(1) receptor. This G-protein-coupled receptor has been shown to be functionally coupled to inhibition of adenylyl cyclase, modulation of ion channels, and activation of extracellular signal-regulated kinase. Using Chinese hamster ovary cells stably transfected with the CB(1) receptor cDNA, we show here that Delta(9)-tetrahydrocannabinol (THC), the major active component of marijuana, induces the activation of c-Jun N-terminal kinase (JNK). Western blot analysis showed that both JNK-1 and JNK-2 were stimulated by THC. The effect of THC was also exerted by endogenous cannabinoids (anandamide and 2-arachidonoylglycerol) and synthetic cannabinoids (CP-55,940, HU-210, and methanandamide), and was prevented by the selective CB(1) antagonist SR141716. Pertussis toxin, wortmannin, and a Ras farnesyltransferase inhibitor peptide blocked, whereas mastoparan mimicked, the CB(1) receptor-evoked activation of JNK, supporting the involvement of a G(i)/G(o)-protein, phosphoinositide 3'-kinase and Ras. THC-induced JNK stimulation was prevented by tyrphostin AG1296, pointing to the implication of platelet-derived growth factor receptor transactivation, and was independent of ceramide generation. Experiments performed with several types of neural cells that endogenously express the CB(1) receptor suggested that long-term JNK activation may be involved in THC-induced cell death. The CB(1) cannabinoid receptor was also shown to be coupled to the activation of p38 mitogen-activated protein kinase. Data indicate that activation of JNK and p38 mitogen-activated protein kinase may be responsible for some of the cellular responses elicited by the CB(1) cannabinoid receptor.
Mol Pharmacol 2000 Oct
PMID:The CB(1) cannabinoid receptor is coupled to the activation of c-Jun N-terminal kinase. 1099 52

The functional activity of Cdc42 is known to be regulated by proteins that control its GDP/GTP-bound state. However, there is still limited information on how Cdc42 is controlled by G-protein-coupled receptors. Adenosine receptors belong to the G-protein-coupled receptor family of cell surface receptors. Human HMC-1 mast cells express the high-affinity A(2A) and the low-affinity A(2B) subtypes of adenosine receptors known to increase intracellular cAMP levels. We found that both subtypes of A(2) adenosine receptors activate Cdc42 in HMC-1 cells. Furthermore, stimulation of adenylate cyclase with forskolin, or loading of HMC-1 with the cell-permeable cAMP analog 8-Br-cAMP, activated Cdc42. Stimulation of Cdc42 by cAMP was also observed in CHO-K1 and COS-7 cells. Protein kinase A (PKA)-mediated phosphorylation is likely involved in cAMP-dependent Cdc42 activation, because transient expression of the PKA catalytic subunit in COS-7 cells activated Cdc42. Inhibition of protein phosphatases 1 and 2A with calyculin A potentiated the effects of 5'-N-ethylcarboxamidoadenosine and 8-Br-cAMP, whereas the selective PKA inhibitor H-89 reversed the activation of Cdc42. We demonstrated that Cdc42 is a poor substrate for PKA phosphorylation in vitro and in intact cells. Our data suggest that PKA does not phosphorylate Cdc42 directly. Instead, the proteins that modulate the GDP/GTP-bound state of Cdc42 may be the primary targets of PKA phosphorylation.
Mol Pharmacol 2000 Nov
PMID:Cyclic AMP and protein kinase A stimulate Cdc42: role of A(2) adenosine receptors in human mast cells. 1104 36

In Saccharomyces cerevisiae, glucose activation of cAMP synthesis requires both the presence of the G-protein-coupled receptor (GPCR) system, Gpr1-Gpa2, and uptake and phosphorylation of the sugar. In a hxt-null strain that lacks all physiologically important glucose carriers, glucose transport as well as glucose-induced cAMP signalling can be restored by constitutive expression of the galactose permease. Hence, the glucose transporters do not seem to have a regulatory function but are only required for glucose uptake. We established a system in which the GPCR-dependent glucose-sensing process is separated from the glucose phosphorylation process. It is based on the specific transport and hydrolysis of maltose providing intracellular glucose in the absence of glucose transport. Preaddition of a low concentration (0.7 mM) of maltose to derepressed hxt-null cells and subsequent addition of glucose restored the glucose-induced cAMP signalling, although there was no glucose uptake. Addition of a low concentration of maltose itself does not increase the cAMP level but enhances Glu6P and apparently fulfils the intracellular glucose phosphorylation requirement for activation of the cAMP pathway by extracellular glucose. This system enabled us to analyse the affinity and specificity of the GPCR system for fermentable sugars. Gpr1 displayed a very low affinity for glucose (apparent Ka = 75 mM) and responded specifically to extracellular alpha and beta D-glucose and sucrose, but not to fructose, mannose or any glucose analogues tested. The presence of the constitutively active Gpa2val132 allele in a wild-type strain bypassed the requirement for Gpr1 and increased the low cAMP signal induced by fructose and by low glucose up to the same intensity as the high glucose signal. Therefore, the low cAMP increases observed with fructose and low glucose in wild-type cells result only from the low sensitivity of the Gpr1-Gpa2 system and not from the intracellular sugar kinase-dependent process. In conclusion, we have shown that the two essential requirements for glucose-induced activation of cAMP synthesis can be fulfilled separately: an extracellular glucose detection process dependent on Gpr1 and an intracellular sugar-sensing process requiring the hexose kinases.
Mol Microbiol 2000 Oct
PMID:Glucose-induced cAMP signalling in yeast requires both a G-protein coupled receptor system for extracellular glucose detection and a separable hexose kinase-dependent sensing process. 1106 60

Sonic hedgehog (Shh) signal transduction via the G-protein-coupled receptor, Smoothened, is required for proliferation of cerebellar granule neuron precursors (CGNPs) during development. Activating mutations in the Hedgehog pathway are also implicated in basal cell carcinoma and medulloblastoma, a tumor of the cerebellum in humans. However, Shh signaling interactions with cell cycle regulatory components in neural precursors are poorly understood, in part because appropriate immortalized cell lines are not available. We have utilized primary cultures from neonatal mouse cerebella in order to determine (i) whether Shh initiates or maintains cell cycle progression in CGNPs, (ii) if G(1) regulation by Shh resembles that of classical mitogens, and (iii) whether individual D-type cyclins are essential components of Shh proliferative signaling in CGNPs. Our results indicate that Shh can drive continued cycling in immature, proliferating CGNPs. Shh treatment resulted in sustained activity of the G(1) cyclin-Rb axis by regulating levels of cyclinD1, cyclinD2, and cyclinE mRNA transcripts and proteins. Analysis of CGNPs from cyclinD1(-/-) or cyclinD2(-/-) mice demonstrates that the Shh proliferative pathway does not require unique functions of cyclinD1 or cyclinD2 and that D-type cyclins overlap functionally in this regard. In contrast to many known mitogenic pathways, we show that Shh proliferative signaling is mitogen-activated protein kinase independent. Furthermore, protein synthesis is required for early effects on cyclin gene expression. Together, our results suggest that Shh proliferative signaling promotes synthesis of regulatory factor intermediates that upregulate or maintain cyclin gene expression and activity of the G(1) cyclin-Rb axis in proliferating granule neuron precursors.
Mol Cell Biol 2000 Dec
PMID:Sonic hedgehog promotes G(1) cyclin expression and sustained cell cycle progression in mammalian neuronal precursors. 1107 3


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