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Query: UMLS:C0043167 (pertussis)
 19,595 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Two cDNAs encoding novel isoforms of Xenopus laevis melatonin receptors were cloned using PCR primers specific for the X. laevis-melanophore Mel1c melatonin receptor described in a recent publication. The novel isoforms were highly homologous to the described frog Mel1c cDNA, although the C-terminal tail of both was shorter by 65 amino acid residues. Nucleotide sequences of these novel isoforms, called Mel1c(alpha) and Mel1c(beta), differed from each other by only 35 nucleotides and six amino acid residues. Studies on several animals of various Xenopus species indicate that Mel1c(alpha) and Mel1c(beta) receptors may correspond to allelic variants of the same locus. Studies on cells transfected with both receptor cDNAs showed the expression of high-affinity 2-[125I]iodomelatonin binding sites. Agonist stimulation of Mel1c(alpha) receptor was associated with the inhibition of cAMP accumulation stimulated by forskolin (IC50 approximately 10(-10) M) in HeLa, Ltk-, and human embryonic kidney 293 (HEK 293) cells. Mel1c(beta) receptor modulated cAMP in HeLa and HEK 293 cells but not in Ltk- cells. Both receptors inhibited, in a dose-dependent manner, cGMP accumulation in all three cell lines incubated with a phosphodiesterase inhibitor. This effect was localized upstream of soluble guanylyl cyclase and was blocked by pertussis toxin treatment. However, IC50 values (approximately 10(-10) M for Mel1c(beta) and 10(-9) to 10(-7) M for Mel1c(alpha)) and maximal inhibition levels showed that Mel1c(alpha) receptors are much less efficiently coupled to the cGMP pathway. Coupling differences may be explained by the fact that five of the six amino acid substitutions between Mel1c(alpha) and Mel1c(beta) receptors are located within cytoplasmic regions potentially involved in signal transduction. The existence of coupling differences is in agreement with the observation that expression of both receptors is evolutionally conserved in native tissue. In conclusion, two novel, potentially allelic, isoforms of Xenopus Mel1c melatonin receptors display identical ligand-binding characteristics, but different potencies in modulating cAMP and cGMP levels through G(i)/G(o)-dependent pathways. Furthermore, to our knowledge, this study provides the first data on the modulation of intracellular cGMP levels by cloned melatonin receptors.
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PMID:Novel isoforms of Mel1c melatonin receptors modulating intracellular cyclic guanosine 3',5'-monophosphate levels. 921 55

Many receptors that couple to heterotrimeric guanine-nucleotide binding proteins (G proteins) have been shown to mediate rapid activation of the mitogen-activated protein kinases Erk1 and Erk2. In different cell types, the signaling pathways employed appear to be a function of the available repertoire of receptors, G proteins, and effectors. In HEK-293 cells, stimulation of either alpha1B- or alpha2A-adrenergic receptors (ARs) leads to rapid 5-10-fold increases in Erk1/2 phosphorylation. Phosphorylation of Erk1/2 in response to stimulation of the alpha2A-AR is effectively attenuated by pretreatment with pertussis toxin or by coexpression of a Gbetagamma subunit complex sequestrant peptide (betaARK1ct) and dominant-negative mutants of Ras (N17-Ras), mSOS1 (SOS-Pro), and Raf (DeltaN-Raf). Erk1/2 phosphorylation in response to alpha1B-AR stimulation is also attenuated by coexpression of N17-Ras, SOS-Pro, or DeltaN-Raf, but not by coexpression of betaARK1ct or by pretreatment with pertussis toxin. The alpha1B- and alpha2A-AR signals are both blocked by phospholipase C inhibition, intracellular Ca2+ chelation, and inhibitors of protein-tyrosine kinases. Overexpression of a dominant-negative mutant of c-Src or of the negative regulator of c-Src function, Csk, results in attenuation of the alpha1B-AR- and alpha2A-AR-mediated Erk1/2 signals. Chemical inhibitors of calmodulin, but not of PKC, and overexpression of a dominant-negative mutant of the protein-tyrosine kinase Pyk2 also attenuate mitogen-activated protein kinase phosphorylation after both alpha1B- and alpha2A-AR stimulation. Erk1/2 activation, then, proceeds via a common Ras-, calcium-, and tyrosine kinase-dependent pathway for both Gi- and Gq/11-coupled receptors. These results indicate that in HEK-293 cells, the Gbetagamma subunit-mediated alpha2A-AR- and the Galphaq/11-mediated alpha1B-AR-coupled Erk1/2 activation pathways converge at the level of phospholipase C. These data suggest that calcium-calmodulin plays a central role in the calcium-dependent regulation of tyrosine phosphorylation by G protein-coupled receptors in some systems.
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PMID:Ras-dependent mitogen-activated protein kinase activation by G protein-coupled receptors. Convergence of Gi- and Gq-mediated pathways on calcium/calmodulin, Pyk2, and Src kinase. 923 1

Dendritic cells are potent antigen-presenting cells involved in the initiation of immune responses. The trafficking of these cells to tissues and lymph nodes is mediated by members of the chemokine family. Recently, a novel CC chemokine known as MIP-3alpha or liver and activation-regulated chemokine has been identified from the EMBL/GenBank/DDBJ expressed sequence tag database. In the present study, we have shown that the messenger RNA for MIP-3alpha is expressed predominantly in inflamed and mucosal tissues. MIP-3alpha produced either synthetically or by human embryonic kidney 293 cells is chemotactic for CD34(+)-derived dendritic cells and T cells, but is inactive on monocytes and neutrophils. MIP-3alpha was unable to displace the binding of specific CC or CXC chemokines to stable cell lines expressing their respective high affinity receptors, namely CCR1-5 and CXCR1 and CXCR2, suggesting that MIP-3alpha acts through a novel CC chemokine receptor. Therefore, we used degenerate oligonucleotide-based reverse transcriptase PCR to identify candidate MIP-3alpha receptors in lung dendritic cells. Our results show that the orphan receptor known as GCY-4, CKRL-3, or STRL-22 is a specific receptor for MIP-3alpha, and that its activation leads to pertussis toxin-sensitive and phospholipase C-dependent intracellular Ca2+ mobilization when it is expressed in HEK 293 cells.
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PMID:Cloning and characterization of a specific receptor for the novel CC chemokine MIP-3alpha from lung dendritic cells. 929 37

The chemokine receptor, CCR-5, a G protein-coupled receptor (GPCR) which mediates chemotactic responses of certain leukocytes, has been shown to serve as the primary co-receptor for macrophage-tropic human immunodeficiency virus type 1 (HIV-1). Here we describe functional coupling of CCR-5 to inhibition of forskolin-stimulated cAMP formation via a pertussis toxin-sensitive G(i) protein mechanism in transfected HEK 293 cells. In response to chemokines, CCR-5 was desensitized, phosphorylated and sequestered like a prototypic GPCR only following overexpression of G protein-coupled receptor kinases (GRKs) and beta-arrestins in HEK 293 cells. The lack of CCR-5 desensitization in HEK 293 cells in the absence of GRK overexpression suggests that differences in cellular complements of GRK and/or beta-arrestin proteins could represent an important mechanism determining cellular responsiveness. When tested, the activity of CCR-5 as an HIV-1 co-receptor was dependent neither upon its ability to signal nor its ability to be desensitized and internalized following agonist stimulation. Thus, while chemokine-promoted cellular signaling, phosphorylation and internalization of CCR-5 may play an important role in regulation of chemotactic responses in leukocytes, these functions are dissociable from its HIV-1 co-receptor function.
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PMID:Molecular mechanism of desensitization of the chemokine receptor CCR-5: receptor signaling and internalization are dissociable from its role as an HIV-1 co-receptor. 930 5

The expression of melatonin receptors (MR) of the Mel1a subtype in basolateral membrane of guinea pig kidney proximal tubule suggests that melatonin plays a role in regulating epithelial functions. To investigate the cellular basis of melatonin action on epithelia, we sought to establish an appropriate in vitro culture model. Epithelial cell lines originating from kidneys of dog (MDCK), pig (LLC-PK1), opossum (OK), and human embryo (HEK-293) were each tested for the presence of MR using 2-[125I]iodomelatonin (125I-MEL) as a radioligand. The HEK-293 cell line exhibited the highest specific 125I-MEL binding. By intermediate filament characterization, the HEK-293 cells were determined to be of epithelial origin. Binding of 125I-MEL in HEK-293 cells demonstrated saturability, reversibility, and high specificity with an equilibrium dissociation constant (Kd) value of 23.8 +/- 0.5 pM and a maximum number of binding sites (Bmax) value of 1.17 +/- 0.11 fmol/mg protein (n = 5), which are comparable with the reported Kd and Bmax values in human kidney cortex. Coincubation with GTPgammaS (10 microM) and pertussis toxin (100 ng/ml) provoked a marked decrease in binding affinity (Kd was increased by a factor of 1.5-2.0), with no significant difference in Bmax. Melatonin (1 microM) decreased the forskolin (10 microM) stimulated cAMP level by 50%. HEK-293 cells do not express dopamine D1A receptor. Following transient transfection of HEK-293 cells with human dopamine D1A receptor (hD1A-R), exposure of the cells to dopamine stimulated an increase in the level of cAMP. Similarly, transient transfection of HEK-293 cells with rat glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic peptide (GIP), and PTH type 1 receptors, each resulted in an hormone inducible increase in cAMP levels. Surprisingly, only the stimulatory effect of dopamine could be inhibited by exposure to melatonin. The inhibitory effect of melatonin on dopamine D1-induced increase in cAMP was completely inhibited by pertussis toxin (100 ng/ml, 18 h). Immunoblot and immunocytochemical studies were carried out using two polyclonal antibodies raised against the extra and cytoplasmic domains of Mel1a receptor. Immunoblot studies using antibody against the cytoplasmic domain of Mel1a receptor confirmed the presence of a peptide blockable 37 kDa band in HEK-293 cells. Indirect immunofluorescent studies with both antibodies revealed staining predominantly at the cell surface, but staining with the antibody directed against the cytoplasmic domain required prior cell permeabilization. By RT-PCR, HEK-293 cells express both Mel1a and Mel1b messenger RNAs, but the messenger RNA level for Mel1b is several orders of magnitude lower than for Mel1a. We conclude that HEK-293 cells express MR predominantly of the Mel1a subtype. Our evidence suggests that one of the ways that melatonin exerts its biological function is through modulation of cellular dopaminergic responses.
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PMID:Studies of melatonin effects on epithelia using the human embryonic kidney-293 (HEK-293) cell line. 934

We have shown previously that metabotropic glutamate receptors with group I-like pharmacology couple to N-type and P/Q-type calcium channels in acutely isolated cortical neurons using G proteins most likely belonging to the Gi/Go subclass. To better understand the potential mechanisms forming the basis for group I mGluR modulation of voltage-gated calcium channels in the CNS, we have examined the ability of specific mGluRs to couple to neuronal N-type (alpha1B-1/alpha2delta/beta1b) and P/Q-type (alpha1A-2/alpha2delta/beta1b) voltage-gated calcium channels in an HEK 293 heterologous expression system. Using the whole cell patch-clamp technique where intracellular calcium is buffered to low levels, we have shown that group I receptors inhibit both N-type and P/Q-type calcium channels in a voltage-dependent fashion. Similar to our observations in cortical neurons, this voltage-dependent inhibition is mediated almost entirely by N-ethylmaleimide (NEM)-sensitive heterotrimeric G proteins, strongly suggesting that these receptors can use Gi/Go-like G proteins to couple to N-type and P/Q-type calcium channels. However, inconsistent with the apparent NEM sensitivity of group I modulation of calcium channels, modulation of N-type channels in group I mGluR-expressing cells was only partially sensitive to pertussis toxin (PTX), indicating the potential involvement of both PTX-sensitive and -resistant G proteins. The PTX-resistant modulation was voltage dependent and entirely resistant to NEM and cholera toxin. A time course of treatment with PTX revealed that this toxin caused group I receptors to slowly shift from using a primarily NEM-sensitive G protein to using a NEM-resistant form. The PTX-induced switch from NEM-sensitive to -resistant modulation was also dependent on protein synthesis, indicating some reliance on active cellular processes. In addition to these voltage-dependent pathways, perforated patch recordings on group I mGluR-expressing cells indicate that another slowly developing, calcium-dependent form of modulation for N-type channels may be seen when intracellular calcium is not highly buffered. We conclude that group I mGluRs can modulate neuronal Ca2+ channels using a variety of signal transduction pathways and propose that the relative contributions of different pathways may exemplify the diversity of responses mediated by these receptors in the CNS.
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PMID:Rat group I metabotropic glutamate receptors inhibit neuronal Ca2+ channels via multiple signal transduction pathways in HEK 293 cells. 942 7

The neuropeptide galanin mediates a diverse spectrum of biological activities by interacting with specific G-protein-coupled receptors. Through expression cloning, human and rat GALR1 receptor cDNA clones have previously been isolated and characterized. In this study, we have used homology screening to isolate a rat brain cDNA clone encoding a second galanin receptor subtype, the GALR2 receptor. The isolated cDNA encodes a 372-amino-acid G-protein-coupled receptor that shares 38% overall amino-acid identity with the rat GALR1 receptor. The pharmacological profile of the rat GALR2 receptor is similar to that of the rat GALR1 receptor. The rat GALR2 receptor binds galanin, N-terminal galanin fragments, and the putative galanin receptor antagonists galantide, C7, M35 and M40 with high affinity but it does not bind C-terminal galanin fragments. Galanin increases intracellular inositol phosphate levels in HEK 293 cells expressing the rat GALR2 receptor via a pertussis toxin-insensitive G-protein. The rat GALR2 receptor mRNA is highly expressed in several brain regions, including hypothalamus and hippocampus as well as the anterior pituitary, with lower levels of expression detected in amygdala, and regions of cortex. It is also highly expressed in the GH3 pituitary cell line and in gut tissues, and to a lower extent in spleen, lung, skeletal muscle, heart, kidney, liver and testis. These results suggest that GALR2 receptor mediates galanin's regulation of pituitary hormone secretion and possibly food intake.
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PMID:Cloning, pharmacological characterization and distribution of a novel galanin receptor. 942 6

We examined the ability of rat Y1, Y2 and Y4 neuropeptide Y (NPY) receptors to regulate K+ and Ca++ channels expressed in Xenopus oocytes and HEK 293 cells, respectively. Stimulation of all three receptors with NPY or related peptides activated inwardly rectifying K+ currents resulting from the expression of rat GIRK1/CIR in frog oocytes. These effects were inhibited by pertussis toxin treatment. The effects of activating Y1 receptors were antagonized competitively by BIBP3226, SR120819A and GW1229. The effects of Y2 receptor activation were not blocked by these drugs, and the effects of Y4 receptor activation were only blocked by GW1229. Activation of all three NPY receptors also inhibited human alpha-1B Ca++ channels stably expressed in HEK293 cells. The effects of agonists at all three receptors were blocked by pertussis toxin treatment and were voltage dependent. Activation of all three types of NPY receptors produced much smaller inhibition of human alpha-1E Ca++ channels also stably expressed in HEK293 cells. These results suggest that NPY receptors can regulate K+ and Ca++ channels and that these effects may be responsible for the observed effects of NPY on neuronal excitability and synaptic transmission.
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PMID:Regulation of K+ and Ca++ channels by a family of neuropeptide Y receptors. 945 7

Sustained stimulation of muscarinic acetylcholine receptors (mAChRs) and other G protein-coupled receptors usually leads to a loss of receptor binding sites from the plasma membrane, referred to as receptor sequestration. Receptor sequestration can occur via endocytosis of clathrin-coated vesicles that bud from the plasma membrane into the cell but may also be accomplished by other, as yet ill-defined, mechanisms. Previous work has indicated that the monomeric GTPase dynamin controls the endocytosis of plasma membrane receptors via clathrin-coated vesicles. To investigate whether mAChRs sequester in a receptor subtype-specific manner via dynamin-dependent clathrin-coated vesicles, we tested the effect of overexpressing the dominant-negative dynamin mutant K44A on m1, m2, m3, and m4 mAChR sequestration in HEK-293 cells. The m1, m2, m3, and m4 mAChRs sequestered rapidly in HEK-293 cells following agonist exposure but displayed dissimilar sequestration pathways. Overexpression of dynamin K44A mutant fully blocked m1 and m3 mAChR sequestration, whereas m2 mAChR sequestration was not affected. Also, m4 mAChRs, which like m2 mAChRs preferentially couple to pertussis toxin-sensitive G proteins, sequestered in a completely dynamin-dependent manner. Following agonist removal, sequestered m1 mAChRs fully reappeared on the cell surface, whereas sequestered m2 mAChRs did not. The distinct sequestration of m2 mAChRs was also apparent in COS-7 and Chinese hamster ovary cells. We conclude that the m2 mAChR displays unique subtype-specific sequestration that distinguishes this receptor from the m1, m3, and m4 subtypes. These results are the first to demonstrate that receptor sequestration represents a new type of receptor subtype-specific regulation within the family of mAChRs.
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PMID:Receptor subtype-specific regulation of muscarinic acetylcholine receptor sequestration by dynamin. Distinct sequestration of m2 receptors. 957 62

While it is well established that adenylyl cyclase and phospholipase C-beta are two proximal signal effectors for the calcitonin receptor, the more distal signaling pathways are less well characterized. G protein-coupled receptors can activate Erk1/2 by Gs-, Gi-, or Gq-dependent signaling pathways, depending on the specific receptor and cell type examined. Since the calcitonin receptor can couple to all three of these G proteins, the ability of calcitonin to activate Erk1/2 was investigated. Calcitonin induced time- and concentration-dependent increases in Shc tyrosine phosphorylation, Shc-Grb2 association and Erk1/2 phosphorylation and activation in a HEK 293 cell line that stably expresses the rabbit calcitonin receptor C1a isoform. Pertussis toxin, which inactivates Gi, and calphostin C, a protein kinase C inhibitor, each partially inhibited calcitonin-induced Shc tyrosine phosphorylation, Shc-Grb2 association, and Erk1/2 phosphorylation. In contrast, neither forskolin nor H89, a protein kinase A inhibitor, had a significant effect on basal or calcitonin-stimulated Erk1/2 phosphorylation. Our results suggest that the calcitonin receptor induces Shc phosphorylation and Erk1/2 activation in HEK293 cells by parallel Gi- and PKC-dependent mechanisms. The calcitonin-induced elevation of cytosolic free Ca2+ was required for Erk1/2 phosphorylation, since preventing any change in cytosolic free Ca2+ by chelating both cytosolic and extracellular Ca2+ abolished the response. However, the change in Ca2+ that is induced by calcitonin is not sufficient to account for the calcitonin-induced Erk1/2 phosphorylation, since treatment with 100 nM ionomycin or 10 microM thapsigargin, each of which induced elevations of Ca2+ comparable to those induced by calcitonin, induced significantly less Erk1/2 phosphorylation than that induced by calcitonin. Erk1/2 may have important roles as downstream effectors mediating cellular responses to calcitonin stimulation.
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PMID:The calcitonin receptor stimulates Shc tyrosine phosphorylation and Erk1/2 activation. Involvement of Gi, protein kinase C, and calcium. 967 14


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