Reduction of G Protein-Coupled Receptor Kinase 2 Expression in U-937 Cells Attenuates H2 Histamine Receptor Desensitization and Induces Cell Maturation

Fernández, Natalia Brenda; Monczor, Federico; Lemos, B; Notcovich, Cintia; Baldi, Alberto; Davio, Carlos; Shayo, Carina

doi:10.1124/mol.62.6.1506

Cited by 27 publications

(31 citation statements)

References 30 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…On the contrary, GRKs seem to be involved in this process, considering that in a stable U937 clone with reduced GRK2 expression (Fernandez et al, 2002) the rate of recovery of CysLT 1 functionality was faster than that in control U937 cells, and a GRK2 inhibitor partially reduced agonist-induced desensitization. However, a 30 minute pretreatment with GFX was able to increase LTD 4 -induced [Ca 2+ ] i levels (data not shown), suggesting a 'tonic' desensitization of the CysLT 1 receptor by PKC.…”

Section: Discussionmentioning

confidence: 99%

“…5B shows that CysLT 1 receptor functionality is recovered faster in a stable U937 clone (D5 clone) with reduced GRK2 expression (Fernandez et al, 2002) than in control dU937 cells after agonistinduced homologous desensitization. In addition, a 30 minute pre-treatment with 1 M of the GRK2 inhibitor (Iino et al, 2002) decreased CysLT 1 homologous desensitization to 78% (data not shown) compared with control cells (>90%, see Fig.…”

Section: Effect Of Cysltmentioning

confidence: 99%

See 1 more Smart Citation

CysLT1 receptor is a target for extracellular nucleotide-induced heterologous desensitization: a possible feedback mechanism in inflammation

Capra

Ravasi

Accomazzo

et al. 2005

Journal of Cell Science

View full text Add to dashboard Cite

Both cysteinyl-leukotrienes and extracellular nucleotides mediate inflammatory responses via specific G-protein-coupled receptors, the CysLT and the P2Y receptors, respectively. Since these mediators accumulate at sites of inflammation, and inflammatory cells express both classes of receptors, their responses are likely to be crossregulated. We investigated the molecular basis of desensitization and trafficking of the CysLT1 receptor constitutively and transiently expressed in the human monocyte/macrophage-like U937 or COS-7 cells in response to LTD4 or nucleotides. Exposure to agonist induced a rapid homologous desensitization of the CysLT1 receptor [as measured by the reduction in the maximal agonist-induced intracellular cytosolic Ca2+ ([Ca2+]i) transient], followed by receptor internalization (as assessed by equilibrium binding and confocal microscopy). Activation of P2Y receptors with ATP or UDP induced heterologous desensitization of the CysLT1 receptor. Conversely, LTD4-induced CysLT1 receptor activation had no effect on P2Y receptor responses, which suggests that the latter have a hierarchy in producing desensitizing signals. Furthermore, ATP/UDP-induced CysLT1 receptor desensitization was unable to cause receptor internalization, induced a faster recovery of CysLT1 functionality and was dependent upon protein kinase C. By contrast, homologous desensitization, which is probably dependent upon G-protein-receptor kinase 2 activation, induced a fast receptor downregulation and, accordingly, a slower recovery of CysLT1 functionality. Hence, CysLT1 receptor desensitization and trafficking are differentially regulated by the CysLT1 cognate ligand or by extracellular nucleotides. This crosstalk may have a profound physiological implication in the regulation of responses at sites of inflammation, and may represent just an example of a feedback mechanism used by cells to fine-tune their responses.

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Effect Of Cysltmentioning

confidence: 99%

CysLT1 receptor is a target for extracellular nucleotide-induced heterologous desensitization: a possible feedback mechanism in inflammation

Capra

Ravasi

Accomazzo

et al. 2005

Journal of Cell Science

View full text Add to dashboard Cite

show abstract

“…Because this experimental procedure may eventually modify the stoichiometry among the components of the signaling pathway and ultimately have an impact on the cellular response, we evaluated the role of arrestin and dynamin in H2r internalization in U937 cells. In this leukemic cell line, we reported previously moderate expression of H2r and its coupling to the Gs pathway, its desensitization mechanism, and its participation in cell maturation (Lemos Legnazzi et al, 2000;Ferná ndez et al, 2002). U937 cells were stably transfected with ␤-arrestin (319-418) or dynaminK44A.…”

Section: Resultsmentioning

confidence: 99%

“…Despite the wide therapeutic use of H2 ligands for gastric ulcers, their cardioprotective effects in patients with chronic heart failure Kim et al, 2006), and their implication in HL-60 and U937 leukemic cell maturation (Tasaka et al, 1994;Ferná ndez et al, 2002), little is known about H2r regulation. H2r internalization was first reported in human embryonic kidney 293 cells in which the authors showed that histamine treatment induces loss of H2r membrane immunoreactivity (Smit et al, 1995).…”

Section: Abbreviationsmentioning

confidence: 99%

Histamine H2 Receptor Trafficking: Role of Arrestin, Dynamin, and Clathrin in Histamine H2 Receptor Internalization

Fernández¹,

Monczor²,

Baldi³

et al. 2008

Mol Pharmacol

Self Cite

View full text Add to dashboard Cite

Agonist-induced internalization of G protein-coupled receptors (GPCRs) has been implicated in receptor desensitization, resensitization, and down-regulation. In the present study, we sought to establish whether the histamine H2 receptor (H2r) agonist amthamine, besides promoting receptor desensitization, induced H2r internalization. We further studied the mechanisms involved and its potential role in receptor resensitization. In COS7 transfected cells, amthamine induced H2r time-dependent internalization, showing 70% of receptor endocytosis after 60-min exposure to amthamine. Agonist removal led to the rapid recovery of resensitized receptors to the cell surface. Similar results were obtained in the presence of cycloheximide, an inhibitor of protein synthesis. Treatment with okadaic acid, an inhibitor of the protein phosphatase 2A (PP2A) family of phosphatases, reduced the recovery of both H2r membrane sites and cAMP response. Arrestin 3 but not arrestin 2 overexpression reduced both H2r membrane sites and H2r-evoked cAMP response. Receptor cotransfection with dominant-negative mutants for arrestin, dynamin, Eps15 (a component of the clathrin-mediated endocytosis machinery), or RNA interference against arrestin 3 abolished both H2r internalization and resensitization. Similar results were obtained in U937 cells endogenously expressing H2r. Our findings suggest that amthamine-induced H2r internalization is crucial for H2r resensitization, processes independent of H2r de novo synthesis but dependent on PP2A-mediated dephosphorylation. Although we do not provide direct evidence for H2r interaction with ␤-arrestin, dynamin, and/or clathrin, our results support their involvement in H2r endocytosis. The rapid receptor recycling to the cell surface and the specific involvement of arrestin 3 in receptor internalization further suggest that the H2r belongs to class A GPCRs.

show abstract

“…Cells were transfected as reported previously (Ferná ndez et al, 2002). Briefly, cells were harvested by centrifugation from cultures in exponential growth phase, washed once in phosphate-buffered saline, and resuspended at 10 6 cells/ml in fresh RPMI 1640 medium on ice.…”

Section: Stable Transfection Of U-937 Cellsmentioning

confidence: 99%

Tiotidine, a Histamine H2 Receptor Inverse Agonist That Binds with High Affinity to an Inactive G-Protein—Coupled Form of the Receptor. Experimental Support for the Cubic Ternary Complex Model

Monczor

Fernández²,

Legnazzi³

et al. 2003

Mol Pharmacol

Self Cite

View full text Add to dashboard Cite

Knowing the importance for research and pharmacological uses of proper ligand classification into agonists, inverse agonists, and antagonists, the aim of this work was to study the behavior of tiotidine, a controversial histamine H2 receptor ligand. We found that tiotidine, described previously as an H2 antagonist, actually behaves as an inverse agonist in U-937 cells, diminishing basal cAMP levels. [3 H]Tiotidine showed two binding sites, one with high affinity and low capacity and the other with low affinity and high capacity. The former site disappeared in the presence of guanosine 5Ј-O-(3-thio)triphosphate, indicating that it belongs to a subset of receptors coupled to G-protein, showing the classic binding profile for an agonist. Considering the occupancy models developed up to now, the only one that explains tiotidine dual behavior is the cubic ternary complex (CTC) model. This model allows Gprotein to interact with the receptor even in the inactive state. We showed by theoretical simulations based on the CTC model of dose-response and binding experiments that tiotidine biases the system to a G-protein-coupled form of the receptor that is unable to evoke a response. This theoretical approach was supported by experimental results in which an unrelated Gprotein-coupled receptor that also signals through G␣ s -protein (␤ 2 -adrenoreceptor) was impeded by tiotidine. This interference clearly implies that tiotidine biases the system to G␣ s -coupled form of the H2 receptor and turns G␣ s -protein less available to interact with ␤ 2 -adrenoreceptor. These findings not only show that tiotidine is an H2 inverse agonist in U-937 cells but also provide experimental support for the CTC model.Receptors coupled to G-proteins (GPCRs) play a major role in signal transduction and are the targets for a large number of therapeutic drugs. Although much is known about signal transduction pathways, the mechanism by which ligands bind to and activate GPCRs remains unclear. In an attempt to understand such mechanisms, several occupancy models have been developed. The first theoretical model of receptor function that included G-proteins was the ternary complex model of De Léan et al. (1980) in which the receptor possesses two binding sites, one for the ligand on the extracellular side and other for the G-protein in the intracellular side. This model is a generalization of former models, in which only the free receptor and the receptor ligand complex existed, allowing

show abstract

Reduction of G Protein-Coupled Receptor Kinase 2 Expression in U-937 Cells Attenuates H2 Histamine Receptor Desensitization and Induces Cell Maturation

Cited by 27 publications

References 30 publications

CysLT1 receptor is a target for extracellular nucleotide-induced heterologous desensitization: a possible feedback mechanism in inflammation

CysLT1 receptor is a target for extracellular nucleotide-induced heterologous desensitization: a possible feedback mechanism in inflammation

Histamine H2 Receptor Trafficking: Role of Arrestin, Dynamin, and Clathrin in Histamine H2 Receptor Internalization

Tiotidine, a Histamine H2 Receptor Inverse Agonist That Binds with High Affinity to an Inactive G-Protein—Coupled Form of the Receptor. Experimental Support for the Cubic Ternary Complex Model

Contact Info

Product

Resources

About