Endocytosis mechanism of P2Y2 nucleotide receptor tagged with green fluorescent protein: clathrin and actin cytoskeleton dependence

Tulapurkar, Mohan E.; Schäfer, Rainer; Hanck, Theodor; Flores, Rosa V.; Weisman, Gary A.; González, Fernando; Reiser, Georg

doi:10.1007/s00018-005-5052-0

Cited by 39 publications

(40 citation statements)

References 40 publications

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“…The cellular localization of hGPR87 seems to be similar to other P2Y family members, as P2Y 1 , P2Y 2 , P2Y 11 and P2Y 12 were described to localize to the cell surface under normal cell culture conditions and internalize after ligand stimulation. [43][44][45][46] As the inducible promoter of our hGPR87-GFP and/or -RFP fusion-proteins allows a dose-dependent expression of different levels of hGPR87, we can exclude that the localization is rendered by the artificial overexpression; the localization is not dependent on the expression level in HEK293 cells. The internalization of hGPR87-GFP/RFP could be observed under specific cell culture conditions (data not shown).…”

Section: Discussionmentioning

confidence: 99%

hGPR87 contributes to viability of human tumor cells

Glatt

Halbauer

Heindl

et al. 2008

Intl Journal of Cancer

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Emerging in vitro and in vivo data underline the crucial role of Gprotein-coupled receptors (GPCRs) in tumorigenesis. Here, we report the contribution of hGPR87, a predicted member of the P2Y subfamily of GPCRs, to proliferation and survival of human tumor cell lines. hGPR87 mRNA transcript was found to be preferentially overexpressed in squamous cell carcinomas (SCCs) of different locations and in their lymph node metastases. Up-regulation of both, transcript and protein, was detected in samples of SCC of the lung, cervix, skin and head and neck (pharynx, larynx and epiglottis). In addition to the expression of hGPR87 in tumors which originate from stratified epithelia, we identified other hGPR87-positive tumor types including subsets of large cell and adenocarcinomas of the lung and transitional cell carcinomas of the urinary bladder. Loss of function studies using siRNA in human cancer cell lines lead to antiproliferative effects and induction of apoptosis. Like other known P2Y receptors, hGPR87 was found to be mainly located on the cell surface. The overexpression of hGPR87 preferentially in SCCs together with our functional data suggests a common molecular mechanism for SCC tumorigenesis and may provide a novel intervention site for mechanismbased antitumor therapies.

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Section: Discussionmentioning

confidence: 99%

hGPR87 contributes to viability of human tumor cells

Glatt

Halbauer

Heindl

et al. 2008

Intl Journal of Cancer

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“…23) is known to become blocked (24). Indeed, this block has been confirmed for other G q -linked P2YR subtypes at 28°C or below, where tested (25,26). Therefore, this effect was applied here to test separation of P2Y 1 R internalization from the other events studied.…”

Section: P2y 1 Receptors With Extracellular Fret Donor or Acceptormentioning

confidence: 99%

Constitutive and Agonist-induced Dimerizations of the P2Y1 Receptor

Choi

Simon

Tsim

et al. 2008

Journal of Biological Chemistry

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In living cells, P2Y 1 receptor dimerization was quantitated by an improved version of fluorescence resonance energy transfer donor photobleaching analysis. 44% of the P2Y 1 receptors expressed in HEK293 cell membranes exist as dimers in the resting state, inducible by agonist exposure to give 85-100% dimerization. Monomer and constitutive dimers are fully active. Agonist-induced dimerization follows desensitization and is fully reversible upon withdrawal of agonist. Receptor dimers are required for internalization at 37°C but are not sufficient; at 20°C dimerization also occurs, but endocytosis is abolished. Removal of the C-terminal 19 amino acids abolished both dimerization and internalization, whereas full activation by agonists was retained up to a loss of 39 amino acids, confirming active monomers. This receptor is known to bind through its last four amino acids (DTSL) to a scaffolding protein, Na/H exchanger regulatory factor-2, which was endogenous here, and DTSL removal blocked constitutive dimerization specifically. Distinction should therefore be made between the following: 1) constitutive dimers tethered to a scaffolding protein, together with effector proteins, within a signaling micro-domain, and 2) free dimers in the cell membrane, which here are inducible by agonist exposure. For the class A G-protein-coupled receptors, we suggest that the percentages of free monomers, and in many cases of induced free dimers, commonly become artifactually increased; this would arise from an excess there of the receptor over its specific scaffold and from a lack of the native targeting of the receptor to that site. Many G-protein-coupled receptors (GPCRs)3 have now been reported to form homo-or heterodimers. In general, the clearest information has come from heterodimers, whose presence may be shown by an expanded phenotype (see Ref. 1 and references therein). More knowledge of homodimers is also needed. For example, does homodimerization compete with heterodimer formation or even usually predominate? The extent of homodimerization of a given GPCR, particularly in the major (rhodopsin-like) class A, and whether functioning GPCR monomers generally co-exist with dimers in that class are still unresolved issues (see Refs. 2-5). Furthermore, the role of a homodimer in the life cycle of the receptor in the cell is as yet unclear, with no consensus on whether such dimers are constitutive nor whether they depend upon agonist activation (5, 6 -9). For these questions, we first need to determine the actual percentage of the membrane population of a receptor under study that is homodimeric in each relevant state, a goal as yet unattained. We have investigated these issues in the case of homodimers of the P2Y receptors for nucleotides.We report here on the P2Y 1 receptor (P2Y 1 R) (10), activated by extracellular ATP, and its product ADP, which are released ubiquitously in animal tissues. P2Y 1 R is expressed very widely therein (11,12). In the nervous system, for example, it contributes to both G q -based signaling (13) an...

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“…Nevertheless, both CD163 and CD5 contain an internalization motif of the type YXXf (where f represents a bulky hydrophobic residue), which has been shown to be associated with clathrin-mediated endocytosis (46,47). The presence of sucrose or chlorpromazine, which are inhibitors of clathrin-mediated endocytosis (48,49), almost completely abolished the internalization of CD163-L1, suggesting that internalization of CD163-L1 in a similar way is mediated through clathrin-coated pits. However, the mechanisms resulting in this internalization seem to differ from those of CD163 and CD5, as the cytoplasmic tails of CD163-L1 do not include the YXXf internalization motif.…”

Section: Discussionmentioning

confidence: 99%

CD163-L1 Is an Endocytic Macrophage Protein Strongly Regulated by Mediators in the Inflammatory Response

Møeller

Nielsen

Reichhardt

et al. 2012

The Journal of Immunology

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CD163-L1 belongs to the group B scavenger receptor cysteine-rich family of proteins, where the CD163-L1 gene arose by duplication of the gene encoding the hemoglobin scavenger receptor CD163 in late evolution. The current data demonstrate that CD163-L1 is highly expressed and colocalizes with CD163 on large subsets of macrophages, but in contrast to CD163 the expression is low or absent in monocytes and in alveolar macrophages, glia, and Kupffer cells. The expression of CD163-L1 increases when cultured monocytes are M-CSF stimulated to macrophages, and the expression is further increased by the acute-phase mediator IL-6 and the anti-inflammatory mediator IL-10 but is suppressed by the proinflammatory mediators IL-4, IL-13, TNF-α, and LPS/IFN-γ. Furthermore, we show that CD163-L1 is an endocytic receptor, which internalizes independently of cross-linking through a clathrin-mediated pathway. Two cytoplasmic splice variants of CD163-L1 are differentially expressed and have different subcellular distribution patterns. Despite its many similarities to CD163, CD163-L1 does not possess measurable affinity for CD163 ligands such as the haptoglobin–hemoglobin complex or various bacteria. In conclusion, CD163-L1 exhibits similarity to CD163 in terms of structure and regulated expression in cultured monocytes but shows clear differences compared with the known CD163 ligand preferences and expression pattern in the pool of tissue macrophages. We postulate that CD163-L1 functions as a scavenger receptor for one or several ligands that might have a role in resolution of inflammation.

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Endocytosis mechanism of P2Y2 nucleotide receptor tagged with green fluorescent protein: clathrin and actin cytoskeleton dependence

Cited by 39 publications

References 40 publications

hGPR87 contributes to viability of human tumor cells

hGPR87 contributes to viability of human tumor cells

Constitutive and Agonist-induced Dimerizations of the P2Y1 Receptor

CD163-L1 Is an Endocytic Macrophage Protein Strongly Regulated by Mediators in the Inflammatory Response

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