D-type prostanoid receptor enhances the signaling of chemoattractant receptor-homologous molecule expressed on T(H)2 cells.

BACKGROUND: Prostaglandin (PG) D(2) is substantially involved in allergic responses and signals through the 7 transmembrane-spanning/G protein-coupled receptors, chemoattractant receptor-homologous molecule expressed on T(H)2 cells (CRTH2), and D-type prostanoid (DP) receptor. OBJECTIVE: Although the proinflammatory function of CRTH2 is well recognized and CRTH2 is hence considered an important emerging pharmacotherapeutic target, the role of the DP receptor in mediating the biological effects of PGD(2) in patients with allergic inflammation has remained unclear. METHODS: The cross-talk of CRTH2 and DP receptors was investigated by using both a recombinant HEK293 cell model and human eosinophils in Ca(2+) mobilization assays, coimmunoprecipitation, Western blotting, radioligand binding, and immunofluorescence. RESULTS: We show that CRTH2 and DP receptors modulate one another's signaling properties and form CRTH2/DP heteromers without altering their ligand-binding capacities. We find that the DP receptor amplifies the CRTH2-induced Ca(2+) release from intracellular stores and coincidentally forfeits its own signaling potency. Moreover, desensitization or pharmacologic blockade of the DP receptor hinders CRTH2-mediated signal transduction. However, CRTH2 internalization occurs independently of the DP receptor. In cells that express both receptors, pharmacologic blockade of Gα(q/11) proteins abolishes the Ca(2+) response to both CRTH2 and DP agonists, whereas inhibition of Gα(i) proteins selectively attenuates the CRTH2-mediated response but not the DP signal. CONCLUSION: Our data demonstrate the capacity of DP receptors to amplify the biological response to CRTH2 activation. Therefore the CRTH2/DP heteromer might not only represent a functional signaling unit for PGD(2) but also a potential target for the development of heteromer-directed therapies to treat allergic diseases.

Prostaglandin H2 induces the migration of human eosinophils through the chemoattractant receptor homologous molecule of Th2 cells, CRTH2.

The major mast cell product PGD2 is released during the allergic response and stimulates the chemotaxis of eosinophils, basophils, and Th2-type T lymphocytes. The chemoattractant receptor homologous molecule of Th2 cells (CRTH2) has been shown to mediate the chemotactic effect of PGD2. PGH2 is the common precursor of all PGs and is produced by several cells that express cyclooxygenases. In this study, we show that PGH2 selectively stimulates human peripheral blood eosinophils and basophils but not neutrophils, and this effect is prevented by the CRTH2 receptor antagonist (+)-3-[[(4-fluorophenyl)sulfonyl] methyl amino]-1,2,3,4-tetrahydro-9H-carbazole-9-acetic acid (Cay10471) but not by the hematopoietic PGD synthase inhibitor 4-benzhydryloxy-1-[3-(1H-tetrazol-5-yl)-propyl]piperidine (HQL79). In chemotaxis assays, eosinophils showed a pronounced migratory response toward PGH2, but eosinophil degranulation was inhibited by PGH2. Moreover, collagen-induced platelet aggregation was inhibited by PGH2 in platelet-rich plasma, which was abrogated in the presence of the D-type prostanoid (DP) receptor antagonist 3-[(2-cyclohexyl-2-hydroxyethyl)amino]-2,5-dioxo-1-(phenylmethyl)-4-imidazolidine-heptanoic acid (BWA868c). Each of these effects of PGH2 was enhanced in the presence of plasma and/or albumin. In eosinophils, PGH2-induced calcium ion (Ca2+) flux was subject to homologous desensitization with PGD2. Human embryo kidney (HEK)293 cells transfected with human CRTH2 or DP likewise responded with Ca2+ flux, and untransfected HEK293 cells showed no response. These data indicate that PGH2 causes activation of the PGD2 receptors CRTH2 and DP via a dual mechanism: by interacting directly with the receptors and/or by giving rise to PGD2 after catalytic conversion by plasma proteins.

Presynaptic Biogenesis Requires Axonal Transport of Lysosome-Related Vesicles.

Nervous system function relies on the polarized architecture of neurons, established by directional transport of pre- and postsynaptic cargoes. While delivery of postsynaptic components depends on the secretory pathway, the identity of the membrane compartment(s) supplying presynaptic active zone (AZ) and synaptic vesicle (SV) proteins is unclear. Live imaging in Drosophila larvae and mouse hippocampal neurons provides evidence that presynaptic biogenesis depends on axonal co-transport of SV and AZ proteins in presynaptic lysosome-related vesicles (PLVs). Loss of the lysosomal kinesin adaptor Arl8 results in the accumulation of SV- and AZ-protein-containing vesicles in neuronal cell bodies and a corresponding depletion of SV and AZ components from presynaptic sites, leading to impaired neurotransmission. Conversely, presynaptic function is facilitated upon overexpression of Arl8. Our data reveal an unexpected function for a lysosome-related organelle as an important building block for presynaptic biogenesis.

A presynaptic role for the cytomatrix protein GIT in synaptic vesicle recycling.

Neurotransmission involves the exo-endocytic cycling of synaptic vesicles (SVs) within nerve terminals. Exocytosis is facilitated by a cytomatrix assembled at the active zone (AZ). The precise spatial and functional relationship between exocytic fusion of SVs at AZ membranes and endocytic SV retrieval is unknown. Here, we identify the scaffold G protein coupled receptor kinase 2 interacting (GIT) protein as a component of the AZ-associated cytomatrix and as a regulator of SV endocytosis. GIT1 and its D. melanogaster ortholog, dGIT, are shown to directly associate with the endocytic adaptor stonin 2/stoned B. In Drosophila dgit mutants, stoned B and synaptotagmin levels are reduced and stoned B is partially mislocalized. Moreover, dgit mutants show morphological and functional defects in SV recycling. These data establish a presynaptic role for GIT in SV recycling and suggest a connection between the AZ cytomatrix and the endocytic machinery.