Identification of TSPAN4 as Novel Histamine H4 Receptor Interactor

Abstract

The histamine H4 receptor (H4R) is a G protein-coupled receptor that is predominantly expressed on immune cells and considered to be an important drug target for various inflammatory disorders. Like most GPCRs, the H4R activates G proteins and recruits -arrestins upon phosphorylation by GPCR kinases to induce cellular signaling in response to agonist stimulation. However, in the last decade, novel GPCR-interacting proteins have been identified that may regulate GPCR functioning. In this study, a split-ubiquitin membrane yeast two-hybrid assay was used to identify H4R interactors in a Jurkat T cell line cDNA library. Forty-three novel H4R interactors were identified, of which 17 have also been previously observed in MYTH screens to interact with other GPCR subtypes. The interaction of H4R with the tetraspanin TSPAN4 was confirmed in transfected cells using bioluminescence resonance energy transfer, bimolecular fluorescence complementation, and co-immunoprecipitation. Histamine stimulation reduced the interaction between H4R and TSPAN4, but TSPAN4 did not affect H4R-mediated G protein signaling. Nonetheless, the identification of novel GPCR interactors by MYTH is a starting point to further investigate the regulation of GPCR signaling.

Single-cell transcriptomics reveals striking heterogeneity and functional organization of dendritic and monocytic cells in the bovine mesenteric lymph node

“…Also tetraspanins showed pronounced cell-type specific expression, for example the macrophage-restricted expression of TSPAN4 , recently shown to interact with Histamin H4 receptor (60), or the high transcription of TSPAN3 in migratory DC, among which migratory cDC1 (c6) were also enriched in CD151 (TSPAN24) and CD81 (TSPAN28) transcripts. Other tetraspanin genes highly expressed in migratory DC included TSPAN13, TSPAN17 , and TSPAN33 , the two latter of which are known to interact with ADAM10, a metalloproteinase mediating ectodomain shedding (61).…”

BRET-Based Biosensors to Measure Agonist Efficacies in Histamine H1 Receptor-Mediated G Protein Activation, Signaling and Interactions with GRKs and β-Arrestins

“…Two days after transfection, basal BRET was measured between H 1 R-Rluc8 and the various eYFP/mVenus-fusion proteins using 5 µM coelenterazine-h in HBSS at 37 °C in the Mithras LB940 multimode microplate reader at 540–40 nm (acceptor) and 480–20 nm (donor) or the PHERAstar FS microplate reader (BMG Labtech; Ortenberg, Germany) at 535–30 nm (acceptor) and 475–30 nm (donor) followed by stimulation with H 1 R ligands, as previously described [ 33 ]. The G protein-activation sensor was measured in the Clariostar microplate reader (BMG Labtech; Ortenberg, Germany) at 535–30 nm monochromator (acceptor) and 470–80 nm monochromator (donor) using 3.2 μL/mL Nanoglo, as previously described [ 22 , 64 ]. The BRET ratio was first calculated for each well by dividing acceptor by donor light emission, and ligand-induced BRET ratio changes (ΔBRET) were subsequently calculated as ΔBRET = (BRET ratio ligand − BRET ratio vehicle )/BRET ratio vehicle [ 7 ].…”

Single-cell transcriptomics reveals striking heterogeneity and functional organization of dendritic and monocytic cells in the bovine mesenteric lymph node

“…Also tetraspanins showed pronounced cell-type specific expression, for example the macrophage-restricted expression of TSPAN4, recently shown to interact with Histamin H4 receptor (66), or the high transcription of TSPAN3 in migratory DC, among which migratory cDC1 (c6) were also enriched in CD151 (TSPAN24) and CD81 (TSPAN28) transcripts. Other tetraspanin genes highly expressed in migratory DC included TSPAN13, TSPAN17, and TSPAN33, the two latter of which are known to interact with ADAM10, a metalloproteinase mediating ectodomain shedding (67).…”