C-terminus of OX2R significantly affects downstream signaling pathways

Abstract

The human orexin 2 receptor (OX2R) is a G-protein-coupled receptor (GPCR) that has been implicated in a number of diverse physiological functions. Recent studies have identified a number of functions of the C-termini of GPCRs. However, the importance of the OX2R C-terminus in regulating signaling and surface expression remains unclear. In the present study, the function of the OX2R C-terminus was investigated using three C-terminal mutants, which were truncated at residues 368, 384 and 414, respectively, and the wild-type control, which expressed the full-length OX2R. HEK-293 cells were transfected with the mutated and control OX2R constructs. ELISA, western blot analysis and calcium assays were used to investigate the effects of the mutations on OX2R function. The present results demonstrated that residues 385–414 and 415–444 exhibited a cumulative effect on the surface expression of OX2R. Residues 369–384 exhibited a significant influence on inositol phosphate production and extracellular signal-regulated kinase 1/2 phosphorylation. Residues 385–414 significantly influenced agonist-induced internalization, whereas residues 369–384 and 385–414 significantly influenced Ca2+ release. The results of the present study suggest that the C-terminus of OX2R is important for its role in various physiological and pathological processes, and may therefore be associated with such disorders as depression and anorexia.

Signaling transduction regulated by 5-hydroxytryptamine 1A receptor and orexin receptor 2 heterodimers

“…Recent studies indicated the interaction of cAMP and calcium as the potential therapeutic targets for the treatment of neurological diseases [37]. As reported, OX2R activation regulated cellular cAMP and calcium levels [18,19]. However, 5-HT1AR activation reduced the cellular cAMP levels, while having no effect on calcium influx [10].…”

“…As mentioned above, 5-HT1AR mainly binds to the Gi protein, leading to the decrease of adenylate cyclase (AC) activation, and further leads to the downregulation of levels of p-CREB and cellular cAMP after ligand stimulation [15]. And OX2R mainly binds to the Gs protein and Gq protein resulting in the activation of adenylate cyclase (AC), and further leads to the upregulation of the expression levels of p-CREB, p-ERK, cellular cAMP and calcium levels after ligand stimulation [18][19][20]. In the study, we found activation of 5-HT1AR-OX2R heterodimers increased the levels of cAMP and calcium, also led to the downregulation of p-ERK and p-CREB ( Figure 6 and Figure 8).…”

“…Analogously, orexin receptor 2 (OX2R) expresses extensively in adult brain including cerebral cortex, hippocampus and cerebellum [17]. As a member of GPCRs, OX2R binds to the Gs protein and Gq protein resulting in the activation of adenylate cyclase (AC), and further leads to the upregulation of the expression levels of p-CREB, p-ERK, cellular cAMP and calcium levels after ligand orexin A or orexin B stimulation [18][19][20]. More interestingly, the expression of OX2R gradually decreased with age [17], indicating the potential role of OX2R in aging.…”

The Effects of Apelin and Elabela Ligands on Apelin Receptor Distinct Signaling Profiles

“…HEK293 cells stably expressing APJ were plated at 10 4 cells per well in 96-well black microplates pretreated with a 0.1 mg/ml solution of poly- d -lysine for 24 h. Calcium fluorescence signals in cells were measured using the Fluo-4 NW calcium assay kit (Thermo Fisher Scientific) according to the manufacturer’s instructions ( Chen et al, 2014 ; Wang et al, 2017 ). Before detection, cells were washed twice with assay buffer (1 × HBSS, 20 mM HEPES).…”