RANTES stimulates Ca²⁺ mobilization and inositol trisphosphate (IP₃) formation in cells transfected with G protein‐coupled receptor 75

Abstract

Background and purpose: RANTES is an inflammatory chemokine with a critical role in T-lymphocyte activation and proliferation. Its effects are mediated through G protein-coupled heptahelical receptors (GPCRs). We show for the first time that RANTES activates the orphan G protein-coupled receptor 75 (GPR75). Experimental approach: To identify a ligand for GPR75 we have used three different and independent methods, namely luciferase assay, bioluminescence assay and IP 3 accumulation assay. Key results: Treatment of cells expressing GPR75 with subnanomolar concentrations of RANTES led to stimulation of the luciferase activity in a reporter-gene assay, an increase in inositol trisphosphate, and intracellular Ca 2 þ . The latter effect was blocked by the phospholipase-C inhibitor (PLC) U73122 indicating that Gq proteins mediate GPR75 signaling. RANTES enhanced the phosphorylation of AKT and mitogen-activated protein kinase (MAPK) in GPR75-transfected cells and this effect was blocked by the PLC inhibitor U73122 and the phosphatidylinositol 3-kinase (PI3K) inhibitor, wortmannin. The hippocampal cell line HT22, which expresses GPR75 endogenously, but not the other known RANTES receptors, was used to study the effects of RANTES and GPR75 on neuronal survival. Treatment of HT22 cells with RANTES significantly reduced the neurotoxicity of amyloid-b peptides, by activating PLC and PI3K. Conclusions and implications: This demonstrate clearly and undoubtedly the ability of RANTES to act on GPR75. Defects in the RANTES/GPR75-signaling pathway may contribute to neuroinflammatory and neurodegenerative processes as observed in Alzheimer's disease.

The novel chemokine receptor, G-protein-coupled receptor 75, is expressed by islets and is coupled to stimulation of insulin secretion and improved glucose homeostasis

“…Consistent with this, CCL5 stimulated IP 3 formation in GPR75-transfected human embryonic kidney cells [10], confirming the Gq-dependent coupling of GPR75 activation. Treatment of GPR75-overexpressing murine hippocampal cells with CCL5 has also been shown to increase cell viability, most likely via PLC and phosphatidylinositol 3-kinase activation [10].…”

“…The identification of GPR75 as the most abundant CCL5 receptor in islets, its localisation to beta cells and previous reports that this receptor couples via Gq to elevate [Ca 2+ ] i [10] led us to investigate whether similar signal transduction occurred in beta cells. Determining the optimal exogenous CCL5 concentration needed to identify whether this chemokine acts via GPR75 to regulate beta cell signalling is challenging.…”

“…Determining the optimal exogenous CCL5 concentration needed to identify whether this chemokine acts via GPR75 to regulate beta cell signalling is challenging. In studies investigating the effects of CCL5 in GPR75-transfected Human embryonic kidney cells and Chinese hamster ovary cells, the EC 50 for effects on IP 3 generation and Ca 2+ mobilisation ranged from 0.1 to 0.3 nmol/l [10], while 1 nmol/l CCL5 has been reported to significantly elevate [Ca 2+ ] i in spinal cord synaptosomes [32]. In addition, the circulating CCL5 concentration in healthy humans is 5.8 to 7.8 nmol/l [33,34], yet as CCL5 is expressed by islet cells, its local concentration may be higher than that reaching islets via the circulation.…”

“…Furthermore, while conventional CCRs are thought to signal predominantly through the Bordetella pertussis toxin (PTX)-sensitive Gi family of G proteins that inhibit cAMP production [13][14][15][16], CCL5 activation of GPR75 transfected into Chinese hamster ovary cells led to phospholipase C (PLC)-mediated elevations of inositol 1,4,5-trisphosphate (IP 3 ) formation and intracellular calcium ([Ca 2+ ] i ), both of which were inhibited by the PLC inhibitor U73122, but unaffected by PTX [10]. Consistent with this, CCL5 stimulated IP 3 formation in GPR75-transfected human embryonic kidney cells [10], confirming the Gq-dependent coupling of GPR75 activation. Treatment of GPR75-overexpressing murine hippocampal cells with CCL5 has also been shown to increase cell viability, most likely via PLC and phosphatidylinositol 3-kinase activation [10].…”

“…It has recently become apparent that CCL5 is also a ligand at G-protein-coupled receptor 75 (GPR75), a novel GPCR that shares only 12 to 16% sequence homology with the chemokine receptor family members [10]. GPR75 is a 540 amino acid protein with seven transmembrane domains, typical of previously characterised GPCRs [11], but its C-terminal tail is considerably longer than that of other chemokine receptors [12]; moreover, its expression has been reported to be limited to the brain, spinal cord and retinal pigment epithelium, rather than the traditional sites of chemokine receptors such as the thymus and spleen [11].…”