Characterization Of A Gonist-mediated Signaling And Internalization Of Human Nicotinic Acid Receptor Gpr109A

Nicotinic acid (niacin) has been widely used as a favourable lipid-lowering drug for50years, and the orphan G protein-coupled receptor GPR109A has been identified to be a receptor for niacin with the ability to lower levels of total plasma cholesterol, free fatty acids and triglycerides and to strongly raise high-density lipoprotein cholesterol (HDL-C). Therefore, GPR109A has been validated as a good target for treatment of lipid disorders and atherosclerosis. However, the underlying molecular mechanisms that regulate signalling and internalization of GPR109A remain largely unknown.Our research indicated that the internalization of GPR109A was mainly regulated by GRK2and arrestin3, and Gβγ subunits recruited GRK2to the membrane. GPR109A internalization was significantly blocked by pretreated with hypertonic sucrose or transfected with clathrin specific SiRNA, indicating that GPR109A internalize via the clathrin-coated pit pathway. Further investigation demonstrated that internalized GPR109A was recycled to the cell surface after the removal of agonist, and recycling of the internalized receptors was not blocked by treatment with acidotropic agents, NH4C1and monensin. Pertussis toxin pretreatment not only inhibited forskolin-induced cAMP accumulation and intracellular Ca2-mobilisation, but it also significantly attenuated agonist-promoted GPR109A internalization.By generating a number of mutatants with deletion or site-directed mutagenesis, we identified different domains at the C-terminal tail responsible for receptor export from the endoplasmic reticulum (ER), internalization and desensitization, and receptor constitutive activation. Deletion of the295-314amino acids completely abolished surface expression of the receptor. This mutant receptors were retained in the ER and failed to response to agonost in signaling. The△315-328mutant was functional in inducing the agonist-mediated signaling as the wild type (WT) receptor, but this mutant exhibited dificiency in agonist-induced internalization and desensitization. By progressively site-directed mutation we found the serine and threonine cluster STS (326-328) plays a key role in receptor internalization and arrestin3association. These data indicate that the STS cluster is the phosphorylation sites after agonist treated. The△329-343mutant underwent rapid agonist-induced internalization in the absence of agonists, showing constitutive activity in signaling and internalization.Almost all GPCRs signal through the mitogen-activated protein kinase (MAPK) cascades, which are traditionally associated with growth factor receptor signaling and are involved in the control of cell proliferation and growth, mobility, differentiation and apoptosis. Using CHO-K1cells stably expressing GPR109A, and A431cells, which is a human epidermoid cell line with high levels of endogenous expression of functional GPR109A receptors, we found that activation of extracellular signal-regulated kinases1and2(ERK1/2) by niacin was rapid, peaking within2-5min, and was significantly blocked by pertussis toxin. Furthermore, time course experiments with different kinase inhibitors demonstrated that GPR109A induced ERK1/2activation via the MMP/EGFR transactivation pathway with a maximum response at2-5min, distinct from the PKC pathway-mediated ERK1/2phosphorylation that occurs at early time points (<2min) in response to niacin. Overexpression of Gβγ subunits scavengers βARK1-CT and a-transducin led to a significant reduction of ERK1/2phosphorylation, suggesting a critical role for Py-subunits in GPR109A-activated ERK1/2phosphorylation. Using arrestin-2/3specific siRNA and an internalization-deficient GPR109A mutant, we found that arrestin-2and arrestin-3were not involved in GPR109A-mediated ERK1/2activation.Through this study, we clarified the detailed mechanism of internalization of human nicotinic receptor GPR109A, and identified the role of its carboxyl terminus on receptor localization, endocytosis, desensitization, arrestin3association and constitutive activation, as well as the regulation of ERK1/2phosphorylation. On this basis, we hope to through follow-up experiments to further elucidate the detailed mechanism of lowering blood lipids and causing skin flushing induced by GPR109A. And our researh provide a theoretical basis for development of more efficient but without causing skin flushing drugs.