Internalization of the Human Nicotinic Acid Receptor GPR109A Is Regulated by Gi, GRK2, and Arrestin3

Abstract

Nicotinic acid (niacin) has been widely used as a favorable lipid-lowering drug for several decades, and the orphan G protein-coupled receptor GPR109A has been identified to be a receptor for niacin. Mechanistic investigations have shown that as a G i -coupled receptor, GPR109A inhibits adenylate cyclase activity upon niacin activation, thereby inhibiting free fatty acid liberation. However, the underlying molecular mechanisms that regulate signaling and internalization of GPR109A remain largely unknown. To further characterize GPR109A internalization, we made a construct to express GPR109A fused with enhanced green fluorescent protein (EGFP) at its carboxyl-terminal end. In stable GPR109A-EGFP-expressing HEK-293 cells, GPR109A-EGFP was mainly localized at the plasma membrane and was rapidly internalized in a dose-and time-dependent manner upon agonist stimulation. GPR109A internalization was completely blocked by hypertonic sucrose, indicating that GPR109A internalizes 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, NH 4 Cl and monensin. Pertussis toxin pretreatment not only inhibited forskolin-induced cAMP accumulation and intracellular Ca 2؉ mobilization; it also significantly attenuated agonist-promoted GPR109A internalization. Moreover, RNA interference experiments showed that knockdown of GRK2 (G protein-coupled receptor kinase 2) and arrestin3 expression significantly impaired receptor internalization. Taken together, these results indicate that the agonist-induced internalization of GPR109A receptors is regulated by GRK2 and arrestin3 in a pertussis toxin-sensitive manner and that internalized receptor recycling is independent of endosomal acidification.

Identification and Characterization of Distinct C-Terminal Domains of the Human Hydroxycarboxylic Acid Receptor-2 That Are Essential for Receptor Export, Constitutive Activity, Desensitization, and Internalization

“…This is followed by recruitment of GRK2 to catalyze phosphorylation of the activated receptors. GRK2-mediated phosphorylation promotes the binding of arrestin3, targeting receptors to clathrin-coated pits and causing their internalization (Li et al, 2010). Using arrestin2/3-specific siRNAs and an internalization-deficient HCA 2 mutant, we found that arrestin2 and arrestin3 are not involved in HCA 2 -mediated ERK1/2 activation (Li et al, 2011).…”

“…For fluorescence detection experiments, the cDNA of the HCA 2 receptor was C-terminally fused to the cDNA of enhanced green fluorescent protein (EGFP). To perform enzyme-linked immunosorbent assays (ELISA) for quantification of receptor expression and internalization, the cDNA of the HCA 2 receptor was inserted between the HindIII and EcoRI sites of the pCMV-FLAG vector as described previously (Li et al, 2010). Mutations and truncations were introduced by overlap extension polymerase chain reaction.…”

Niacin and biosynthesis of PGD2 by platelet COX-1 in mice and humans

“…The GPR109A receptor for niacin was identified on human platelets by flow cytometry (Supplemental Figure 8A) and immunofluorescent staining, predominately of the cell membrane (Supplemental Figure 8, B and C). Niacin-induced degradation of its receptor, as observed in other cell types (43), was apparent on Western blotting of platelet cell membranes (Supplemental Figure 8D). Niacin dose-dependently decreased platelet cyclic AMP, as expected, upon activation of this Gi-linked receptor (ref.…”

International Union of Basic and Clinical Pharmacology. LXXXII: Nomenclature and Classification of Hydroxy-carboxylic Acid Receptors (GPR81, GPR109A, and GPR109B)

“…The full agonist nicotinic acid induced internalization, whereas a partial agonist (which, unlike nicotinic acid, did not induce ERK phosphorylation) had no effect . Internalization of heterologously expressed HCA 2 activated by nicotinic acid was shown to occur in a manner dependent on G-protein-coupled receptor kinase 2 (GRK2) and arrestin 3, whereas the recycling of the internalized receptor was independent of endosomal acidification (Li et al, 2010).…”

“…However, whether these desensitization phenomena are due to effects on the receptor itself or are at the level of downstream signaling processes is not clear. For heterologously expressed HCA 1 and HCA 2 receptors, internalization of the receptor in response to ligand application has been described previously Liu et al, 2009;Li et al, 2010). In the case of heterologously expressed HCA 2 receptor, internalization is dependent on the type of ligand.…”