The psoriasis drug monomethylfumarate is a potent nicotinic acid receptor agonist

Nicotinic acid has been used for several decades to treat dyslipidemia. In mice, the lipid-lowing effect of nicotinic acid is mediated by the Gi coupled receptor PUMA-G. In humans, high (GPR109A) and low (GPR109B) affinity nicotinic acid receptors have been characterized. Here we identify monomethylfumarate as a GPR109A agonist. Monomethylfumarate is the active metabolite of the psoriasis drug Fumaderm. We show that monomethylfumarate activates GPR109A in a calcium based aequorin assay, cAMP assay and demonstrate competitive binding with nicotinic acid. We show that GPR109A is highly expressed in neutrophils and epidermal keratinocytes, and that its expression is increased in human psoriatic lesions. Our findings provide evidence that GPR109A is a target for the drug Fumaderm and suggest that niacin should be investigated to treat psoriasis in addition to its role in treating lipid disorders.     

Nicotinic acid receptor agonists differentially activate downstream effectors

Nicotinic acid remains the most effective therapeutic agent for the treatment and prevention of atherosclerosis resulting from low high density lipoprotein cholesterol. The therapeutic actions of nicotinic acid are mediated by GPR109A, a Gi protein-coupled receptor, expressed primarily on adipocytes, Langerhans cells, and macrophage. Unfortunately, a severe, cutaneous flushing side effect limits its use and patient compliance. The mechanism of high density lipoprotein elevation is not clearly established but assumed to be influenced by an inhibition of lipolysis in the adipose. The flushing side effect appears to be mediated by the release of prostaglandin D2 from Langerhans cells in the skin. We hypothesized that the signal transduction pathways mediating the anti-lipolytic and prostaglandin D2/flushing pathways are distinct and that agonists may be identified that are capable of selectively eliciting the therapeutic, anti-lipolytic pathway while avoiding the activation of the parallel flush-inducing pathway. We have identified a number of GPR109A pyrazole agonists that are capable of fully inhibiting lipolysis in vitro and in vivo and not only fail to elicit a flushing response but can antagonize the ability of nicotinic acid to elicit a flush response in vivo. In contrast to flushing agonists, exposure of cells expressing GPR109A to the non-flushing agonists fails to induce internalization of the receptor or to activate ERK 1/2 mitogen-activated protein kinase phosphorylation.     

Non-genomic oestrogen receptor signal in B lymphocytes: An approach towards therapeutic interventions for infection,autoimmunity and cancer

The non-genomic membrane bound oestrogen receptor (mER) regulates intracellular signals through receptor-ligand interactions. The mER, along with G-protein coupled oestrogen receptor GPR 30 (GPER), induces diverse cell signalling pathways in murine lymphocytes. The mER isoform ER-alpha46 has recently been demonstrated in human B and T lymphocytes as an analogue receptor for chemokine CCL18, the signalling events of which are not clearly understood. Ligand-induced mER and GPER signalling events are shared with BCR, CD19 mediated intracellular signalling through phospholipase C, PIP2/IP3/PI3 mediated activation of Akt, MAP kinase, and mTOR. Oestrogen has the ability to induce CD40-mediated activation of B cells. The complete signalling pathways of mER, GPR30 and their interaction with other signals are targeted areas for novel drug development in B cells during infection, autoimmunity and cancer. Therefore, an in depth investigation is critical for determining shared signal outputs during B cell activation. Here, we focus on the mode of action of membrane bound ER in B cells as therapeutic checkpoints.     

Differential tissue and ligand-dependent signaling of GPR109A receptor: Implications for anti-atherosclerotic therapeutic potential

Until recently, the anti-atherosclerotic effects of niacin were attributed primarily to its lipid modification properties mediated by adipocyte G-protein coupled receptor GPR109A, though recent studies have raised significant doubts about this mechanism. In fact, in rodents it has recently been demonstrated that niacin inhibits progression of atherosclerosis through actions on immune cells, particularly via macrophage-expressed GPR109A, independent of lipid-modifying properties. Here, we studied GPR109A signal transduction in human Langerhans cells, macrophages and adipocytes. We find that the consequences of receptor activation are profoundly influenced by cellular context and that ligand-biased signaling significantly impacts functionally relevant signaling. In Langerhans cells, niacin initiates GPR109A-mediated signaling pathways (Erk1/2 and Ca^2 +) responsible for the release of vasodilatory prostanoids, while the synthetic GPR109A agonist MK-0354 fails to elicit any signaling, providing a mechanistic basis for the latter compound's inability to cause flushing. While GPR109A mediates inhibition of cAMP in adipocytes, in macrophages GPR109A signaling via G_βγ subunits results in paradoxical augmentation of intracellular cAMP levels. Also, in macrophages niacin and GPR109A full agonists induce Erk1/2 and Ca^2 + signaling, release of prostanoids, upregulation of cholesterol transporters ABCA1 and ABCG1 and stimulation of reverse cholesterol transport in GPR109A dependent manner. A mechanism is presented in which signals from the autocrine action of released prostanoids and G_i protein mediated cAMP augmentation are integrated leading to modulation of reverse cholesterol transport regulatory components. These studies provide key insights into mechanisms by which GPR109A may influence cholesterol efflux in macrophages; a process that may be at least partially responsible for niacin's anti-atherosclerotic activity. MK-0354 does not induce niacin-like GPR109A signaling in macrophages, suggesting that biased agonists devoid of the flushing side-effect may also lack properties required for macrophage-mediated anti-atherosclerotic effects.     

Nrt1 and Tna1-independent export of NAD+ precursor vitamins promotes NAD+ homeostasis and allows engineering of vitamin production

NAD(+) is both a co-enzyme for hydride transfer enzymes and a substrate of sirtuins and other NAD(+) consuming enzymes. NAD(+) biosynthesis is required for two different regimens that extend lifespan in yeast. NAD(+) is synthesized from tryptophan and the three vitamin precursors of NAD(+): nicotinic acid, nicotinamide and nicotinamide riboside. Supplementation of yeast cells with NAD(+) precursors increases intracellular NAD(+) levels and extends replicative lifespan. Here we show that both nicotinamide riboside and nicotinic acid are not only vitamins but are also exported metabolites. We found that the deletion of the nicotinamide riboside transporter, Nrt1, leads to increased export of nicotinamide riboside. This discovery was exploited to engineer a strain to produce high levels of extracellular nicotinamide riboside, which was recovered in purified form. We further demonstrate that extracellular nicotinamide is readily converted to extracellular nicotinic acid in a manner that requires intracellular nicotinamidase activity. Like nicotinamide riboside, export of nicotinic acid is elevated by the deletion of the nicotinic acid transporter, Tna1. The data indicate that NAD(+) metabolism has a critical extracellular element in the yeast system and suggest that cells regulate intracellular NAD(+) metabolism by balancing import and export of NAD(+) precursor vitamins.     

GPER促进乳腺癌相关成纤维细胞的有氧糖酵解

为探讨GPER基因的活化对人乳腺癌相关成纤维细胞(CAF)有氧糖酵解的影响,本研究构建GPERsi RNA慢病毒,转染CAF细胞,以构建GPER敲低的稳定细胞系;对照组(CAF-shNC)和GPER-shRNA慢病毒感染组(CAF-shGPER组),经嘌呤霉素筛选后,实时荧光定量PCR和蛋白印迹法检测GPER的m RNA及蛋白的表达;用GPER特异性激动剂G1 (1μmol/L)处理以上两组细胞,得到G1+CAF-shNC和G1+CAFsh GPER,应用蛋白印迹法分别检测以上4组细胞中GPER下游基因p-PKA和p-CREB的表达;采用实时荧光定量PCR和蛋白印迹法检测CREB糖酵解相关靶基因PDK4和LDHB的表达,应用葡萄糖、乳酸检测试剂盒检测4组细胞中葡萄糖消耗,乳酸生成情况。最终,成功构建GPER敲低的CAF稳定细胞系;GPER特异性激动剂G1可明显上调CAF-shNC组中GPER mRNA和蛋白水平;G1+CAF-shNC与CAF-shNC和G1+CAFsh GPER组相比,GPER下游基因PKA和CREB的磷酸化水平显著增加(p<0.05);CREB糖酵解相关靶基因PDK4和LDHB的mRNA和蛋白水平明显上调(p<0.05);葡萄糖消耗量和乳酸生成量明显增加(p<0.05)。由此可得,在人乳腺癌相关成纤维细胞中,GPER特异性激动剂G1活化GPER促进CAF的有氧糖酵解。     

Deciphering the GPER/GPR30-agonist and antagonists interactions using molecular modeling studies,molecular dynamics,and docking simulations

The G-protein coupled estrogen receptor 1 GPER/GPR30 is a transmembrane seven-helix (7TM) receptor involved in the growth and proliferation of breast cancer. Due to the absence of a crystal structure of GPER/GPR30, in this work, molecular modeling studies have been carried out to build a three-dimensional structure, which was subsequently refined by molecular dynamics (MD) simulations (up to 120 ns). Furthermore, we explored GPER/GPR30’s molecular recognition properties by using reported agonist ligands (G1, estradiol (E2), tamoxifen, and fulvestrant) and the antagonist ligands (G15 and G36) in subsequent docking studies. Our results identified the E2 binding site on GPER/GPR30, as well as other receptor cavities for accepting large volume ligands, through GPER/GPR30 π–π, hydrophobic, and hydrogen bond interactions. Snapshots of the MD trajectory at 14 and 70 ns showed almost identical binding motifs for G1 and G15. It was also observed that C107 interacts with the acetyl oxygen of G1 (at 14 ns) and that at 70 ns the residue E275 interacts with the acetyl group and with the oxygen from the other agonist whereas the isopropyl group of G36 is oriented toward Met141, suggesting that both C107 and E275 could be involved in the protein activation. This contribution suggest that GPER1 has great structural changes which explain its great capacity to accept diverse ligands, and also, the same ligand could be recognized in different binding pose according to GPER structural conformations.     

Hetero-oligomeric Complex between the G Protein-coupled Estrogen Receptor 1 and the Plasma Membrane Ca2+-ATPase 4b

The new G protein-coupled estrogen receptor 1 (GPER/GPR30) plays important roles in many organ systems. The plasma membrane Ca²⁺-ATPase (PMCA) is essential for removal of cytoplasmic Ca²⁺ and for shaping the time courses of Ca²⁺-dependent activities. Here, we show that PMCA and GPER/GPR30 physically interact and functionally influence each other. In primary endothelial cells, GPER/GPR30 agonist G-1 decreases PMCA-mediated Ca²⁺ extrusion by promoting PMCA tyrosine phosphorylation. GPER/GPR30 overexpression decreases PMCA activity, and G-1 further potentiates this effect. GPER/GPR30 knockdown increases PMCA activity, whereas PMCA knockdown substantially reduces GPER/GPR30-mediated phosphorylation of the extracellular signal-related kinase (ERK1/2). GPER/GPR30 co-immunoprecipitates with PMCA with or without treatment with 17β-estradiol, thapsigargin, or G-1. Heterologously expressed GPER/GPR30 in HEK 293 cells co-localizes with PMCA4b, the main endothelial PMCA isoform. Endothelial cells robustly express the PDZ post-synaptic density protein (PSD)-95, whose knockdown reduces the association between GPER/GPR30 and PMCA. Additionally, the association between PMCA4b and GPER/GPR30 is substantially reduced by truncation of either or both of their C-terminal PDZ-binding motifs. Functionally, inhibition of PMCA activity is significantly reduced by truncation of GPER/GPR30''s C-terminal PDZ-binding motif. These data strongly indicate that GPER/GPR30 and PMCA4b form a hetero-oligomeric complex in part via the anchoring action of PSD-95, in which they constitutively affect each other''s function. Activation of GPER/GPR30 further inhibits PMCA activity through tyrosine phosphorylation of the pump. These interactions represent cross-talk between Ca²⁺ signaling and GPER/GPR30-mediated activities.     

Impedance measurement: a new method to detect ligand-biased receptor signaling

Ligand-biased receptor signaling has been proposed for several G-protein coupled receptors including the niacin receptor GPR109A. Coupling to the G_i/o pathway has been shown to be responsible for the well described triglyceride lowering effect of nicotinic acid in mice, while activation of the β-arrestin pathway has been suggested to be responsible for its peripheral vasodilatory effect that causes cutaneous flushing. Several ligands have been described to selectively induce triglyceride lowering without inducing flushing.Cellular impedance has been demonstrated to determine G-protein coupled receptors activation in a G-protein specific manner. Agonists, which induce triglyceride lowering, but not flushing show a profile in cellular impedance that is distinct from the one induced by niacin and those compounds that induce triglyceride lowering as well as flushing. The strength of the signal correlates with the activation of β-arrestin.     

Retrograde transport of the transmembrane estrogen receptor, G-protein-coupled-receptor-30 (GPR30/GPER) from the plasma membrane towards the nucleus

G-protein-coupled receptor 30 (GPR30/GPER) belongs to the seven transmembrane receptor (7TMR) superfamily, the most common class of surface receptor with approximately 800 known members. GPER promotes estrogen binding and rapid signaling via membrane-associated enzymes resulting in increased cAMP and release of heparan bound epidermal growth factor (proHB-EGF) from breast cancer cells. However, GPER is predominately localized intracellularly in breast cancer cells with minor amounts of receptor on the cell surface, an observation that has caused some controversy regarding its potential role as a plasma membrane estrogen receptor. Using the widely employed approach of tracking recombinant 7TMRs by surface labeling live cells, we have begun to characterize and compare the endocytic fate of GPER to other similarly labeled 7TMRs. Upon ectopic expression in human embryonic kidney HEK-293 cells, functional GPER is generated as these cells acquire the capacity to stimulate cAMP and activate cyclic AMP responsive binding protein in response to estradiol-17 beta stimulation. GPER is detectable on the cell surface by immunofluorescent analysis using HA-specific antibodies, albeit the bulk of the receptor is located intracellularly. Like β1AR (beta 1 adrenergic receptor) and CXCR4 (C-X-C chemokine receptor 4), GPER exits the plasma membrane via clathrin-coated pits and enters early endosomes. Interestingly, GPER has a destination that is uncommon among 7TMRs, as it accumulates in a perinuclear compartment. Like many 7TMRs (approximately one-third), GPER trafficking from the plasma membrane is constitutive (occurs in the absence of agonist). However, its route of intracellular trafficking is highly unusual, as 7TMRs typically recycle to the plasma membrane (e.g. β1AR) or are degraded in lysosomes (e.g. CXCR4). The accumulation of GPER in the perinuclear space and its possible significance for attenuating estrogen action via this newly recognized membrane estrogen receptor is discussed herein.     

Discovery of pyrazolopyrimidines as the first class of allosteric agonists for the high affinity nicotinic acid receptor GPR109A

Pyrazolopyrimidines were discovered as the first class of allosteric agonists for the high affinity nicotinic acid receptor GPR109A. In addition to its intrinsic activity, compound 9n significantly enhances nicotinic acid binding to the receptor, thereby potentiating the functional efficacy of nicotinic acid.     

Neutrophil apoptosis mediated by nicotinic acid receptors (GPR109A)

G protein-coupled receptor (GPR)109A (HM74A) is a G(i) protein-coupled receptor, which is activated by nicotinic acid (NA), a lipid-lowering drug. Here, we demonstrate that mature human neutrophils, but not eosinophils, express functional GPR109A receptors. The induction of the GPR109A gene appears to occur late in the terminal differentiation process of neutrophils, since a mixed population of immature bone marrow neutrophils did not demonstrate evidence for its expression. NA accelerated apoptosis in cultured neutrophils in a concentration-dependent manner, as assessed by phosphatidylserine redistribution, caspase-3 activation, and DNA fragmentation assays. The pro-apoptotic effect of NA was abolished by pertussis toxin, which was used to block G(i) proteins, suggesting a receptor-mediated mechanism. Activation of GPR109A by NA resulted in decreased levels of cyclic adenosine monophosphate (cAMP), most likely due to G(i)-mediated inhibition of adenylyl cyclase activity. NA-induced apoptosis was reversed by the addition of cell-permeable cAMP, pointing to the possibility that reduced cAMP levels promote apoptosis in neutrophils. Distal mechanism involved in this process may include the post-translational modification of members of the Bcl-2 family, such as dephosphorylation of pro-apoptotic Bad and antiapoptotic Mcl-1 proteins. Taken together, following maturation in the bone marrow, neutrophils express functional GPR109A receptors, which might be involved in the regulation of neutrophil numbers. Moreover, this study identified a new cellular target of NA and future drugs activating GPR109A receptors, the mature neutrophil.     

Targeting multiple tyrosine kinase receptors with Dovitinib blocks invasion and the interaction between tumor cells and cancer-associated fibroblasts in breast cancer

A constitutive and dynamic interaction between tumor cells and their surrounding stroma is a prerequisite for tumor invasion and metastasis. Fibroblasts and myofibroblasts (collectively called cancer associated fibroblasts, CAFs) often represent the major cellular components of tumor stroma. Tumor cells secret different growth factors which induce CAFs proliferation and differentiation, and, consequently, CAFs secrete different chemokines, cytokines or growth factors which induce tumor cell invasion and metastasis. In this study we showed here that CAFs from breast cancer surgical specimens significantly induced the invasion of breast cancer cells in vitro. Most interestingly, the novel multiple tyrosine kinase inhibitor Dovitinib significantly blocked the CAFs-induced invasion of breast cancer cells by, at least in part, inhibition of the expression and secretion of CCL2, CCL5 and VEGF in CAFs. Inhibition of PI3K/Akt/mTOR signaling could be responsible for the effects of Dovitinib, since Dovitinib antagonized the promoted phosphorylated Akt after treatment with PDGF, FGF or breast cancer cell-conditioned media. Treatment with Dovitinib in combination with PI3K/Akt/mTOR signaling inhibitors Ly294002 or RAD001 resulted in additive inhibition of cell invasion. This is the first in vitro study to show that the multiple tyrosine kinase inhibitor has therapeutic activities against breast cancer metastasis by targeting both tumor cells and CAFs.     

Identification of Isn1 and Sdt1 as Glucose- and Vitamin-regulated Nicotinamide Mononucleotide and Nicotinic Acid Mononucleotide 5��-Nucleotidases Responsible for Production of Nicotinamide Riboside and Nicotinic Acid Riboside

Recently, we discovered that nicotinamide riboside and nicotinic acid riboside are biosynthetic precursors of NAD⁺, which are utilized through two pathways consisting of distinct enzymes. In addition, we have shown that exogenously supplied nicotinamide riboside is imported into yeast cells by a dedicated transporter, and it extends replicative lifespan on high glucose medium. Here, we show that nicotinamide riboside and nicotinic acid riboside are authentic intracellular metabolites in yeast. Secreted nicotinamide riboside was detected with a biological assay, and intracellular levels of nicotinamide riboside, nicotinic acid riboside, and other NAD⁺ metabolites were determined by a liquid chromatography-mass spectrometry method. A biochemical genomic screen indicated that three yeast enzymes possess nicotinamide mononucleotide 5′-nucleotidase activity in vitro. Metabolic profiling of knock-out mutants established that Isn1 and Sdt1 are responsible for production of nicotinamide riboside and nicotinic acid riboside in cells. Isn1, initially classified as an IMP-specific 5′-nucleotidase, and Sdt1, initially classified as a pyrimidine 5′-nucleotidase, are additionally responsible for dephosphorylation of pyridine mononucleotides. Sdt1 overexpression is growth-inhibitory to cells in a manner that depends on its active site and correlates with reduced cellular NAD⁺. Expression of Isn1 protein is positively regulated by the availability of nicotinic acid and glucose. These results reveal unanticipated and highly regulated steps in NAD⁺ metabolism.     

Oleate promotes the proliferation of breast cancer cells via the G protein-coupled receptor GPR40

Evidence from epidemiological studies and animal models suggests a link between high levels of dietary fat intake and risk of breast cancer. In addition, obesity, in which circulating lipids are elevated, is associated with increased risk of various cancers. Relative to this point, we previously showed that oleate stimulates the proliferation of breast cancer cells and that phosphatidylinositol 3-kinase plays a role in this process. Nonetheless, questions remain regarding the precise mechanism(s) by which oleate promotes breast cancer cell growth. Pharmacological inhibitors of the GTP-binding proteins G(i)/G(o), phospholipase C, Src, and mitogenic-extracellular signal-regulated kinase 1/2 (MEK 1/2) decreased oleate-induced [3H]thymidine incorporation in the breast cancer cell line MDA-MB-231. In addition, oleate caused a rapid and transient rise in cytosolic Ca2+ and an increase in protein kinase B phosphorylation. Overexpressing in these cells the G protein-coupled receptor GPR40, a fatty acid receptor, amplified oleate-induced proliferation, whereas silencing the GPR40 gene using RNA interference decreased it. Overexpressing GPR40 in T47D and MCF-7 breast cancer cells that are poorly responsive to oleate allowed a robust proliferative action of oleate. The data indicate that the phospholipase C, MEK 1/2, Src, and phosphatidylinositol 3-kinase/protein kinase B signaling pathways are implicated in the proliferative signal induced by oleate and that these effects are mediated at least in part via the G protein-coupled receptor GPR40. The results suggest that GPR40 is implicated in the control of breast cancer cell growth by fatty acids and that GPR40 may provide a link between fat and cancer.     

Human breast cancer associated fibroblasts exhibit subtype specific gene expression profiles

ABSTRACT: BACKGROUND: Breast cancer is a heterogeneous disease for which prognosis and treatment strategies are largely governed by the receptor status (estrogen, progesterone and Her2) of the tumor cells. Gene expression profiling of whole breast tumors further stratifies breast cancer into several molecular subtypes which also co-segregate with the receptor status of the tumor cells. We postulated that cancer associated fibroblasts (CAFs) within the tumor stroma may exhibit subtype specific gene expression profiles and thus contribute to the biology of the disease in a subtype specific manner. Several studies have reported gene expression profile differences between CAFs and normal breast fibroblasts but in none of these studies were the results stratified based on tumor subtypes. METHODS: To address whether gene expression in breast cancer associated fibroblasts varies between breast cancer subtypes, we compared the gene expression profiles of early passage primary CAFs isolated from twenty human breast cancer samples representing three main subtypes; seven ER+, seven triple negative (TNBC) and six Her2+. RESULTS: We observed significant expression differences between CAFs derived from Her2+ breast cancer and CAFs from TNBC and ER + cancers, particularly in pathways associated with cytoskeleton and integrin signaling. In the case of Her2+ breast cancer, the signaling pathways found to be selectively up regulated in CAFs likely contribute to the enhanced migration of breast cancer cells in transwell assays and may contribute to the unfavorable prognosis of Her2+ breast cancer. CONCLUSIONS: These data demonstrate that in addition to the distinct molecular profiles that characterize the neoplastic cells, CAF gene expression is also differentially regulated in distinct subtypes of breast cancer.     

The G protein-coupled receptor GPR30 mediates c-fos up-regulation by 17beta-estradiol and phytoestrogens in breast cancer cells

A growing body of evidence concerning estrogen effects cannot be explained by the classic model of hormone action, which involves the binding to estrogen receptors (ERs) alpha and ERbeta and the interaction of the steroid-receptor complex with specific DNA sequences associated with target genes. Using c-fos proto-oncogene expression as an early molecular sensor of estrogen action in ERalpha-positive MCF7 and ER-negative SKBR3 breast cancer cells, we have discovered that 17beta-estradiol (E2), and the two major phytoestrogens, genistein and quercetin, stimulate c-fos expression through ERalpha as well as through an ER-independent manner via the G protein-coupled receptor homologue GPR30. The c-fos response is repressed in GPR30-expressing SKBR3 cells transfected with an antisense oligonucleotide against GPR30 and reconstituted in GPR30-deficient MDA-MB 231 and BT-20 breast cancer cells transfected with a GPR30 expression vector. GPR30-dependent activation of ERK1/2 by E2 and phytoestrogens occurs via a Gbetagamma-associated pertussis toxin-sensitive pathway that requires both Src-related and EGF receptor tyrosine kinase activities. The ability of E2 and phytoestrogens to regulate the expression of growth-related genes such as c-fos even in the absence of ER has interesting implications for understanding breast cancer progression.     

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

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, NH₄Cl and monensin. Pertussis toxin pretreatment not only inhibited forskolin-induced cAMP accumulation and intracellular Ca²⁺ 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.