Distinct Kinetic and Spatial Patterns of Protein Kinase C (PKC)- and Epidermal Growth Factor Receptor (EGFR)-dependent Activation of Extracellular Signal-regulated Kinases 1 and 2 by Human Nicotinic Acid Receptor GPR109A

Abstract

Nicotinic acid (niacin) has been widely used as a lipid-lowering drug for several decades, and recently, orphan G proteincoupled receptor GPR109A has been identified as a receptor for niacin. Mechanistic investigations have shown that, upon niacin activation, GPR109A couples to a G i protein and inhibits adenylate cyclase activity, leading to inhibition of liberation of free fatty acid. However, the underlying molecular mechanisms for GPR109A signaling remain largely unknown. Using CHO-K1 cells stably expressing GPR109A and A431 cells, which are a human epidermoid cell line with high levels of endogenous expression of functional GPR109A receptors, we found that activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) by niacin was rapid, peaking at 5 min, and was significantly blocked by pertussis toxin. Furthermore, time course experiments with different kinase inhibitors demonstrated that GPR109A induced ERK1/2 activation via the matrix metalloproteinase/epidermal growth factor receptor transactivation pathway at both early and later time points (2-5 min); this pathway was distinct from the PKC pathway-mediated ERK1/2 phosphorylation that occurs at early time points (<2 min) in response to niacin. Overexpression of G␤␥ subunit scavengers ␤ARK1-CT and the G␣ subunit of transducin led to a significant reduction of ERK1/2 phosphorylation, suggesting a critical role for ␤␥ subunits in GPR109A-activated ERK1/2 phosphorylation. Using arrestin-2/3-specific siRNA and an internalization-deficient GPR109A mutant, we found that arrestin-2 and arrestin-3 were not involved in GPR109A-mediated ERK1/2 activation. In conclusion, our findings demonstrate that upon binding to niacin GPR109A receptors initially activate G i , leading to dissociation of the G␤␥ subunit from activated G i , and subsequently induce ERK1/2 activation via two distinct pathways, one PKC-dependent pathway occurring at a peak time of <2 min and the other matrix metalloproteinase-dependent growth factor receptor transactivation occurring at both early and later time points (2-5 min).Nicotinic acid (niacin), a B group vitamin, has been demonstrated to be the first pharmacologic agent that is able to clinically modify plasma lipids favorably (1, 2). Previous clinical studies have revealed that niacin has the abilities to lower levels of total plasma cholesterol, free fatty acids, and triglycerides and to strongly raise high density lipoprotein cholesterol compared with other lipid-lowering drugs (3, 4). In addition, niacin has been shown to effectively reduce the progression of atherosclerosis and mortality from coronary heart disease. However, its exact mechanism was not understood until the orphan receptor GPR109A (HM74a in human and protein up-regulated in macrophages by IFN-␥ (PUMA-G) in mice) was identified as a receptor for niacin in 2003 (5-7). Niacin-mediated activation of GPR109A functions in a G protein-coupled manner to decrease cAMP production, resulting in decreased hormone-sensitive lipase activity and reduced hydrolysis of trigl...

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

“…4c). These results are consistent with our previous observation that the ⌬315-328 mutant exhibited sustained activation of ERK1/2 from 10 to 30 min compared with wild-type HCA 2 (Li et al, 2011). From our current data, we cannot exclude a potential role for PKA and PKC in regulating HCA 2 desensitization.…”

“…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). In addition, mutagenesis and modeling studies have shown that the arginine residue at position 111 in transmembrane helix (TM) 3, together with Phe180 (in extracellular loop 2) and Phe276 and Tyr284 (both present in TM7) are responsible for HCA 2 binding with niacin (Tunaru et al, 2005).…”

“…For GPCRs, phosphorylation of the PKA and PKC sites within the third intracellular loop and the carboxyl-terminal tail has been found to contribute to agonist-dependent desensitization (Yuan et al, 1994;Moffett et al, 1996;Liang et al, 1998;Tang et al, 1998), whereas mutation of all of the serine and threonine residues within the C-terminal tail of the ␤ 2 -adrenergic receptor and the third intracellular loop of the m2 muscarinic acetylcholine receptor prevents the induction of GRK-mediated desensitization (Bouvier et al, 1988;Nakata et al, 1994). Results from our previous mutagenesis experiments demonstrated that a sequence of 14 amino acids, from residues 315 to 328 (⌬315-328) at the C-terminal end of the HCA 2 receptor is responsible for GRK-mediated phosphorylation and arrestin recruitment and internalization (Li et al, 2011). In the present study, mutant receptors with C-terminal deletions between Arg315 to Ser328 (⌬315-328) or with alanine substitutions for the three Ser/Thr residues in this region (Ser326, Thr327, and Ser328) not only exhibited deficiencies in arrestin3 binding and receptor internalization and phosphorylation (Figs.…”

“…The fluorescence detection experiment of arrestin translocation was performed as described previously (Li et al, 2011). The method of quantitative arrestin3 membrane translocation was described previously (Li et al, 2010).…”

“…All constructs were sequenced to verify sequences and orientations. The arrestin3-EGFP was generated as described previously (Li et al, 2011).…”