The human hydroxycarboxylic acid receptor 2 (HCA 2 ), also known as GPR109A and HM74a, was first identified as a niacin receptor and has recently received significant attention because of its potential to clinically modify plasma lipids in a favorable manner. Our recent studies have demonstrated that the niacin-induced internalization of HCA 2 receptors is regulated by G protein-coupled receptor kinase (GRK) 2 and arrestin3 and that internalized receptors rapidly recycle back to the cell surface. The investigation presented here used a combination of amino acid deletion and site-directed mutagenesis to identify structural and functional domains within the HCA 2 C terminus and explore their potential roles in receptor phosphorylation, desensitization, and internalization. We first constructed four mutants with deletions of 10 to 15 amino acids each that were distinct from truncated mutants. We successfully identified different domains responsible for receptor export, constitutive activity, desensitization, phosphorylation, and internalization. We also generated a comprehensive series of alanine substitution mutants, replacing conserved serine and threonine residues in the C terminus with alanine residues to pinpoint the key residues that are essential for GRK2-mediated phosphorylation and arrestin3 association. Moreover, we found that a sequence from residues 329 to 343 in the C-terminal tail of HCA 2 plays a crucial role in keeping HCA 2 in an inactive conformation. These data demonstrate the importance of distinct domains within the C terminus of HCA 2 for receptor cell surface expression, desensitization, and internalization and phosphorylation and stabilization of an inactive receptor conformation.
The short neuropeptide F (sNPF) neuropeptides, closely related to vertebrate neuropeptide Y (NPY), have been suggested to exert pleiotropic effects on many physiological processes in insects. In the silkworm () two orphan G protein-coupled receptors, neuropeptide G protein-coupled receptor (BNGR) A10 and A11, have been identified as cognate receptors for sNPFs, but other sNPF receptors and their signaling mechanisms in remain unknown. Here, we cloned the full-length cDNA of the orphan receptor BNGR-A7 from the brain of larvae and identified it as a receptor for sNPFs. Further characterization of signaling and internalization indicated that BNGR-A7, -A10, and -A11 are activated by direct interaction with synthetic sNPF-1 and -3 peptides. This activation inhibited forskolin or adipokinetic hormone-induced adenylyl cyclase activity and intracellular Ca mobilization via a G-dependent pathway. Upon activation by sNPFs, BNGR-A7, -A10, and -A11 evoked ERK1/2 phosphorylation and underwent internalization. On the basis of these findings, we designated the receptors BNGR-A7, -A10, and -A11 as -sNPFR-1, -2, and -3, respectively. Moreover, the results obtained with quantitative RT-PCR analysis revealed that the three sNPF receptor subtypes exhibit differential spatial and temporal expression patterns, suggesting possible roles of sNPF signaling in the regulation of a wide range of biological processes. Our findings provide the first in-depth information on sNPF signaling for further elucidation of the roles of the sNPF/sNPFR system in the regulation of physiological activities.
Niacin has been demonstrated to activate a PI3K/Akt signaling cascade to prevent brain damage after stroke and UV-induced skin damage; however, the underlying molecular mechanisms for HCA2-induced Akt activation remain to be elucidated. Using CHO-K1 cells stably expressing HCA2 and A431 cells, a human epidermoid cell line with high levels of endogenous expression of functional HCA2 receptors, we first demonstrated that niacin induced a robust Akt phosphorylation at both Thr308 and Ser473 in a time-dependent fashion, with a maximal activation at 5 min and a subsequent reduction to baseline by 30 min through HCA2, and that the activation was significantly blocked by pertussis toxin. The HCA2-mediated activation of Akt was also significantly inhibited by the PKC inhibitors GF109203x and Go6983 in both cell lines, by the PDGFR-selective inhibitor tyrphostin A9 in CHO-HCA2 cells and by the MMP inhibitor GM6001 and EGFR-specific inhibitor AG1478 in A431 cells. These results suggest that the PKC pathway and PDGFR/EGFR transactivation pathway play important roles in HCA2-mediated Akt activation. Further investigation indicated that PI3K and the Gβγ subunit were likely to play an essential role in HCA2-induced Akt activation. Moreover, Immunobloting analyses using an antibody that recognizes p70S6K1 phosphorylated at Thr389 showed that niacin evoked p70S6K1 activation via the PI3K/Akt pathway. The results of our study provide new insight into the signaling pathways involved in HCA2 activation.
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