title: 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.
abstract: The human hydroxycarboxylic acid receptor 2 (HCA₂), 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 HCA₂ 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 HCA₂ 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 HCA₂ plays a crucial role in keeping HCA₂ in an inactive conformation. These data demonstrate the importance of distinct domains within the C terminus of HCA₂ for receptor cell surface expression, desensitization, and internalization and phosphorylation and stabilization of an inactive receptor conformation.
authors.affiliation: College of Life Sciences, Zhejiang University, Zijingang Campus, Hangzhou Zhejinag, China.
authors.forename: Guo
authors.initials: G
authors.lastname: Li
authors.forename: Qi
authors.initials: Q
authors.lastname: Zhou
authors.forename: Yena
authors.initials: Y
authors.lastname: Yu
authors.forename: Linjie
authors.initials: L
authors.lastname: Chen
authors.forename: Ying
authors.initials: Y
authors.lastname: Shi
authors.forename: Jiansong
authors.initials: J
authors.lastname: Luo
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Benovic
authors.forename: Jianxin
authors.initials: J
authors.lastname: Lu
authors.forename: Naiming
authors.initials: N
authors.lastname: Zhou
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000235:genetics / Q000378:metabolism; D019390:Arrestins / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000235:genetics / Q000378:metabolism; D054769:G-Protein-Coupled Receptor Kinase 2 / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000379:methods; D009154:Mutation; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D043562:Receptors, G-Protein-Coupled / Q000235:genetics / Q000378:metabolism*; D011978:Receptors, Nicotinic / Q000235:genetics / Q000378:metabolism*
chemical_list: D000596:Amino Acids; D019390:Arrestins; C530636:HCAR2 protein, human; D043562:Receptors, G-Protein-Coupled; D011978:Receptors, Nicotinic; C082333:arrestin3; C496567:GRK2 protein, human; D054769:G-Protein-Coupled Receptor Kinase 2