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
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
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DOI：
10.1124/mol.112.081307
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发表时间：
2012-12-01
影响因子：
3.6
通讯作者：
Zhou, Naiming
中科院分区：
文献类型：
--
作者：
Li, Guo;Zhou, Qi;Zhou, Naiming
人羟基羧酸受体2（HCA（2）），也称为GPR 109 A和HM 74 a，首先被鉴定为烟酸受体，最近受到了极大的关注，因为它可能以有利的方式在临床上改变血脂。我们最近的研究表明，烟酸诱导的HCA（2）受体的内化受G蛋白偶联受体激酶（GRK）2和arrestin 3的调节，内化的受体迅速再循环回细胞表面。本研究采用氨基酸缺失和定点突变相结合的方法来鉴定HCA（2）C末端的结构和功能域，并探索它们在受体磷酸化、脱敏和内化中的潜在作用。我们首先构建了四个突变体，每个突变体缺失10至15个氨基酸，与截短突变体不同。我们成功地确定了不同的领域负责受体的出口，组成活动，脱敏，磷酸化和内化。我们还产生了一个全面的系列丙氨酸取代突变体，取代保守的丝氨酸和苏氨酸残基的C末端的丙氨酸残基，以查明关键残基，是必不可少的GRK 2介导的磷酸化和arrestin 3协会。此外，我们还发现HCA（2）C-末端尾区329 - 343位残基的序列在保持HCA（2）处于非活性构象中起着至关重要的作用。这些数据证明了HCA（2）C末端内的不同结构域对于受体细胞表面表达、脱敏、内化、磷酸化和稳定无活性受体构象的重要性。
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 success-fully 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.