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.
Footnotes
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The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.
This work was supported by the Ministry of Science and Technology [Grants 2012CB910402, 2012AA020303-05]; National Natural Science Foundation of China [Grant 81173106]; Zhejiang Natural Science Foundation [Grant Z2080207]; and National Institutes of Health National Institute of General Medical Sciences [Grant GM44944].
Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
ABBREVIATIONS:
- GRK
- G protein-coupled receptor kinase
- ERK1/2
- extracellular signal-regulated kinase1/2
- siRNA
- small interfering RNA
- GPCR
- G protein-coupled receptor
- DiI
- 1,1′-Dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate
- FITC
- fluorescein isothiocyanate
- HRP
- horseradish peroxidase
- HEK
- human embryonic kidney
- EGFP
- enhanced green fluorescent protein
- ELISA
- enzyme-linked immunosorbent assay
- WT
- wild-type
- TBS
- Tris-buffered saline
- BSA
- bovine serum albumin
- DMEM
- Dulbecco's modified Eagle's medium
- PBS
- phosphate-buffered saline
- CRE
- cAMP response element
- ER
- endoplasmic reticulum
- MβCD
- methyl-β-cyclodextrin
- PKA
- protein kinase A
- PKC
- protein kinase C
- 5-HT
- 5-hydroxytryptamine.
- Received July 18, 2012.
- Accepted September 7, 2012.
- Copyright © 2012 The American Society for Pharmacology and Experimental Therapeutics
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