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.

Fig. 1.

Expression and functional characterization of C-terminal deletion mutants of human HCA₂. a, sequence comparison of the COOH domains of the wild-type and mutant HCA₂ constructs. A series of mutants were made in the carboxyl terminus of HCA₂ using overlap polymerase chain reaction as described under Materials and Methods. The deleted amino acids are indicated with a dash (–). b, HEK-293 cells stably expressing WT or mutant HCA₂-EGFP (green) were stained with DiI (5 μM, red), fixed, and examined by confocal microscopy. c and d, HEK-293 cells transiently transfected with WT or mutant FLAG-HCA₂ were used to assess cell surface receptor levels and total cell receptor levels by ELISA (see Materials and Methods). e, cAMP accumulation in HEK-293 cells transiently cotransfected with HCA₂ or HCA₂ mutants and pCRE-Luc was determined in response to treatment with forskolin and niacin. f, activation of ERK1/2 in HEK-293 cells stably expressing HCA₂ or HCA₂ mutants by a challenge with 200 μM niacin for 5 min. Error bars represent the S.E.M. for four replicates. Data were analyzed using Student's t test (*, p < 0.05; ***, p < 0.001). All data shown are representative of at least three independent experiments. IB, immunoblot; CTL, control.

Fig. 2.

The C-terminal domain between residues Y295 and Q314 plays an essential role in the regulation of HCA₂ export from the endoplasmic reticulum to the cell surface. a, HEK-293 cells stably expressing EGFP-tagged wild type HCA₂ (HCA₂-EGFP) or Δ295–314 (Δ295–314-EGFP) were stimulated with 200 μM niacin for 40 min. b, HEK-293 cells were transiently cotransfected with HCA₂-EGFP or Δ295–314-EGFP (green) and with pDsRed2-ER (red). After 24 h, cells were fixed and examined by confocal microscopy as described under Materials and Methods. All images are representative of at least three independent experiments.

Fig. 3.

The C-terminal Ser/Thr cluster of 326TST328 is involved in the regulation of receptor internalization. HEK-293 cells stably expressing HCA₂ or HCA₂ mutants were stimulated with 200 μM niacin for 1 h and examined with confocal microscopy (a and c) or ELISA (b and d). e, cAMP accumulation in HEK-293 cells transiently cotransfected with HCA₂ or HCA₂ mutants and with pCRE-Luc was determined in response to treatment with forskolin and niacin. The dose-dependent inhibition of forskolin-induced cAMP accumulation in cells expressing HCA₂ or HCA₂ mutants was measured. f, Activation of ERK1/2 after a 5-min challenge with 200 μM niacin was assessed by Western blot in HEK-293 cells stably expressing HCA₂ or HCA₂ mutants starved in serum-free DMEM media for 2 h as described under Materials and Methods. Error bars represent S.E.M.s for four replicates. Data were analyzed using Student's t test (**, p < 0.01, *** p < 0.001). All data shown are representative of at least three independent experiments. FSL, forskolin; IB, immunoblot; CTL, control.

Fig. 4.

The C-terminal Ser/Thr cluster of 326TST328 is involved in regulation of arrestin3 recruitment and desensitization of HCA₂. a, HEK-293 cells stably expressing HCA₂ or HCA₂ mutants were transiently transfected with arrestin3-EGFP. Then cells were treated with 200 μM niacin for 8 min, and confocal images were taken on a Zeiss LSM 510 microscope. b, HEK-293 cells transfected with Flag-HCA₂ or STS/A mutant were stimulated with 200 μM niacin for 10 min. An equal volume of whole-cell lysate was separated by 10% SDS-polyacrylamide gel electrophoresis, and receptor phosphorylation was detected by the mobility shift of receptor with anti-Flag antibody as described under Materials and Methods. c, HEK-293 cells stably expressing HCA₂, Δ315–328, or STS/A mutant were pretreated with water (control) or 200 μM niacin for 1 h, washed three times with ice-cold PBS, and stimulated with 10 μM forskolin and 200 μM niacin for 20 min. The reaction was stopped by a quick wash with ice-cold PBS followed by the addition of cell lysis buffer. The cAMP concentration was assessed using a commercially available kit. Data were analyzed using Student's t test (**, p < 0.01). All images and data are representative of at least three independent experiments.

Fig. 5.

Cytoplasmic localization of the Δ329–343 mutant receptor. a, HEK-293 cells were transiently cotransfected with HCA₂-EGFP or Δ329–343-EGFP (green) and pDsRed2-ER (red). After 24 h, cells were fixed and examined by confocal microscopy. b, HEK-293 cells stably expressing Δ329–343-EGFP were treated (1, 2, and 4 h) with cycloheximide (CHX; 25 μM). c, HEK-293 cells stably expressing Δ329–343-EGFP (green) were incubated with either 100 μg/ml Alexa Fluor 546-labeled transferrin (red) or 50 nM LysoTracker DND-99 (red) for 40 min. All images are representative of at least three independent experiments. CTL, control.

Fig. 6.

Deletion of the C-terminal domain from Val329 to Glu343 leads to constitutive activation of HCA₂. a and b, HEK-293 cells stably expressing Flag-HCA₂ or Flag-Δ329–343 were prelabeled with anti-FLAG-FITC (a) or anti-FLAG (b) antibody (1:250 dilution) for 1 h at 4°C. Cells were next incubated in media alone (vehicle) or with 200 μM niacin for 30 min at 37°C. Receptor-antibody complexes were then analyzed by confocal microscopy (a) or ELISA (b). c and d, HEK-293 cells stably expressing Δ329–343-EGFP or FLAG-Δ329–343 were incubated in media alone or with cycloheximide (CHX) or with cycloheximide and MβCD (5 mM) or sucrose (450 mM) for 2 h and then were analyzed by confocal microscopy (c) or ELISA (d). e and f, arrestin and clathrin expression was measured as described under Materials and Methods. g, The role of arrestin2/3 and clathrin in the regulation of Δ329–343 constitutive endocytosis was measured by confocal microscopy. h, HEK-293 cells transiently cotransfected with HCA₂ or Δ329–343 mutant and pCRE-Luc were stimulated with media alone or with 10 μM forskolin for 4 h at 37°C. Luciferase activity was detected by a firefly luciferase kit. i, HEK-293 cells stably expressing wild-type HCA₂ or Δ329–343 mutant were stimulated with media alone or with 10 μM forskolin for 20 min. The cAMP concentration was assessed with a commercially available kit. Data were analyzed by using Student's t test (*, p < 0.05; **, p < 0.01; ***, p < 0.001). All images and data shown are representative of at least three independent experiments. IB, immunoblot; CTL, control.