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First published online 26 September 2006
doi: 10.1242/jcs.03189

Journal of Cell Science 119, 4276-4284 (2006)
Published by The Company of Biologists 2006
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Role of the C-terminal di-leucine motif of 5-HT1A and 5-HT1B serotonin receptors in plasma membrane targeting

Damien Carrel, Michel Hamon and Michèle Darmon*

INSERM, U677, University Pierre et Marie Curie, Paris 75013, France


Figure 1
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  		Fig. 1. Cytoplasmic C-terminal domains of 5-HT1AR and 5-HT1BR contain a dileucine motif and palmitoylated cysteines. For both receptors, the C-terminal tail amino acid sequence is indicated, with the residues of interest in bold italics.

 

Figure 2
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  		Fig. 2. Relative surface labeling of 5-HT1AR and related mutants in transfected COS-7 cells, LLC-PK1 cells and hippocampal neurons. Transfection of COS-7 cells (A) and hippocampal neurons (B) with Flag-5-HT1A (wild-type), Flag-I414/415A-5-HT1A or Flag-C417/420S-5-HT1A. Surface staining with mouse monoclonal anti-Flag M2 antibody and intracellular labeling with rabbit anti-Flag after plasma membrane permeabilization are shown. Bars, 10 µm. (C) Quantification of the percentage of surface labeling of each construct in transfected COS-7 cells, LLC-PK1 cells and neurons. Data are expressed as mean relative surface labeling ± s.e.m. of 10-15 transfected cells per construct. ***P<0.001, when compared with levels in the wild type.

 

Figure 3
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  		Fig. 3. Substitution of the dileucine motif of 5-HT1AR results in ER sequestration. (A) Transfection of COS-7 cells with Flag-5-HT1A (wild type), Flag-I414/415A-5-HT1A or Flag-C417/420S-5-HT1A. Cells were permeabilized and anti-Flag labeling is shown in green and anti-calregulin or anti-giantin labeling is shown in red. Bar, 10 µm. (B) Glycosylation state of receptors was analyzed by western blotting of crude membranes from transfected COS-7 cells with anti-5-HT1A polyclonal antibody. Membranes were treated with Endo H, PNGaseF or untreated. Similar results were obtained in three independent experiments.

 

Figure 4
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  		Fig. 4. Roles of dileucine motif and palmitoylated cysteines in 5-HT1AR binding capacity and coupling. (A) Specific binding of [3H]LSD (1.6 nM) to Flag-5-HT1A (wild-type), Flag-I414/415A-5-HT1A or Flag-C417/420S-5-HT1A receptors in membranes from transfected LLC-PK1 cells. Specific binding, corrected according to transfection efficiency, is expressed as a percentage of the wild-type value. Each bar is the mean ± s.e.m. of three independent determinations performed in triplicate. (B) 5-CT-stimulated [35S]GTP{gamma}S binding to membranes from LLC-PK1 cells transfected with Flag-5-HT1A (wild-type), Flag-I414/415A-5-HT1A or Flag-C417/420S-5-HT1A. Basal binding determined in the absence of 5-CT (set to 100%) did not differ from non-specific binding determined in the presence of both 5-CT and WAY 100,635 (1 µM). Each bar is the mean ± s.e.m. of three independent experiments performed in triplicate. (C,D) 8-OH-DPAT-stimulated increase of ERK phosphorylation in LLC-PK1 cells transfected with Flag-5-HT1A (wild-type), Flag-I414/415A-5-HT1A or Flag-C417/420S-5-HT1A. Basal phosphorylation was determined in the absence of ligand. Equal amounts of cell lysate were separated by SDS-PAGE, blotted and revealed with anti-ERK or anti-PERK antibody (C). PERK2 and ERK2 signals were quantified and ERK phosphorylation is expressed as fold increase over basal levels after normalization with total ERK2 signal (D). ERK phosphorylation in stimulated untransfected (LLC-PK1) cells did not differ from the basal signal. Each bar is the mean ± s.e.m. of three independent experiments. *P<0.05 and ***P<0.001, when compared with the wild type; ns, not significant.

 

Figure 5
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  		Fig. 5. Relative surface labeling of 5-HT1BR and related mutants in transfected COS-7, LLC-PK1 cells and hippocampal neurons. Transfection of COS-7 cells (A) and hippocampal neurons (B) with Flag-5-HT1B (wild type), Flag-LI379/380A-5-HT1B or Flag-C384S-5-HT1B. Surface staining with mouse monoclonal anti-Flag M2 antibody and intracellular labeling with rabbit anti-Flag after plasma membrane permeabilization are shown. Bars, 10 µm. (C) Quantification of the percentage of surface labeling of each construct in transfected COS-7 cells, LLC-PK1 cells and neurons. Data are expressed as mean relative surface labeling ± s.e.m. of 10-15 transfected cells per construct. **P<0.01, when compared with levels in the wild-type.

 

Figure 6
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  		Fig. 6. Relative surface labeling of 1BctA and 1ActB chimeras and related mutants in transfected COS-7 cells, LLC-PK1 cells and hippocampal neurons. Transfection of COS-7 cells (A) and hippocampal neurons (B) with Flag-1BctA (wild type), Flag-I379/380A-1BctA or Flag-C382/385S-1BctA. Surface staining with mouse monoclonal anti-Flag M2 antibody and intracellular labeling with rabbit anti-Flag after plasma membrane permeabilization are shown. Bars, 10 µm. (C) Quantification of the percentage of surface labeling of each construct in transfected COS-7 cells, LLC-PK1 cells and neurons. Data are expressed as mean relative surface labeling ± s.e.m. of 10-15 transfected cells per construct. No significant differences were noted between the two mutants and the wild-type 1BctA chimera. Transfection of COS-7 cells (D) and hippocampal neurons (E) with Flag-1ActB (wild type), Flag-LI414/415A-1ActB or Flag-C419S-1ActB. Bars, 10 µm. (F) Quantification of the percentage of surface labeling of each construct in transfected COS-7 cells, LLC-PK1 cells and neurons. Data are expressed as mean relative surface labeling ± s.e.m. of 10-15 transfected cells per construct. ***P<0.001, when compared with levels in the wild type.

 

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