QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 1 November 2005
doi: 10.1242/jcs.02634

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Related articles in JCS Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Urs, N. M. Articles by Radhakrishna, H. Search for Related Content PubMed PubMed Citation Articles by Urs, N. M. Articles by Radhakrishna, H.

A requirement for membrane cholesterol in the ß-arrestin- and clathrin-dependent endocytosis of LPA₁ lysophosphatidic acid receptors

¹ School of Biology, Georgia Institute of Technology, 315 Ferst Drive, Atlanta, GA 30332, USA
² School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA 30332, USA
³ Department of Pharmacology, University of North Carolina, Chapel Hill, NC 27599, USA
⁴ Petit Institute for Bioengineering and Biosciences, Georgia Institute of Technology, 315 Ferst Drive, Atlanta, GA 30332, USA

View larger version (48K): [in a new window]   Fig. 1. siRNA-mediated reduction of clathrin inhibits agonist-induced endocytosis of LPA1. (A) Stably-transfected LPA1/HeLa cells were incubated in the presence or absence of 10 µM LPA for 30 minutes, fixed and processed for immunofluorescence detection of FLAG-tagged LPA1 with M1 mouse anti-FLAG antibodies and fluorescently-labeled secondary antibodies. (B) Cell lysates were prepared from stably-transfected LPA1/HeLa cells, which were either mock transfected (siControl) or transfected with clathrin siRNA (siClathrin) for 48 hours, separated by SDS-PAGE and immunoblotted for clathrin heavy chain (CHC) or actin. (C) Stably-transfected LPA1/HeLa cells grown in 24-well plates were either mock transfected (siControl) or transfected with clathrin siRNA (siClathrin) for 48 hours prior to treatment with or without 10 µM LPA for 45 minutes. The cells were fixed and processed for whole-cell ELISA to quantify surface LPA1 receptors as described in Materials and Methods. LPA1 internalization is expressed as the percentage difference in surface LPA1 between unstimulated cells and agonist-stimulated cells. The data are the mean±s.e.m. of six replicates/siRNA sample combined from two independent experiments. **P<0.01 compared to levels in the siControl. (D) Stably transfected LPA1/HeLa cells were treated with clathrin siRNA for 48 hours prior to incubation with FITC-labeled mouse anti-CD59 and Alexa 594-Tfn for 30 minutes and fluorescence visualization of anti-CD59 and Alexa 594-Tfn labeling. Bar, 10 µm.

View larger version (53K): [in a new window]   Fig. 2. Agonist-induced endocytosis of LPA1 is inhibited in ß-arrestin 1/2 double knockout mouse embryo fibroblasts. (A) Wild-type MEFs were transiently transfected with plasmids encoding either FLAG-tagged LPA1 or HA-tagged ß2AR and then incubated in the presence or absence of agonist (10 µM LPA or 20 µM isoproterenol, respectively) for 30 minutes prior to indirect immunofluorescence localization of the receptor proteins either in the presence or absence of detergent permeabilization. (B) ß-arrestin 1/2 double knockout MEFs were transiently transfected with plasmids encoding either FLAG-tagged LPA1 or HA-tagged ß2ARs and incubated in the presence or absence of agonist, as above, prior to indirect immunofluorescence localization of the receptor proteins. Bar, 10 µm.

View larger version (42K): [in a new window]   Fig. 3. Re-expression of wild-type ß-arrestin 2 GFP in ß-arrestin knockout MEFs restores LPA1 signal attenuation and receptor endocytosis. (A) ß-arrestin 1/2 double knockout MEFs were transiently transfected with plasmids encoding LPA1 and wild-type ß-arrestin-2-GFP. Cells were then incubated with 10 µM LPA for 0, 2, or 30 minutes prior to fixation and indirect immunofluorescence. The inset shows a magnified image of the boxed region and the arrows indicate punctate structures that co-label for both LPA1 and ß-arrestin-2-GFP. Note that the recruitment of ß-arrestin2-GFP to these punctate structures is transient, observable after 2 minutes of LPA treatment but not after 30 minutes of LPA treatment. (B) MEFs derived from wild type (WT) or ß-arrestin 1/2 null (ßArr 1/2KO) mice were transfected with plasmid encoding wild-type LPA1 receptors; ß-arrestin 1/2 null MEFs were also co-transfected with plasmids encoding LPA1 and wild-type ß-arrestin 2 (ßArr 1/2KO + LPA1 + WT ßArr2). Cells were then labeled with [3H]myo-inositol overnight in serum-free medium and incubated for 1 hour in the absence (Untreated) or presence of 10 µM LPA prior to analysis of phosphoinositide hydrolysis, as described in Materials and Methods. The radioactivity recovered in the different samples was normalized to total cellular protein and the data are presented as the mean±s.e.m. of triplicate measurements from a representative experiment that was repeated three times. **P<0.01, comparison of LPA-stimulated phosphoinositide hydrolysis in ß-arrestin 1/2 KO MEFs to that observed in WT MEFs. Bar, 10 µm.

View larger version (22K): [in a new window]   Fig. 4. Stimulation of phosphoinositide hydrolysis by LPA1 receptors is inhibited by cholesterol extraction with methyl-ß-cyclodextrin. (A) Either native HeLa cells or stably transfected LPA1/HeLa cells were labeled overnight with [3H] myo-inositol and then either left untreated (HeLa and lane 1) or pre-incubated with 5 mM MßCD for 1 hour (lane 2), 50 µg/ml nystatin for 1 hour (lane 4), 5 mM MßCD for 1 hour followed by 10 mM cholesterol/MßCD complexes for 60 minutes (lane 3), or 50 µg/ml nystatin for 1 hour followed by 10 mM cholesterol/MßCD complexes for 60 minutes (lane 5) prior to an additional 1 hour treatment with 10 µM LPA. Cells were then solubilized and the total accumulation of labeled inositol phosphates was determined. The radioactivity recovered in the different samples was normalized to total cellular protein and the data are presented as the mean±s.e.m. of triplicate measurements from a representative experiment that was repeated four times. **a, P<0.01, comparison of LPA-stimulated phosphoinositide hydrolysis in MßCD-treated LPA1/HeLa cells to that observed in non-MßCD-treated LPA1/HeLa cells. **b, P<0.01, comparing phosphoinositide hydrolysis in MßCD-treated or nystatin-treated LPA1/HeLa cells that were incubated with water-soluble cholesterol to that observed in unstimulated LPA1/HeLa cells. (B) HeLa cells were transiently transfected with plasmids encoding either vector alone (lanes 1 and 2), LPA1 (lanes 3 and 4), or M1 mAChRs (lanes 5 and 6). The cells were incubated in the absence (–) or presence (+) of 5 mM MßCD for 1 hour prior to a subsequent 1 hour incubation with agonist (10 µM LPA or 1 mM carbachol). After solubilization, the radioactively-labeled inositol phosphates were isolated as described. The radioactivity recovered in the different samples was normalized to total cellular protein and the data are presented as the mean±s.e.m. of triplicate measurements from a representative experiment that was repeated three times. **P<0.01, comparison of LPA-stimulated phosphoinositide hydrolysis in MßCD-treated LPA1-transfected HeLa cells to that observed in non-MßCD-treated cells.

View larger version (71K): [in a new window]   Fig. 5. Cholesterol extraction inhibits the agonist-induced endocytosis of LPA1 but not M1 mAChRs. (A) HeLa cells were transiently transfected with plasmids encoding either LPA1 or M1 mAChRs and were incubated in the absence (Untreated) or presence of agonist (10 µM LPA or 1 mM carbachol, respectively) and 50 µg/ml Alexa 594-Tfn for 30 minutes and then processed for indirect immunofluorescence localization of the transfected receptors. Bar, 10 µm. (B and C) HeLa cells were transfected as described above and were pre-incubated for 1 hour in the absence (Control) or presence of 5 mM MßCD or MßCD and 10 mM cholesterol/MßCD complexes prior to a subsequent incubation in the presence or absence of 10 µM LPA and 50 µg/ml Alexa 594-Tfn. The cells were fixed and processed for immunofluorescence localization of the transfected receptors. The extent of colocalization between LPA1 (B) or M1 mAChRs (C) and the internalized Alexa 594-Tfn was quantified using Metamorph image analysis as described in Materials and Methods. The data are expressed as the mean±s.e.m. of 20 cells/condition from a representative experiment that was performed three times with similar results. **P<0.01 compared with control, LPA-treated cells.

View larger version (66K): [in a new window]   Fig. 6. LPA1 receptors localize to detergent-resistant cellular domains upon agonist stimulation. (A) LPA1/HeLa cells were incubated with 10 µM LPA for different times and subsequently treated with 1% cold Triton X-100 on ice for 3 minutes, fixed and processed for indirect immunofluorescence localization of LPA1. (B) Quantitative analysis of receptor expression after detergent extraction was performed by MetaMorph image analysis as described in Materials and Methods. Cells were either untreated, treated with 5 µM cytochalasin D (Cyto. D) for 30 minutes, or treated with 5 mM MßCD for 1 hour, prior to incubation with 10 µM LPA for the indicated times. The LPA1 labeling in detergent-extracted cells was normalized to the amount of LPA1 labeling observed in non-agonist-treated cells, which had not been subjected to detergent extraction. The data are presented as the mean±s.e.m. of five to six cells per time point and are from a representative experiment that was repeated twice with similar results. (C) LPA1/HeLa cells were incubated with 10 µM LPA for different times and incubated with mouse anti-FLAG antibody on ice for 30 minutes prior to extraction with ice-cold 1% Triton X-100, to label surface LPA1 receptors. Cells were then processed for indirect immunofluorescence localization of surface LPA1. (D) Quantitative analysis of surface LPA1 receptor expression after detergent extraction was performed by MetaMorph image analysis as described in Materials and Methods. The LPA1 labeling in detergent-extracted cells was normalized to the amount of LPA1 labeling observed in non-agonist treated cells, which had not been subjected to detergent extraction. The data are presented as the mean±s.e.m. of five to six cells per time point and are from a representative experiment that was repeated twice with similar results. **P<0.01, comparison of the amount of detergent-resistant surface LPA1 staining after the indicated time of agonist treatment with that observed in unstimulated cells. Bar, 10 µm.

View larger version (75K): [in a new window]   Fig. 7. LPA stimulation leads to the colocalization of ß-arrestin-2-GFP with clathrin AP2 adaptors. LPA1/HeLa cells were transiently-transfected with plasmids encoding ß-arrestin-2-GFP and incubated with 10 µM LPA for either 0 minute or 2 minutes prior to fixation. Endogenous clathrin AP2 was localized in permeabilized cells using mouse anti-AP2 antibodies. The inset shows a high magnification image of the boxed region and the arrows indicate structures where ß-arrestin-2-GFP colocalized with AP2. Bar, 10 µm.

View larger version (68K): [in a new window]   Fig. 8. MßCD extraction prevents recruitment of ß-arrestin-2-GFP to the plasma membrane by LPA1 but not by ß2AR. (A,B) HeLa cells were transiently transfected with plasmid encoding either LPA1 (A) or ß2AR (B) along with ß-arrestin-2-GFP. The cells were then left untreated, pre-treated with 5 mM MßCD for 1 hour, or treated sequentially with 5 mM MßCD for 1 hour and 10 mM water-soluble cholesterol for 1 hour prior to incubation with 10 µM LPA for 2 minutes. The cells were then fixed and processed for indirect immunofluorescence microscopy. The inset shows a magnified image of the region of the cell indicated by the arrow. (C) The percentage of cells exhibiting recruitment of ß-arrestin-2-GFP to punctate plasma membrane spots after 2 minutes agonist stimulation was determined by scoring 100 cells per condition for cells expressing LPA1 and ß2AR. Bar, 10 µm.