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First published online 26 March 2003
doi: 10.1242/jcs.00397

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Agonist-induced endocytosis of lysophosphatidic acid-coupled LPA₁/EDG-2 receptors via a dynamin2- and Rab5-dependent pathway

Mandi M. Murph^1,*, Launa A. Scaccia^1,*, Laura A. Volpicelli² and Harish Radhakrishna^1,

¹ School of Biology and Petit Institute for Biosciences and Bioengineering, Georgia Institute of Technology, Atlanta, GA 30332-0363, USA
² Department of Neurology, Emory University School of Medicine, Atlanta, GA 30322, USA

View larger version (48K): [in a new window]   Fig. 1. Stable expression of human LPA1 in HeLa cells and LPA stimulation of ERK1/2 activity. (A) Cell extracts were prepared from either untransfected HeLa cells (H) or from stably transfected HeLa cells expressing LPA1 (E2). Various amounts of extracts were separated by 10% SDS-PAGE, transferred to nitrocellulose and probed with rabbit anti-FLAG antibodies and processed for chemiluminescence detection. A single band of approximately 43 kDa was detected by anti-FLAG antibodies in extracts prepared from stably transfected LPA1-expressing cells, but not from untransfected HeLa cells. (B) Stable LPA1-expressing HeLa cells or untransfected HeLa cells were stimulated with 10 µM LPA for the indicated times and washed at 4°C prior to detergent solubilization. Equal amounts of cell extracts (30 µg) were separated by SDS-PAGE and probed with rabbit antibodies against dually phosphorylated ERK1/2 or total ERK1/2 as described in the Materials and Methods. LPA-stimulated ERK activity is maximal between 5 and 10 minutes and then declines by 30 minutes in the FLAG-LPA1 stable transfectants.

View larger version (78K): [in a new window]   Fig. 2. LPA induces the time-dependent internalization of LPA1 in HeLa cells. Stably transfected HeLa cells expressing LPA1 were incubated for various times with 10 µM LPA, fixed and processed for indirect immunofluorescence localization of LPA1 using mouse anti-FLAG antibodies and Alexa594-labeled goat anti-mouse secondary antibodies. LPA1+ endosomal structures are first observed after 10 minutes of LPA treatment. Bar, 10 µm.

View larger version (61K): [in a new window]   Fig. 3. Quantitation of LPA1 internalization. LPA1-transfected cells were incubated with 10 µM LPA for various times and then fixed and processed for quantitation of receptor internalization by laser scanning confocal microscopy as described in the Materials and Methods. (A) A representative confocal image is shown of untreated and LPA-treated cells stained with ConA to label the PM, and anti-FLAG antibodies to label FLAG-tagged LPA1. Note that LPA1 extensively co-localizes with ConA in untreated cells, but localizes to punctate fluorescent structures after LPA treatment. Bar, 10 µm. (B) Quantitation of internalization showed that approximately 40% of LPA1 is internalized within 15 minutes after LPA treatment. The data is presented as the mean ± s.e.m. at each time point (n=24 cells analyzed). ***P<0.0001 compared with untreated cells.

View larger version (96K): [in a new window]   Fig. 4. Internalized LPA1 co-localizes with the clathrin-dependent endosomal markers, EEA-1 and transferrin receptor (TfR). Stably transfected HeLa cells expressing LPA1 were treated with 10 µM LPA for 30 minutes, fixed, processed for double-label indirect immunofluorescence and analyzed by confocal microscopy. The endosomal markers EEA-1 and TfR, and the lysosomal marker LAMP-2, were localized with mouse monoclonal antibodies followed by Alexa488-labeled goat anti-mouse secondary antibodies. LPA1 was localized using rabbit anti-FLAG antibodies followed by Alexa594-labeled goat anti-rabbit secondary antibodies. The arrows in the upper panels indicate endosomal structures that contain both LPA1 and TfR. The arrow in the bottom panel indicates a structure that is LAMP2+, but does not contain LPA1. Bar, 10 µm.

View larger version (49K): [in a new window]   Fig. 5. Agonist removal stimulates the recycling of internalized LPA1 back to the PM. (A) Stably transfected HeLa cells expressing LPA1 were incubated in the absence (Untreated) or presence (+LPA) of 10 µM LPA for 30 minutes. (B) The cells were then rinsed, incubated in serum-free medium for the indicated times and processed for indirect immunofluorescence localization of LPA1. Bar, 10 µm.

View larger version (39K): [in a new window]   Fig. 6. Concentration dependence of LPA-induced LPA1 internalization. Stably transfected HeLa cells expressing LPA1 were incubated for 30 minutes with different concentrations of LPA, fixed and processed for indirect immunofluorescence localization of LPA1. Endosomal labeling of LPA1 can be observed even after treatment with 0.01 µM LPA. Bar, 10 µm.

View larger version (76K): [in a new window]   Fig. 7. Lipid specificity of LPA1 internalization. LPA1-expressing HeLa cells were incubated in serum-free medium for 16 hours prior to a 30 minutes incubation with no lipid (Control), 10 µM LPA, 1 µM LPC, or 10 µM S1P. Note: compare 1 µM LPC-treated cells with 1 µM LPA-treated cells in Fig. 6. The cells were then fixed and processed for indirect immunofluorescence localization of LPA1. Bar, 10 µm.

View larger version (61K): [in a new window]   Fig. 8. Dominant-inhibitory dynamin2 K44A inhibits LPA1 internalization. (A) LPA1-expressing HeLa cells were incubated for 30 minutes in the absence (Untreated) or presence of 10 µM LPA prior to indirect immunofluorescence localization of LPA1. (B) Stable LPA1 transfectants were transiently transfected with plasmids encoding either WT Dyn2-GFP or dominant-inhibitory Dyn2-GFP K44A. The cells were then incubated with 10 µM LPA for 30 minutes, fixed and processed for indirect immunofluorescence localization of LPA1 using mouse anti-FLAG antibodies followed by Alexa594-labeled goat anti-mouse IgG. Dynamin localization was determined by direct visualization of GFP fluorescence. Bar, 10 µm.

View larger version (39K): [in a new window]   Fig. 9. Dominant-inhibitory GFP-Rab5a S34N inhibits LPA1 internalization. (A) LPA1-expressing HeLa cells were incubated for 30 minutes in the absence (Untreated) or presence of 10 µM LPA prior to indirect immunofluorescence localization of LPA1. (B) Stable LPA1 transfectants were transiently transfected with plasmids encoding either WT GFP-Rab5a or dominant-inhibitory GFP-Rab5a S34N. The cells were then incubated with 10 µM LPA for 30 minutes, fixed and processed for indirect immunofluorescence localization of LPA1 using mouse anti-FLAG antibodies followed by Alexa594-labeled goat anti-mouse IgG. Bar, 10 µm. (C) Quantitation of inhibitory phenotype of dominant-negative dynamin2 and Rab5 mutants on LPA1 internalization. Stable LPA1 transfectants that were transiently transfected with no plasmid, Dyn2-GFP K44A, or GFP-Rab5a S34N were then incubated with 10 µM LPA for 30 minutes. The cells were fixed and processed for indirect immunofluorescence localization of LPA1. Two hundred cells per sample were scored for the presence of endocytic vesicles that contained LPA1 in an experiment. The data from three independent experiments were expressed as the mean ± s.d. of the percentage of cells that contained LPA1+ endosomal structures under each transfection condition (n=3).

View larger version (18K): [in a new window]   Fig. 10. Effects of WT and mutant Rab5 and dynamin2 on LPA1 stimulation of SRF-mediated transcription. (A) HepG2 cells were transiently transfected in serum-free medium with plasmids encoding SRE-luciferase, pRL-TK alone or with an expression vector for FLAG-tagged LPA1. Cells were incubated in the absence or presence of 1 µM LPA for 16 hours in serum-free medium prior to determination of luciferase activity (see Materials and Methods). Normalized luciferase activity is first calculated as the ratio of SRE-encoded firefly luciferase activity to TK-encoded Renilla luciferase activity. Next, the activities of all the samples are expressed as a fraction of the data collected from cells expressing LPA1 and SRE-luciferase, which had not been treated with LPA. Note that, in the absence of LPA1 expression, LPA treatment does not induce the SRE-luciferase construct. The data are expressed as the mean ± s.e.m. from two independent experiments that were performed in triplicate. (B) HepG2 cells were transiently transfected with plasmids encoding SRE-luciferase, pRL-TK and FLAG-LPA1 alone or with either GFP-tagged WT or GFP-tagged mutant Rab5 or Dyn2. The data represent the means ± s.e.m. of three measurements from a representative experiment that was repeated three times. (C) The data shown in B above was re-analyzed to determine the fold induction of luciferase activity by LPA treatment. Fold induction was calculated by first dividing the normalized luciferase data from each LPA-treated sample by the luciferase data of the corresponding untreated samples and then averaging these ratios. The data shown is the average fold induction ratios ± s.e.m. *P<0.05 compared with LPA1 alone.

View larger version (61K): [in a new window]   Fig. 11. LPA1 is constitutively internalized and recycled in serum-containing medium in the absence of added LPA. (A) Stable LPA1-transfected HeLa cells were incubated in serum-containing medium with 10 µM LPA for 30 minutes prior to fixation and indirect immunofluorescence. Alternatively, cells were incubated in medium containing 10% FBS alone (No Treatment) or incubated in this same medium with 25 µM monensin for the indicated times prior to fixation and indirect immunofluorescence localization of LPA1. Bar, 10 µm. (B) LPA1-expressing cells were incubated in serum-free medium for 16 hours and then incubated with either 25 µM monensin alone or 25 µM monensin and 10 µM LPA for 15 minutes prior to fixation and indirect immunofluorescence. Note that there was no vesicular labeling by LPA1 in the absence of LPA. Bar, 10 µm.