Supporting Online Material


Abstract
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Modulation of Postendocytic Sorting of G Protein-Coupled Receptors
Jennifer L. Whistler, Johan Enquist, Aaron Marley, Jamie Fong, Fredrik Gladher, Pamela Tsuruda, Stephen R. Murray, and Mark von Zastrow

Supporting Online Material

Materials and Methods

Biotin protection-degradation assay
Stably transfected cells expressing FLAG-tagged receptors were grown to 80% confluency washed 2 times with cold phosphate buffered saline (PBS) then incubated in 3 Greek Letter Mug/ml disulfide cleavable (sulfo-NHS S-S biotin, Pierce) in PBS at 4°C for 30 minutes with gentle agitation. Cells were washed 2 times with Tris buffered saline and placed back into medium for treatment. Cells labeled 100% biotinylated were left on ice in PBS. Cells labeled 100% stripped were also left on ice in PBS then stripped as described below. Cells were treated with 5 Greek Letter MuM agonist for 30 minutes or 3 hours, washed 2 times with cold PBS and the remaining cell surface biotinylated receptors were stripped in 50 mM glutathione, 0.3 M NaCl, 75 mM NaOH, 1% fetal bovine serum at 4°C for 30 minutes. Glutathione was quenched with a 20 minute wash of PBS with 50 mM iodoacetamide, 1% bovine serum albumin. Cells were extracted in 0.1% Triton X-100, 150 mM NaCl, 25 mM KCl, 10 mM Tris-HCl pH 7.4 containing 1 Greek Letter MuM leupeptin, 1 Greek Letter MuM pepstatin A, 1 Greek Letter MuM aprotinin, 2.5 Greek Letter MuM Pefabloc SC, and cell debris was removed by centrifugation at 10,000 x g for 10 minutes at 4°C. Receptors were immunoprecipitated using M2 mouse anti-FLAG antibody (Sigma), rabbit anti-mouse linker (Jackson Immunoresearch) and protein A Sepharose (Pharmacia) overnight. Precipitates were extensively washed, and deglycosylated for 2 hours at 37°C with PNGase F (New England Biolabs). Proteins were denatured in SDS sample buffer with no reducing agent and separated by SDS-PAGE. Proteins were transferred to nitrocellulose and the membrane blocked in Tris buffered saline containing 0.1% Tween and 5% nonfat milk for 1 hour. Biotinylated proteins were visualized by incubating with the Vectastain ABC immunoperoxidase reagent (Vector Laboratories), followed by development with ECL reagents (Amersham).

Fluorescence microscopy
Cells were grown on glass coverslips and incubated in media containing 3.5 Greek Letter Mug/ml M1 mouse anti-FLAG monoclonal antibody (Sigma) to label surface receptors. Cells were then treated as described with 5 Greek Letter MuM of the alkaloid agonist etorphine for the indicated time periods. For recycling experiments (Figure 4A), cells were incubated for 30 minutes with etorphine followed by a wash and incubation for an additional 30 minutes with 10 Greek Letter MuM of the opiate antagonist naloxone. Cells were fixed using 4% formaldehyde in phosphate-buffered saline and permeabilized using 0.1% Triton X100. For visualization of receptor and GFP tagged GASP, fixed specimens were then incubated with Texas Red conjugated donkey anti-mouse antibody (Jackson Immunoresearch) to detect M1 antibodies bound to tagged receptors. For visualization of FLAG-tagged receptors relative to (amino-terminally) HA-tagged GASP, a mouse anti-HA IgG1 (HA.11, clone 16B12, Covance) was used. Selective detection of anti-FLAG (IgG2b) and anti-HA (IgG1) was accomplished using subsequent incubation of specimens with a rabbit anti-mouse IgG2b linker antibody (Zymed), then with FITC-conjugated goat anti-mouse IgG1 (Boeringer Mannheim) to visualize HA and Texas Red-conjugated donkey anti-rabbit serum (Jackson Immunoresearch) to visualize FLAG. For co-localization of FLAG-tagged receptors with LAMP1 and LAMP2, H4A3 and H4B4 mouse IgG1 reagents (Developmental Studies Hybridoma Data Bank) were used with the same series of detection steps. Epifluorescence microscopy was performed using an inverted Nikon microscope fitted with a Nikon 60XNA1.4 objective, standard filter sets (Omega Optical) and a cooled CCD camera (Princeton Instruments). Confocal fluorescence microscopy was performed using a Zeiss LSM510 microscope fitted with a Zeiss 63XNA1.4 objective in single photon mode with standard filter sets and standard (1 Airy disc) pinhole.

Co-immunoprecipitation
Cells were grown to confluency, washed 2 times with PBS and lysed in 0.1% Triton X-100, 150 mM NaCl, 25 mM KCl, 10 mM Tris-HCl pH 7.4 containing 1 Greek Letter MuM leupeptin, 1 Greek Letter MuM pepstatin A, 1 Greek Letter MuM aprotinin, 2.5 Greek Letter MuM Pefabloc SC, and cell debris was removed by centrifugation at 10,000 x g for 10 minutes at 4°C. An aliquot of lysate was removed for GASP control blot. Lysate was incubated with M2 anti-FLAG affinity resin (Sigma) overnight. Pellets were extensively washed and deglycosylated with PNGase F (New England Biolabs) for 2 hours at 37°C and then eluted in SDS sample buffer. For co-immunoprecipitation with endogenous GASP, one fourth of eluate was separated by SDS-PAGE on a 12% gel (receptor blot), three quarters on a 7% gel (GASP blot). For co- immunoprecipitation with cGASP eluate was run on a 12 % gel that was later cut and blotted separately for cGASP and receptor. Proteins were transferred to nitrocellulose. GASP blots were incubated for 2 hours with rabbit anti-GASP (1:4000). cGASP blots were incubated with rabbit anti-GFP antibodies for 2 hours (1:200) (Clontech). Both were followed by 1 hour with HRP-conjugated anti-rabbit antibody (NEB) (1:3000), and visualized with ECL plus (Amersham). Receptor blots were incubated with biotinylated M2 (1:250) (Sigma) for two hours, followed by visualization with Vectastain ABC reagents (Vector) and ECL plus. HA-11 (Covance) was used for blotting at 1:1000 for 2 hours for HA-GASP blots.