Volume 8 issue 7 July 2007
The conserved IVF motif couples activation state and endocytic functions of β-arrestin
A Burtey, EM Schmid, MGJ Ford, JZ Rappoport, MGH Scott, S Marullo, SM Simon, HT McMahon and A Benmerah

Supplemental Figure 1: AP-2 binding is required for constitutive CCP targeting of the C-terminal tail of barrestins.  above panel, Schematic representation of barrestins and C-terminal tail C1 fragments. lower panel, Hela cells were transiently transfected with GFP-tagged constructs encoding for the wild type C-terminal tail domains of either human βarr1 (residues 315-418; A) or of rat βarr2 (C1: residues 317-410; B) or with mutants of rat βarr2 fragment C5 (C) and C6 (D).  After fixation and permeabilization, cells were processed for immunofluorescence microscopy using an anti-AP-2 antibody and revealed by an Alexa594-labelled secondary antibody. The focus was made on the planar plasma membrane adherent to the coverslip to better visualize the localization of the constructs in CCPs. Insets show higher magnifications of representative areas. To help to visualize colocalizing of arrestin spots (GFP, green) AP-2 spots (Alexa594, red) were shifted from several pixels on the left.

Figure 1 (.jpg)

Supplemental Figure 2: CCP localization of IVF mutants. (A) Hela cells were transiently transfected with GFP-tagged constructs corresponding to AVF (A and B), or IVA (C and D) mutants of the IVF motif in the full length context. After fixation and permeabilization, cells were processed for fluorescence microscopy using the M300 mouse anti-AP-2 antibody and revealed by an Alexa594-labelled secondary antibody. Insets show higher magnifications of representative areas and colocalizing spots are indicated by arrows. (A and C) green fluorescence emitted by GFP (βarr2). (B and D) red fluorescence emitted by Alexa594 (AP-2).

Figure 2 (.jpg)

Supplemental Figure 3: The AAF mutant does not localize to caveolae. Hela cells were transiently co-transfected with the GFP-tagged AAF mutant (A) and mRFP-tagged caveolin1 (B). Live cells were directly imaged by TIR-FM at 37°C. (C) overlay image. Inset shows higher magnification of a representative area. (D) Quantification of GFP and mRFP fluorescence in spots corresponding to caveolin1 and AAF mutant respectively. Fluorescence intensity of each spot was normalized by substracting the background fluorescence intensity. (E) The fluorescence intensities of GFP and mRFP were measured inside and outside caveolin1 and AAF mutant spots respectively. The fluorescence inside spots is expressed as a percentage of the total fluorescence inside and outside the spot.

Figure 3 (.jpg)

Supplemental Figure 4: The AAF mutant constitutively shuttles in and out of the nucleus. Hela cells were transiently transfected with the GFP-tagged AAF mutant and treated (D-F) or not (A-C) with leptomycin B (LMB) for 45 minutes. Cells were then fixed and nuclei were stained with DAPI (C and F). Focus was made on the adherent surface of the cells (A, D) to show CCP localization or on a medial section of the cells (B, C, E and F) to show nuclear translocation upon leptomycin B treatment.

Figure 4 (.jpg)

Supplemental Figure 5: Activated mutants of βarr does not bypass agonist activation requirement for GPCR endocytosis.  HeLa cells were transiently transfected with GFP-tagged wild type or AAF mutant of βarr2 together with a TRH-R construct with extracellular VSV tag. Cell surface TRH-R were stained with a Cy3 conjugated anti-VSV antibody at 4°C and then either directly fixed or processed for immunofluorescence (A-D) or stimulated with TRH for 20 minutes in the presence of Alexa647-labelled transferrin (E-H). Cells were then washed and fixed directly observed by epifluorescence microscopy. (A, C, E) green fluorescence emitted by GFP (βarr). (B, D, F) red fluorescence emitted by Cy3 (TRH). (G) far red fluorescence emitted by Alexa647 (transferrin). (H) combined image from E, F and G.

Figure 5 (.jpg)