Figure 5.

The recycling activity of SNX27 requires both its PDZ domain–mediated interaction with cargo and PX domain–mediated association with endosomes. (A) Fluorescence polarization analysis demonstrating the ability of the H112A mutation of the SNX27 PDZ domain to disrupt binding to the wild-type β₂AR-derived PDZ motif. Representative saturation plots for the wild-type PDZ domain (solid line) and H112A mutant PDZ domain (broken line) are shown. (B) Representative examples of fluorescence localization patterns of PDZ mutant (H112A) and PX mutant (ΔPX) versions of SNX27, relative to FLAG-β₂AR and EEA1, verifying that the PX domain is specifically required for early endosome localization of SNX27, whereas the PDZ domain is not. Bar, 5 µm. (C) Flow cytometric analysis of FLAG-β₂AR recycling in SNX27 knockdown cells also transfected with a GFP, SNX27a-GFP, or SNX27a-GFP containing a mutated PDZ domain (H112A) or deleted PX domain (ΔPX). Error bars indicate SEM. P-values: Student’s t test comparison to the empty GFP control; n = 4. (D) Flow cytometric analysis showing that SNX27 depletion specifically prevents PDZ motif–directed recycling of FLAG-β₂AR (first and second bars from the left; these data are replotted from Fig. 3 for comparison) without detectably affecting recycling directed by a distinct, non-PDZ sorting sequence (FLAG-β2-mrs, rightmost two bars). The inset shows a representative immunoblot confirming efficient knockdown of SNX27 in the FLAG-β2-mrs–expressing HEK293 cells used in the recycling assays (left lane, negative control; right lane, SNX27 siRNA transfection). K, kilodaltons. (E) SNX27-GFP and FLAG-β₂AR were coexpressed in A10 aortic smooth muscle cells. FLAG-β₂AR present in the plasma membrane was labeled and internalized as described in Materials and methods. Representative confocal images showing SNX27-GFP (top), FLAG-β₂AR (middle), and a merged image (bottom). Colocalization of SNX27-GFP with β₂AR-containing endosomes are enlarged 2× in the inset. Bar, 20 µm. (F) The effect of SNX27 depletion on FLAG-β₂AR or FLAG-β2-mrs recycling was measured in A10 cells by antibody efflux, as described in Materials and methods. Bars represent the reduction of recycling efficiency produced by SNX27 knockdown, measured 50 min after agonist removal from the culture medium. The third bar from the right shows a rescue condition, where the relative effect of SNX27 siRNA on FLAG-β₂AR recycling was assessed in the presence of recombinant SNX27. Error bars indicate SEM of recycling differences. P-values: paired t test of recycling percentage in negative control versus SNX27 siRNA-transfected conditions; n = 3–7.