The P2Y₂ nucleotide receptor requires interaction with v integrins to access and activate G₁₂

JCS003441 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. UTP induces a dose-dependent activation of Rho in 1321N1 cells expressing P2Y₂ receptors. Human 1321N1 cells expressing the WT P2Y₂R were treated with the indicated concentration of UTP for 5 minutes. Rho activity was then measured as described in Materials and methods. UTP-induced Rho activation is expressed as a percentage of maximum response levels. Data points represent the mean ± s.e.m. of results from three experiments.

• Supplemental Figure S2 -

  Fig. S2. Effects of anti-αvβ5 integrin antibody on UTP- and FBS-stimulated Rho activation. Human 1321N1 cells expressing the WT P2Y₂R were incubated overnight at 37°C in serum-free medium with 10 μg/ml of anti-IgG or anti-αvβ5 antibody. Cells were then treated with or without 100 μM UTP or 10% (v/v) fetal bovine serum (FBS) for 5 minutes prior to analysis of Rho activity. Representative blots and relative intensities as means ± s.e.m. of results from three experiments are shown.

  Supplemental Movie 1

• Movie 1 -

  Movie 1. Distribution of P2Y₂R in response to UTP gradient. Human 1321N1 cells expressing GFP-tagged P2Y₂Rs were plated on chambered coverslips and incubated overnight in serum-free medium. In this movie, 1 μl of 100 mM UTP was added at the top left and fluorescent microscopy images were captured every 0.5 minutes for 45 minutes.