Figure 4.

β-Arrestin-dependent PTHR ubiquitination. Wildtype or β-arrestin1/2 double KO MEF were transiently transfected with TAP(HA)-PTHR, Myc-ubiquitin, and FLAG-β-arrestin1 or FLAG-β-arrestin2 using Lipofectamine 3000. After 48 h, cells were serum-starved for 2 h and treated with 10 μm MG132 for 30 min followed by a 30-min challenge with 100 nm PTH(1–34). PTHR was isolated and detected by immunoblotting with antibodies against ubiquitin (P4D1) (top) and HA for receptor expression (middle). Expression of β-arrestin1 or -2 from cell lysates was detected with an anti-β-arrestin1/2 antiserum (bottom). The positions of molecular weight standards are marked at the right. Experiments performed in triplicate yielded similar results.