Saturation BRET of Y2R-eYFP with Rluc8-arrestin3 and kinetic parameters of recruitment

BRET of WT and mutant receptors were analyzed against an arrestin3 gradient (columns 1 and 2). Detectable BRET only occurred upon NPY stimulation, and maximal arrestin3 recruitment was significantly reduced for the W6.48 variants. Kinetic profiles of recruitment (columns 3 and 4) recorded at saturating F/L ratios specify that this was not due to slowed complex formation. All values are given as the mean (95% CI) corrected for multiple comparisons. Statistical significance of netBRET changes relative to the WT Y2R was assessed by one-way analysis of variance and Dunnett’s post-test.

Arrestin3-Rluc Recruitment, 1 µM NPY10 µM NPY
WT Y2R0.0011 (0.0005–0.0017)0.429 (0.384–0.474)0.286 (0.210–0.362)0.292 (0.234–0.351)
W6.48Y0.0020 (0–0.0045)0.241 (0.137–0.345)***0.618 (0.391–0.845)0.518 (0.232–0.805)
W6.48H0.0044 (0.0004–0.0084)0.176 (0.100–0.251)***0.357 (0.233–0.480)0.323 (0.219–0.427)
W6.48T0.0022 (0.007–0.0037)0.222 (0.161–0.283)***0.452 (0.258–0.645)0.391 (0.271–0.511)
  • *** P < 0.001.