TABLE 3

Saturation BRET of Y₂R-Rluc8 variants with Gα_i1- and Gα_Δ6qi4myr-Venus

BRET of WT and mutant receptors was analyzed against a gradient of G protein, with and without ligand stimulation. Significant and saturable BRET was present also in the absence of NPY, suggesting preassembly of the complex. NPY stimulation increased total BRET, but a half-maximal signal occurred at similar F/L ratios (BRET₅₀), indicative of structural reorganization rather than additional recruitment upon NPY activation. This mechanism was preserved for the W^6.48 variants. However, stimulation with 1 µM was not sufficient to induce WT-like netBRET of W^6.48 mutants with Gα_Δ6qi4myr-Venus (compare with EC₅₀ values of activation in Table 5). 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.

	Gα_i1-Venus					Gα_Δ6qi4myr-Venus
	Unstimulated		Stimulated 1 µM NPY			Unstimulated		Stimulated 1 µM NPY
	BRET₅₀	Max. BRET	BRET₅₀	Max. BRET	netBRET	BRET₅₀	Max. BRET	BRET₅₀	Max. BRET	netBRET
WT Y₂R	0.007 (0.005–0.009)	0.189 (0.171–0.206)	0.009 (0.007–0.012)	0.245 (0.220–0.271)	0.046 (0.038–0.0587)	0.012 (0–0.028)	0.081 (0.064–0.098)	0.010 (0.006–0.014)	0.122 (0.107–0.136)	0.034 (0.027–0.041)
W^6.48Y	0.007 (0–0.014)	0.156 (0.119–0.192)	0.010 (0.001–0.019)	0.194 (0.160–0.230)	0.039 (0.008–0.069)ns	0.012 (0–0.029)	0.126 (0.091–0.161)	0.008 (0–0.019)	0.138 (0.106–0.169)	0.014 (0.003–0.025)^**
W^6.48H	0.008 (0.004–0.012)	0.206 (0.175–0.237)	0.010 (0.006–0.015)	0.257 (0.222–0.292)	0.042 (0.034–0.050)ns	0.007 (0–0.017)	0.099 (0.068–0.130)	0.009 (0–0.023)	0.101 (0.064–0.139)	0.003 (−0.015 to 0.022)^***
W^6.48T	0.007 (0.003–0.010)	0.170 (0.140–0.200)	0.008 (0.004–0.012)	0.226 (0.188–0.264)	0.040 (0.023–0.056)ns	0.006 (0–0.013)	0.071 (0.048–0.094)	0.007 (0–0.015)	0.094 (0.062–0.126)	0.020 (0.002–0.038)^*

Max., maximum.
↵* P < 0.05;
↵** P < 0.01;
↵*** P < 0.001; ns, not significant.