A new loss-of-function mutants for activity which retains trafficking.
(A) A complete deletion of the Ct of mβ2AR to R328 blocks proper trafficking of the protein, which appears to be retained in the periphery of the nucleus (ΔCt➔R328, 9/10 cells) and does not traffic to filopodia (magnified pictures). (B) The mutation Y326A rescues trafficking of the protein in the cell (Y326A/ΔCt➔R328, 10/10 cells) and to filopodia (arrow head in magnified pictures). (C and D) This rescue is not observed anymore when the 2 N-linked glycosylation sites located in the Nt are invalidated (no NxS / Y326A/ΔCt➔R328) or when the Nt is replaced by the one of M71 (M71 Nt / Y326A/ΔCt➔R328) (respectively 3/10 cells and 8/10 cells). In both cases the protein fails to traffic to filopodia (magnified pictures). (E) Dose response curves to isoprenaline obtained by FLIPR analysis are showed as normalized relative fluorescent units (RFUs) as a function of Log [isoprenaline] (M). The same color code is used as in F. (F) The average number of GFP-labeled filopodia per cell is showed (bars in graph, mean +s.d.) as well as the number of cells with ≤1 GFP-labeled filopodium (blue diamonds). ΔCt➔R328 labels only 0.5 ±1.6*** filopodia per cell, whereas Y326A/ΔCt➔R328 26.4 ±15.2 n.s., NtΔ9AA/Y326A/ΔCt➔R328 14.7 ±9.5 n.s., no NxS / Y326A/ΔCt➔R328 4.2 ±4.0***, M71 Nt / Y326A/ΔCt➔R328 0.5 ±1.1*** and Y326A 14.9 ±7.3 n.s.. ***means significantly different from WT, one-way ANOVA 6 degrees of freedom followed by Scheffe tests, p<0.001. n.s. means not significantly different from WT, p>0.001. EC50 (nM) and maximum response (expressed as a % of mβ2AR::GFP’s maximum response) for isoprenaline are indicated in the table. (G) Snake plot of the 7th and last transmembrane domain and the Ct of the protein showing the localization of the NPxxY region as well as R328.