A new loss-of-function mutants for activity which retains trafficking.

<p>(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 <sup>n.s.</sup>, NtΔ9AA/Y326A/ΔCt➔R328 14.7 ±9.5 <sup>n.s.</sup>, no NxS / Y326A/ΔCt➔R328 4.2 ±4.0***, M71 Nt / Y326A/ΔCt➔R328 0.5 ±1.1*** and Y326A 14.9 ±7.3 <sup>n.s.</sup>. ***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 7<sup>th</sup> and last transmembrane domain and the Ct of the protein showing the localization of the NPxxY region as well as R328.</p>