The N- and C- termini of the odorant receptor M71 impair trafficking.

<p>(A and B) The replacement of either the Ct or the Nt of mβ2AR with the one of M71 does not block proper trafficking of the protein inside the cell (both 10/10 cells) and to filopodia (arrow head in magnified pictures). (C) On the contrary the double swaps of both the Nt and Ct of mβ2AR with the Nt and Ct of M71 blocks proper trafficking of the protein, which accumulates in the periphery of the nucleus (9/10 cells) and does not locate to filopodia (magnified pictures). (D) This is not observed for a mutant with a shorter Nt and the Ct of M71 (10/10 cells) and the protein successfully traffics to the filopodia (arrow head in 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). M71-Nt/mβ2AR labels 32.1 ±24.2 ^n.s. filopodia per cell, mβ2AR/M71-Ct 10.3 ±5.2 ^n.s., M71-Nt/mβ2AR/M71-Ct 1.6 ±5.1*** and NtΔ9AA/mβ2AR/M71-Ct 13.2 ±11.3 ^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) Top pair is the alignment between the Nt of M71 and the truncated version of the Nt of mβ2AR (NtΔ9AA) and on the bottom pair is the alignment between the Ct of M71 and the truncated version of the Ct of mβ2AR (ΔCt➔Y350). Conserved residues are in violet and sequences present in the chimera are in the red boxes. (H) The chimera M71-Nt/mβ2AR::GFP shows normal localization in cells and localize to filopodia (arrow head in magnified picture) when co-expressed with mβ2AR/M71-Ct::mCherry. (I) mβ2AR/M71-Ct::GFP also locates as the WT in cell including to filopodia (arrow head in magnified pictures) when co-expressed with M71-Nt/mβ2AR::mCherry. (J) 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). M71-Nt/mβ2AR::GFP labels 32.1 ±24.2 ^n.s. filopodia per cell and 14.8 ±7.3 ^n.s. when co-expressed with mβ2AR/M71-Ct::mCherry. mβ2AR/M71-Ct::GFP labels 10.3 ±5.2 ^n.s. filopodia per cell and 10.7 ±9.7 ^n.s. when co-expressed with M71-Nt/mβ2AR::mCherry. M71-Nt/mβ2AR/M71-Ct::GFP labels only 1.6 ±5.1*** filopodia per cell and 2.2 ±2.6*** when co-expressed with mβ2AR/M71-Ct::mCherry.