Colocalization of β₂AR::XFP and transferrin after isoprenaline exposure.

<p>Single OP 6 cells expressing each β₂AR::XFP construct were incubated with 20µg/ml of Alexa Fluor transferrin 647 (A-E) or Alexa Fluor transferrin 488 (F, G) for 30 minutes and then exposed to 10µM isoprenaline for 20 minutes. β₂AR::XFP is diffusely expressed at the plasma membrane, and transferrin is localized in the endosomes (A-G, 0) before isoprenaline exposure. After 20 minutes, β₂AR::XFP is internalized and becomes punctate, colocalizing with transferrin (arrowheads). ICQ values for all fusions reflect a significant increase (P<0.0001) in dependency after exposure to isoprenaline. ICQ values for 0 minute and 20 minute isoprenaline stimulation: (A) 0.086, 0.186 (B) 0.046, 0.187 (C) 0.116, 0.227 (D) -0.006, 0.212 (E) 0.132, 0.319 (F) 0.013, 0.254 (G) 0.047, 0.133.</p

Filopodia count in OP 6 cells transiently expressing untagged fluorescent proteins, gap::XFPs, or β₂AR::XFPs.

<p>Filopodia on N=10 cells were counted for cells expressing each untagged fluorescent protein, tagged with gap, or fused to the β₂AR. gap::XFPs featured 78-141 filopodia per 10 cells, and β₂AR::XFPs featured 97-167 filopodia per 10 cells. Cells expressing the untagged fluorescent proteins featured between 7-16 filopodia. Expression in filopodia of gap::XFPs and β₂AR::XFPs was significantly different from the untagged XFPs, Fisher’s exact test P<0.0001.</p

Dose response curves for β₂AR::XFPs.

<p>Cells expressing each β₂AR::XFP and human Gα15 were exposed to concentrations of isoprenaline and analyzed using the FLIPR assay. Normalized curves (A) show an EC50 between 2.6-5.2 x 10^-9 for all fusions. Unnormalized curves (B) show a difference in maximum RFUs for individual fluorophore fusions. Transient expression of β₂AR::GFP truncated reveals no membrane expression (C) whereas β₂AR::GFP human localizes to filopodia (D, arrowhead).</p

Visualization of OP 6 cells transiently expressing individual β₂AR::XFPs and mixed.

<p>Mixed OP 6 cells expressing [β₂AR::Cerulean, β₂AR::Venus, β₂AR::mCherry] in (A), [β₂AR::Teal, β₂AR::Venus, β₂AR::mCherry] in (B) and [β₂AR::GFP, β₂AR::mCherry, β₂AR::AFP] in (C). Each β₂AR::XFP fusion was solely identified in its respective channel (Ai, Cerulean, Bi, Venus, Ci, mCherry), (Aii, Teal, Bii, Venus, Cii, mCherry), (Aiii, GFP, Biii, mCherry, Ciii, AFP).</p

M71::GFP directed mutations that do not traffic to plasma membrane.

<p>(A) M71::GFP mutations F12D;I13H;L14D;G15V;G16T (FILGG to DHDGG), Y35A, P58V;M59T;Y60N (PMY to VTN), M98E, C169A, Y176T;F177C (FY to TC), C178A and Y217A. None of these mutations gave raise to any GFP-labeled filopodia. (B) M71::GFP mutations with N-linked glycosylation modifications: 4x NQS, 4x NSS, NQS to NGT, NQS to NAT, Nt to K4 Nt. (C) Addition of leader sequences (Kirrel2, 5HT3, Calcumenin, Rhodopsin, Endothelin, LUCY and LUCY-FLAG) to M71::GFP did not relocate GFP expression into the filopodia. (A-C) Co-expression with RTP1S for all M71::GFP mutations did not increase the presence of any GFP-labeled filopodia.</p

Conserved OR sequences revealed by logo plot shared with M71 and mβ2AR.

<p>(A) OR logo using mouse Class I and II odorant receptor sequences. Most highly conserved OR residues are depicted with black circles. M71 and mβ2AR sequences are delineated underneath logo plot; Residues with greater than 50% bit conservation to OR logo are underlined. Conserved residues between M71 and mβ2AR are in bold and red. M71 residue swaps between M71 and mβ2AR are delineated in purple and green, respectively. M71 residues converted to alanine are described with bold blue A. Five conserved methionine residues, three conserved histidine residues and one weakly conserved tryptophan residue marked with asterisk. Two of these residues are also found in OSN expressed TAARs marked with red asterisk. (B) Schematic of OR seven transmembrane structure with conserved methionine and histidine residues depicted in linear and predicted 3 dimensional state. Methionine and histidine residues in non-transmembrane might be in close enough proximity to coordinate a metal group, like copper. Methionine residues in TM3 and TM7 may form bridge if modified.</p

ORs and TAARs have greater hydrophobic character than Adrenergic and Amine receptors.

<p>The percent difference in ORs and TAARs compared to Amine receptors was multiplied by the hydropathy index (HI) of each residue. Positive percent differences x positive HI values and negative percent differences x negative HI values increase the hydrophobic character to ORs. Many residues had increased the hydrophobic character or no effect. Histidine showed the most significant difference overall and in relation to TAARs. Amino acids are ordered according to their hydropathy index from 4.5 to -4.5 (L to R). (Inset) The positive and negative hydrophobic character of Adrenergic, Amine, ORs and TAARs wre calculated by adding together all residues that contribute to non-polar/neutral or polar/positive-negative hydropathy index and calculating the percentage relative to the total residues. There was a greater percentage of positive hydropathy and reduced negative hydropathy values for ORs and TAARs compared to Adrenergic and Amine receptors. This translated into higher ratios of polar/neutral to polar/positive-negative residues.</p

Expression of GFP and GPCR::GFP in OP6 cells.

<p>(A) Cytosolic GFP shows diffuse fluorescence in the cytoplasm of an OP6 cell and does not co-localize with the plasma membrane staining CellMask Deep Red in filopodia (A’, arrow head in magnified image). (B) On the contrary, gap::GFP, shows a sharp edge staining of a whole OP6 cell and co-localize with the plasma membrane staining CellMask Deep Red in filopodia (B’, arrow head in magnified image). (C) The M71 coding sequence missing a stop codon (pink box) was cloned followed by a linker sequence (yellow box) and the coding sequence of GFP (green box). The peGFP-N1 backbone includes the cytomegalovirus promoter (CMV, arrow), a 5’ untranslated region (5’UTR, blue box) and a polyadenylation sequence (pA, black box). The resulting 2D structure of the fusion protein is showed, the M71 odorant receptor has a putative 7 transmembrane domains structure and is glycosylated at its N-terminus (pink diagram). The GFP protein, composed of eleven β-barrels, is fused to the Ct of M71 (green diagram) after a 9 amino acids linker (in yellow). M71::GFP shows intense perinuclear localization in an OP6 cell (see picture) and does not localize to filopodia. (D) Using the same strategy the mβ2AR sequence (teal box) was cloned in the same backbone. mβ2AR has also 7 transmembrane domains but 2 N-linked glycosylation sites located in its Nt (see 2D topology). When express in OP6 cells (see picture) mβ2AR::GFP stains homogeneously the whole cell and locates in filopodia (arrow head in magnified image), like gap::GFP. (E) Cells expressing GFP and M71::GFP have very low number of GFP-labeled filopodia (gray bars, 2.8 ±2.9 and 0.0 ±0.0 GFP-labeled filopodia per cell respectively), whereas cells expressing gap::GFP and mβ2AR::GFP show the same numerous number of filopodia. In addition many cells expressing GFP and M71::GFP show ≤1 GFP-labeled filopodium whereas all cells expressing gap::GFP or mβ2AR::GFP have more filopodia (blue diamonds, 2^nd y axis in graph). (F) Counts of GFP- and CellMask Deep Red-labeled filopodia for cells expressing GFP or gap::GFP. The number of filopodia revealed by plasma membrane staining is the same for both type of cells (44.7 ±15.9 and 33.6 ±9.9 respectively), only in cells expressing gap::GFP-filopodia are also GFP-labeled (orange bars in graph, 2.6 ±3.2 and 33.2 ±9.8 co-labeled filopodia per cell respectively). All filopodia counts are represented as average for 10 cells ± standard deviation. ***means significantly different from mβ2AR::GFP, one-way ANOVA followed by Scheffe tests, p<0.001. n.s. means not significantly different from mβ2AR::GFP, p>0.001.</p

M71::GFP chimeras and truncations that do not traffic to plasma membrane.

<p>(A) M71::GFP chimeras with Nt-mβ2AR, Ct-mβ2AR, Nt-and Ct- mβ2AR. None of these chimeras led to GFP-labeled filopodia. (B) Altered NPxxY motif, Y289A M71::GFP mutants, with altered Nt and Ct: WT, ΔCt, Nt-mβ2AR, Nt-mβ2AR/ΔCt, and Nt- and Ct-mβ2AR. None of these chimeras led to GFP labeled filopodia. (A and B) Co-expression with RTP1S for all M71::GFP mutations did not increase the presence of any GFP-labeled filopodia.</p

Amino acid residue composition of Adrenergic, Amine, Odorant and Trace amine-associated receptors.

<p>(A) Amino acid percentages for Adrenergic, Amine, Odorant (ORs) and Trace amine-associated Receptors (TAARs). ORs and TAARs profiles differ from Adrenergic and Amine receptors. (B) Amino acid differences for ORs and TAARs expressed as percentage difference from Amine receptors. Many residues show an increase in proportion (positive percentage) and decrease in proportion (negative percentage). Three residues for ORs show a large, 60% difference (methionine-M, histidine-H and tryptophan-W). These three residues also show large differences from TAARs with methionine and histidine comprising the bulk of the statistical difference between them. Results did not change if TAARs and Adrenergic receptors were removed from the Amine receptor superfamily. Amino acids are ordered according to their hydropathy index from 4.5 to -4.5 (L to R). It appears that most of the residue differences favor ORs and TAARs to be more hydrophobic.</p