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

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

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

Detailed time course analysis of β₂AR::Teal and β₂AR::Venus.

<p>ICQ values were used to measure colocalization events. (A) Single OP 6 cells (n=2) expressing the β₂AR::Teal or β₂AR::Venus construct were incubated with 20µg/ml of Alexa Fluor transferrin 647 for 30 minutes and then exposed to 10µM isoprenaline and imaged live every 4 minutes until 20 minutes. (B) Single OP 6 cells (n=3,4) expressing the β₂AR::Teal or β₂AR::Venus construct and myc-tagged β-Arrestin2 were fixed after 24 hours and incubated with rabbit polyclonal antibody for Myc after exposure to 10µM isoprenaline in 4 minute intervals between 0 and 20 minutes and were visualized with Anti-rabbit Alexa Fluor 546 antibody. Fusions of Teal and Venus to the β₂AR follow the same time course for internalization into the early endosomes (A) and the same time course regulation by β-Arrestin2 (B). Error bars represent standard error.</p

OP 6 cells transiently expressing gap::XFPs and β₂AR::XFPs.

<p>Single cells expressing each gap tagged fluorescent protein localizes to the plasma membrane (A-G). gap::XFPs also target to the filopodia (arrowheads, Ai-Gi magnified images). Single cells expressing each β₂AR fusion (β₂AR::XFP) (H-N) exhibit diffuse protein expression at the plasma membrane defined by localization at the filopodia (arrowheads, Hi-Ni magnified images). The ability of β₂AR to traffic to the plasma membrane is unaffected by fusion to the seven tested fluorescent proteins.</p