Fig. 1. Membrane topology of receptor truncations and variants. (a) Membrane topology of the β₂AR protein sequence with numbers and pins indicating the location of truncations for the 14 constructs used in this experimental series. Numbers and pins in red indicate the mutations focused on for further study as highlighted in Fig. 3 and Fig. 4. (b) Membrane topology of a Cx26 subunit. The four transmembrane domains (M1–M4) are denoted with gray boxes. Areas that were mutated are the N-terminus (red), extracellular loop 1 (yellow), the intracellular M2–M3 loop (green), extracellular loop 2 (blue) and the C-terminus (magenta).

Fig. 2. Example of β₂AR cell surface expression. (a) Histogram of cell count (events) vs. mean fluorescence intensity (MFI). For cases in which the expressing cells are not clearly delineated close to the limit of detection of 10 MFI, the final reported MFI is derived from the mean fluorescence of the highest 70% of the total cell population (top panel, expressing cell population framed by solid line). For cases in which a clearly expressing population of cells is detectable (bottom panel, expressing cell population framed by solid line), the reported MFI is derived from all cells in the expressing population, which is frequently much greater than 70%. (b) Cell surface expression of β₂AR arranged from the longest construct (1–408) to the shortest (1–343). Both MFI and %CV are calculated from two protocols described in (a). The latter protocol was preferred and utilized when possible. In general, as the MFI decreases the %CV increases indicating a progressive loss of a clearly expressing cell population.

Fig. 3. Saturation ligand binding data for β₂AR. (a) [³H]DHA isothermal binding curves to a selected set of C-terminal β₂AR truncation variants compared to wild-type. The data represent five repetitions of three assay replicates. (b) Protein immunoblot of isolated Sf9 cell membranes.

Fig. 4. Expression comparison of β₂AR truncations relative to wild-type. Relative expression levels of three selected truncation variants of β₂AR relative to wild-type as represented by the maximal receptor expression levels (B_max) of the crude membrane fraction (striped), cell surface fluorescence (gray) and by protein immunoblot analysis (dotted).

Fig. 5. Cx26 parallel expression in Sf9 cells using the Vertiga-IM. Estimated relative expression levels of expressed variants on a relative scale of 1–10, as determined by protein immunoblot analysis. The associated regions of the protein as described in Fig. 1b are indicated. Full definition of the constructs are described in Supplementary Fig. 1.

B_max and K_d for β₂AR wild-type and truncations

Correlation table for relative expression levels