Nanoscale organization of b2-adrenergic receptor-Venus fusion protein domains on the surface of mammalian cells

Adrenergic receptors are a key component of nanoscale multiprotein complexes that are responsible for controlling the beat rate in a mammalian heart. We demonstrate the ability of near-field scanning optical microscopy (NSOM) to visualize b2-adrenergic receptors (b2AR) fused to the GFP analogue Venus at the nanoscale on HEK293 cells. The expression of the b2AR-Venus fusion protein was tightly controlled using a tetracycline-induced promoter. Both the size and density of the observed nanoscale domains are depen- dent on the level of induction and thus the level of protein expression. At concentrations between 100 and 700 ng/ml of inducer doxycycline, the size of domains containing the b2AR-Venus fusion protein appears to remain roughly constant, but the number of domains per cell increase. At 700 ng/ml doxycy- cline the functional receptors are organized into domains with an average diameter of 150 nm with a density similar to that observed for the native protein on primary murine cells. By contrast, larger micron-sized domains of b2AR are observed in the membrane of the HEK293 cells that stably overexpress b2AR-GFP and b2AR-eYFP. We conclude that precise chemical control of gene expression is highly advan- tageous for the use b2AR-Venus fusion proteins as models for b2AR function. These observations are crit- ical for designing future cell models and assays based on b2AR, since the receptor biology is consistent with a relatively low density of nanoscale receptor domains.