Nanoscale organization of β2-adrenergic receptor-Venus fusion protein domains on the surface of mammalian cells

Abstract

a b s t r a c tAdrenergic 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 b 2 -adrenergic receptors (b 2 AR) fused to the GFP analogue Venus at the nanoscale on HEK293 cells. The expression of the b 2 AR-Venus fusion protein was tightly controlled using a tetracycline-induced promoter. Both the size and density of the observed nanoscale domains are dependent 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 b 2 AR-Venus fusion protein appears to remain roughly constant, but the number of domains per cell increase. At 700 ng/ml doxycycline 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 b 2 AR are observed in the membrane of the HEK293 cells that stably overexpress b 2 AR-GFP and b 2 AR-eYFP. We conclude that precise chemical control of gene expression is highly advantageous for the use b 2 AR-Venus fusion proteins as models for b 2 AR function. These observations are critical for designing future cell models and assays based on b 2 AR, since the receptor biology is consistent with a relatively low density of nanoscale receptor domains.Ó 2009 Elsevier Inc. All rights reserved. IntroductionThe beating rate in the mammalian heart is strongly influenced by the binding of catecholamines to b-adrenergic G-protein coupled receptors (bARs) in cardiac myocytes, initiating an adrenergic response [1][2][3]. The signaling cascade initiated by catecholamine binding requires the association of bARs into multiprotein complexes called signalosomes [4] that include the a, b and c G-protein subunits. Changes in the molecular composition of signalosomes are associated with receptor desensitization, sequestration of the receptor to subcellular membranous compartments and internalization, all of which strongly influence bAR function [5][6][7][8][9][10][11]. Signaling has also been shown to depend on differential interactions with scaffolding proteins in signaling complexes [12]. Recently we have used near field scanning optical microscopy (NSOM) to demonstrate that functional receptors are organized into multi-protein domains of $140 nm average diameter on murine neonatal and embryonic cardiac myocytes [13]. Colocalization experiments in these primary cells at the nanometer scale show that 15-20% of receptors are pre-associated in caveolae, an important component of signalosomes [13]. bAR signaling represents an important pharmacological target [14] and many assays have been developed to aid in the drug discovery process [15][16][17][18]. Herein we investigate the conditions required for mimicking the nanoscale distributions and local environment observed in primary cel...

Cell Type-specific β2-Adrenergic Receptor Clusters Identified Using Photoactivated Localization Microscopy Are Not Lipid Raft Related, but Depend on Actin Cytoskeleton Integrity

“…S1. PALM images yielded a number of ␤2 cluster features in the ϳ0.1-0.2/ m 2 range, which compares with ϳ1-1.4/m 2 from previous NSOM data (7,15). The presence of the endogenous ␤2 receptor in H9C2 cells in the clustered fraction, undetected by PALM, can lead to an underestimation in our data of the real clustering fraction.…”

“…Because our goal was to compare ␤2 receptor clustering in different cell types, we also considered cardiomyocytes, such as H9C2 cells, derived from the embryonic rat heart, where the physiological role of the ␤2 receptor is well documented (31,32). In addition, in this cell line, a very high degree of receptor clustering was reported by means of NSOM measurements (7,15). When comparing the results from PALM with those from NSOM, the following considerations are taken into account.…”

“…Atomic force microcopy is particularly suited for highly packed receptors in native cell membranes, such as rhodopsin and opsin, whereas its resolution can be severely affected in sparser samples. Another report, focusing on ␤1-and ␤2-adrenergic receptors in rat cardiac myocytes and HEK293 cells, used near-field scanning optical microscopy (NSOM) to show that the vast majority of receptors, detected via fluorescent antibodies, are imaged as clusters (7,15). However, it is still technically challenging for NSOM to generate high spatial resolution images of plasma membrane proteins.…”

Fluorescence Imaging on the Nanoscale: Bioimaging Using Near-field Scanning Optical Microscopy

“…These constraints have been addressed by expressing a fluorescent protein fusion (Venus-  AR) in HEK293 cells at variable expression levels. 37 The results showed that the number of small nanometer-sized clusters increased by a factor of ~3 with increased Venus-  AR expression level, but that their size was roughly constant with an average diameter of ~150 nm and with a   AR distribution in small nanometer-sized clusters. A second detailed quantitative study of membrane protein clustering visualized the distribution of a C-type lectin, DC-SIGN, that is involved in pathogen recognition for several viruses and other microorganisms.…”

Labeling and Single-Molecule Methods To Monitor G Protein-Coupled Receptor Dynamics

“…It was found that these receptors were organized in nanodomains with a diameter of ∼150 nm and did not reorganize upon agonist binding. 157,582 NSOM is nonetheless particularly difficult to implement in living cells. More recently, SMLM was used to re-evaluate the molecular density of the β 2 AR in cardiomyocytes, HeLa cells, and CHO cells.…”