Human ADRB2-SNAP Stable Cell Line-HEK293

The concept that agonist-independent signaling can be inhibited by inverse agonists is a fundamental element of the "cubic ternary complex model" of G protein-coupled receptor (GPCR) activation. This model reveals that, in the absence of ligands, GPCRs coexist in two conformational states-the inactive R state and the active R* state-which exhibit distinct affinities for agonists, inverse agonists, and G protein α subunits. In the absence of agonists, the proportion of receptors in the R* state determines the level of constitutive receptor activity. This study demonstrates that mechanical stimulation induces agonist-independent, Gs protein-mediated cAMP signaling by the β2-adrenergic receptor (β2AR), a process that can be inhibited by inverse agonists such as ICI-118551 and propranolol. The magnitude of this mechanosensitive response depends on the surface expression level of the receptor in HEK293G cells; the response is observed even in the ligand-binding-deficient β2AR mutant (D113A); furthermore, the response is attenuated by site-directed mutagenesis of extracellular N-glycosylation sites located on the N-terminus and the second extracellular loop of the β2AR. A similar mechanosensitive, agonist-independent response was also observed in HEK293G cells overexpressing the A2A adenosine receptor. These data provide new insights into how mechanical stimulation enhances agonist-independent constitutive receptor activity and how inverse agonists modulate this process.

Figure 1 illustrates the time course of GloSensor^TM luminescence responses elicited by increasing concentrations of isoproterenol (Figure 1a) and salbutamol (Figure 1b) in a HEK293G clonal cell line overexpressing the human β2-adrenergic receptor (tagged with an N-terminal Twin-Strep affinity tag and an N-terminal SNAP tag; TS-SNAP-β2AR). Peak responses were observed within 5 to 10 minutes of agonist addition, followed by a gradual decline to basal levels over the course of 60 minutes. Analysis of the concentration-response characteristics for four different β2-agonists (Figure 1c) revealed EC50 values ​​significantly lower than those previously measured for endogenous β2AR expression in HEK293G cells; this is consistent with the substantial increase in "spare receptor reserve" expected in this recombinant cell line. For instance, the EC50 value for isoproterenol in TS-SNAP-β2AR-expressing cells was two orders of magnitude lower than that measured in the parental HEK293G cell line. Correspondingly, the relative maximal responses (normalized to the full agonist isoproterenol) obtained with lower-efficacy agonists (salmeterol and salbutamol) were also markedly higher than those observed in cells expressing endogenous β2AR (Figure 1c). However, a notable feature of the kinetic profiles shown in Figure 1a and 1b is the substantial basal response to the vehicle (HBSS), which peaked at 4 to 5 minutes. Across the five experiments shown in Figure 1a, the peak basal (HBSS) response amounted to 26.9 ± 2.9% (n = 5) of the maximal response elicited by 1 nM isoproterenol in the same experiment.

Figure 1. Basal and agonist-induced GloSensor^TM luminescence responses in HEK293G cells expressing TS-SNAP-β2ARs or endogenous β2ARs. (Cullum S A, et al., 2024)