Özlem Uğur

Cell Contact-dependent Functional Selectivity of β2-Adrenergic Receptor Ligands in Stimulating cAMP Accumulation and Extracellular Signal-regulated Kinase Phosphorylation

Background: β2-Adrenegic receptor (β2-AR) mediates cAMP accumulation and ERK phosphorylation via different transducers. Results: Some β2-AR ligands selectively activate cAMP and ERK responses in HEK-293 cells. Selectivity is cell contact-dependent. Conclusion: β2-AR stimulates ERK phosphorylation by cAMP-dependent and Gi/Go-mediated pathways. Cell contact inhibits the cAMP-dependent pathway. Significance: Cell adherence/contact is an important modulatory factor in biased signaling by GPCR ligands. Activation of β2-adrenegic receptor (β2-AR) leads to an increase in intracellular cAMP and activation of ERK. These two signals are activated by the interaction of the receptor with different transducer partners. We showed that the intrinsic activities of β2-AR ligands for stimulating cAMP production and ERK phosphorylation responses in HEK-293 cells were not correlated. The lack of correlation resulted mainly from the discrepancy between the intrinsic activities of two groups of ligands for these two responses: The first group consisted of clenbuterol, cimaterol, procaterol, and terbutaline which acted as full agonists for cAMP production but displayed very weak effect on ERK phosphorylation. The second group comprised adrenaline and noradrenaline which displayed higher intrinsic activity for the ERK phosphorylation than for the cAMP response. Thus, both groups behaved as functionally selective ligands. The functional selectivity of the first group was observable only in adherent cells when confluence was approximately 100%. When cell-cell contact was minimized either by decreasing the density of the adherent cells or by bringing the cells into suspension, the first group of ligands gained the ability to stimulate ERK phosphorylation without a change in their effect on cAMP production. In contrast, selectivity of the second group was independent of the adherence state of the cells. Our results show that the inherent “bias” of ligands in coupling a G protein-coupled receptor to different transducers may not always be revealed as functional selectivity when there is a “cross-talk” between the signaling pathways activated by the same receptor.

Exploring allosteric coupling in the α-subunit of Heterotrimeric G proteins using evolutionary and ensemble-based approaches

BackgroundAllosteric coupling, which can be defined as propagation of a perturbation at one region of the protein molecule (such as ligand binding) to distant sites in the same molecule, constitutes the most general mechanism of regulation of protein function. However, unlike molecular details of ligand binding, structural elements involved in allosteric effects are difficult to diagnose. Here, we identified allosteric linkages in the α-subunits of heterotrimeric G proteins, which were evolved to transmit membrane receptor signals by allosteric mechanisms, by using two different approaches that utilize fundamentally different and independent information.ResultsWe analyzed: 1) correlated mutations in the family of G protein α-subunits, and 2) cooperativity of the native state ensemble of the Gαi1 or transducin. The combination of these approaches not only recovered already-known details such as the switch regions that change conformation upon nucleotide exchange, and those regions that are involved in receptor, effector or Gβγ interactions (indicating that the predictions of the analyses can be viewed with a measure of confidence), but also predicted new sites that are potentially involved in allosteric communication in the Gα protein. A summary of the new sites found in the present analysis, which were not apparent in crystallographic data, is given along with known functional and structural information. Implications of the results are discussed.ConclusionA set of residues and/or structural elements that are potentially involved in allosteric communication in Gα is presented. This information can be used as a guide to structural, spectroscopic, mutational, and theoretical studies on the allosteric network in Gα proteins, which will provide a better understanding of G protein-mediated signal transduction.

Systematic errors in detecting biased agonism: Analysis of current methods and development of a new model-free approach

Discovering biased agonists requires a method that can reliably distinguish the bias in signalling due to unbalanced activation of diverse transduction proteins from that of differential amplification inherent to the system being studied, which invariably results from the non-linear nature of biological signalling networks and their measurement. We have systematically compared the performance of seven methods of bias diagnostics, all of which are based on the analysis of concentration-response curves of ligands according to classical receptor theory. We computed bias factors for a number of β-adrenergic agonists by comparing BRET assays of receptor-transducer interactions with Gs, Gi and arrestin. Using the same ligands, we also compared responses at signalling steps originated from the same receptor-transducer interaction, among which no biased efficacy is theoretically possible. In either case, we found a high level of false positive results and a general lack of correlation among methods. Altogether this analysis shows that all tested methods, including some of the most widely used in the literature, fail to distinguish true ligand bias from “system bias” with confidence. We also propose two novel semi quantitative methods of bias diagnostics that appear to be more robust and reliable than currently available strategies.

Long and short distance movements of β2-adrenoceptor in cell membrane assessed by photoconvertible fluorescent protein dendra2–β2-adrenoceptor fusion

Local movements of receptors in the plasma membrane have been extensively studied, as it is generally believed that the dynamics of membrane distribution of receptors regulate their functions. However, the properties of large-scale (>5μm) receptor movements in the membrane are relatively obscure. In the present study, we addressed the question as to whether the large-scale movement of receptor in the plasma membrane at the whole cell level can be explained quantitatively by its local diffusive properties. We used HEK 293 cells transfected with human β2-adrenoceptor fused to photoconvertible fluorescent protein dendra2 as a model system; and found that 1) functional integrity of the dendra2-tagged receptor remains apparently intact; 2) in a mesoscopic scale (~4μm), ~90% of the receptors are mobile on average, and receptor influx to, and out-flux from a membrane area can be symmetrically explained by a diffusion-like process with an effective diffusion coefficient of ~0.1μm(2)/s; 3) these mobility parameters are not affected by the activity state of the receptor (assessed by using constitutively active receptor mutants); 4) in the macroscopic scale (4-40μm), although a slowly diffusing fraction of receptors (with D<0.01μm(2)/s) is identifiable in some cases, the movement of the predominant fraction is perfectly explained by the same effective diffusion process observed in the mesoscopic scale, suggesting that the large scale structure of the cell membrane as felt by the receptor is apparently homogeneous in terms of its mesoscopic properties. We also showed that intracellular compartments and plasma membrane are kinetically connected even at steady-state.

Β2-Adrenoceptor, Gs and adenylate cyclase coupling in purified detergent-resistant, low density membrane fractions

Membrane rafts and caveolae are specialized microdomains of the cell membrane that form physical platforms for compartmentalization of signalling molecules. Here, we intended to gain insight into the consequences of caveolar localization in G protein-coupled receptor function. We analysed beta(2)-adrenoceptor signalling in purified CRLDF (caveolin-rich low density fractions) of beta(2)-adrenoceptor-overexpressing HEK-293 cells. beta(2)-adrenoceptor and Gs immunoreactivities and forskolin-stimulated adenylate cyclase activity were all detected in CRLDF obtained by the conventional raft purification method that uses Triton X-100 solubilization. However, Triton X-100 caused a complete loss of the functional coupling between beta(2)-adrenoceptor, Gs and adenylate cyclase. Therefore, we developed an optimized purification method based on n-octyl-beta-d-glucopyranoside solubilization, where the functional properties of beta(2)-adrenoceptor, Gs and adenylate cyclase were preserved in the CRLDF. Using this method, we showed that isoproterenol-stimulated adenylate cyclase activity was similar in CRLDF and bulk membrane preparations of HEK-293 cells that overexpress beta(2)-adrenoceptor or beta(2)-adrenoceptor-Gs fusion. Accordingly, treatment of cells with methyl-beta-cyclodextrin, a caveola-disrupting agent, did not affect beta(2)-adrenoceptor-induced cAMP response. Likewise, these responses were insensitive to caveolin 1 and 2 overexpression. On the other hand, methyl-beta-cyclodextrin treatment did decrease beta(2)-adrenoceptor-induced ERK phosphorylation. However, the latter effect of methyl-beta-cyclodextrin could be attributed to a non-specific effect rather than its ability to disrupt membrane microdomains. We showed that localization in the raft microdomains did not affect the signalling efficiency of beta(2)-adrenoceptor-Gs-adenylate cyclase pathway, and that methyl-beta-cyclodextrin may inhibit signalling by directly affecting the signalling system independently of its caveola-disrupting property.

Ca2+-Induced Inhibition of Adenylyl Cyclase in Turkey Erythrocyte Membranes

We investigated the effects of calcium ions (Ca2+) on the adenylyl cyclase activity in purified turkey erythrocyte membranes. Results showed the following: (i) Ca2+ inhibits cAMP accumulation stimulated by isoproterenol (1 µmol/l), NaF + AlCl3 (10 mmol/l + 20 µmol/l) or forskolin (10 µmol/l) in EGTA-washed turkey erythrocyte membranes. IC50 of free [Ca2+] is approximately 0.1 mmol/l in the presence of Mg2+ (2.5 mmol/l) and isobutylmethylxanthine (1 mmol/l). (ii) The potency of Ca2+ to inhibit cAMP accumulation is independent of the type of stimulus used to activate the adenylyl cyclase. We also evaluated the calcium sensitivity of the basal cAMP accumulation in the presence of GTP (10 µmol/l) and Mg2+ (2.5 mmol/l) which was also inhibited by Ca2+ with the same potency. (iii) The inhibition pattern of cAMP accumulation is not affected by the presence of added calmodulin (100 nmol/l). (iv) Ca2+ is ineffective on the binding of isoproterenol to the β-adrenoceptors. (v) Increasing the concentration of Ca2+ does not induce an observable activation of cyclic nucleotide phosphodiesterase in the present experimental conditions. Thus, we concluded that the inhibition of cAMP accumulation is due to an inhibition of the adenylyl cyclase rather than the activation of phosphodiesterase(s). The presence of a yet unidentified isoform of adenylyl cyclase that can be directly inhibited by Ca2+ or a Gi protein that can be activated by Ca2+ seems to explain these results. In either case, these results provide an additional mode of cross-talk that can take place between the Ca2+- and cAMP-signaling systems.

Cell adhesion modulates 5-HT1D and P2Y receptor signal trafficking differentially in LTK-8 cells

In this study, we investigated adhesion-induced changes in cellular responses to serotonin 5-HT(1D) and purinergic P2Y receptor stimulation. We demonstrated that detachment of LTK-8 cells increased 5-HT(1D) receptor-mediated intracellular Ca(2+) and extracellular signal regulated kinase (ERK) phosphorylation responses without affecting the adenylate cyclase response. Additionally, detachment enabled 5-HT(1D) receptor stimulation to inhibit P2Y receptor-induced [Ca(2+)](i) mobilization. Such a cross talk between the two receptor systems was not observed in attached cells. P2Y receptor-induced Ca(2+) response was insensitive to adhesion state of the cells, while ERK phosphorylation response was enhanced upon detachment. Integrity of the actin cytoskeleton did not appear to play a role in adhesion sensitivity of 5-HT(1D)-mediated responses, as treatment of attached cells with cytochalasin D did not mimic detachment-induced effects. Effects of detachment were reversed immediately after re-attachment of the suspended cells on poly-l-lysine coated cover slips, suggesting that the involvement of integrins or focal adhesion complexes is unlikely. Taken collectively, our results demonstrate that not only cellular responses induced by different G protein-coupled receptors, but also different responses induced by a particular G protein-coupled receptor, can be affected differentially by the adhesion status of cells. This suggests an important role for cell adhesion in controlling the coupling of a single G protein-coupled receptor to different intracellular responses.

In memoriam R.J. Domenjoz

On March 7 this year, Prof. Robert J. Domenjoz died at the age of 89 years. Pharmacology is extremely indebted to him. He founded the journal in 1959, which was named Medicina Experimentalis and some years later renamed Medicina et Pharmacologia Experimentalis still in the tradition that Latin was the medical language.