Tae Weon Lee

Functionally Different Agonists Induce Distinct Conformations in the G Protein Coupling Domain of the β2Adrenergic Receptor

G protein-coupled receptors represent the largest class of drug discovery targets. Drugs that activate G protein-coupled receptors are classified as either agonists or partial agonists. To study the mechanism whereby these different classes of activating ligands modulate receptor function, we directly monitored ligand-induced conformational changes in the G protein-coupling domain of the β2 adrenergic receptor. Fluorescence lifetime analysis of a reporter fluorophore covalently attached to this domain revealed that, in the absence of ligands, this domain oscillates around a single detectable conformation. Binding to an antagonist does not change this conformation but does reduce the flexibility of the domain. However, when the β2 adrenergic receptor is bound to a full agonist, the G protein coupling domain exists in two distinct conformations. Moreover, the conformations induced by a full agonist can be distinguished from those induced by partial agonists. These results provide new insight into the structural consequence of antagonist binding and the basis of agonism and partial agonism.

Sequential Binding of Agonists to the β2 Adrenoceptor KINETIC EVIDENCE FOR INTERMEDIATE CONFORMATIONAL STATES

The β2 adrenoreceptor (β2AR) is a prototypical G protein-coupled receptor (GPCR) activated by catecholamines. Agonist activation of GPCRs leads to sequential interactions with heterotrimeric G proteins, which activate cellular signaling cascades, and with GPCR kinases and arrestins, which attenuate GPCR-mediated signaling. We used fluorescence spectroscopy to monitor catecholamine-induced conformational changes in purified β2AR. Here we show that upon catecholamine binding, β2ARs undergo transitions to two kinetically distinguishable conformational states. Using a panel of chemically related catechol derivatives, we identified the specific chemical groups on the agonist responsible for the rapid and slow conformational changes in the receptor. The conformational changes observed in our biophysical assay were correlated with biologic responses in cellular assays. Dopamine, which induces only a rapid conformational change, is efficient at activating Gs but not receptor internalization. In contrast, norepinephrine and epinephrine, which induce both rapid and slow conformational changes, are efficient at activating Gs and receptor internalization. These results support a mechanistic model for GPCR activation where contacts between the receptor and structural determinants of the agonist stabilize a succession of conformational states with distinct cellular functions.

Different Effects of Gsα Splice Variants on β2-Adrenoreceptor-mediated Signaling THE β2-ADRENORECEPTOR COUPLED TO THE LONG SPLICE VARIANT OF Gsα HAS PROPERTIES OF A CONSTITUTIVELY ACTIVE RECEPTOR

The β2-adrenoreceptor (β2AR) couples to the G-protein Gs to mediate adenylyl cyclase activation. The splice variants of Gsα differ by a 15-amino acid insert between the Ras-like domain and the α-helical domain. The long splice variant of Gsα (GsαL) binds GDP with lower affinity than the short splice variant (GsαS), but the impact of this difference on the interaction of Gsα with the β2AR is not known. We studied the β2AR/Gsα interaction using receptor/G-protein fusion proteins (β2ARGsαS and β2ARGsαL) expressed in Sf9 cells. Fusion of the β2AR to Gsα promotes efficient coupling as shown by high-affinity agonist binding and GTPase and adenylyl cyclase activation and ensures fixed stoichiometry between receptor and G-protein. Importantly, fusion does not change the fundamental properties of the β2AR or Gsα. The β2AR in β2ARGsαL showed hallmarks of constitutive activity (increased potency and intrinsic activity of partial agonists, increased efficacy of inverse agonists, and increased basal GTPase activity) compared with the β2AR in β2ARGsαS. The apparent constitutive activity of the β2AR in β2ARGsαL may be due to the lower GDP affinity of GsαL compared with GsαS, i.e.GsαL is more often nucleotide-free than GsαS and, therefore, more frequently available to stabilize the β2AR in the active (R*) state. This study demonstrates that subtle structural differences between closely related G-protein α-subunits can have important consequences for the functional properties of a G-protein-coupled receptor.

The Effect of pH on β2 Adrenoceptor Function EVIDENCE FOR PROTONATION-DEPENDENT ACTIVATION

The transition of rhodopsin from the inactive to the active state is associated with proton uptake at Glu134 (1), and recent mutagenesis studies suggest that protonation of the homologous amino acid in the α1Badrenergic receptor (Asp142) may be involved in its mechanism of activation (2). To further explore the role of protonation in G protein-coupled receptor activation, we examined the effects of pH on the rate of ligand-induced conformational change and on receptor-mediated G protein activation for the β2adrenergic receptor (β2AR). The rate of agonist-induced change in the fluorescence of NBD-labeled, purified β2AR was 2-fold greater at pH 6.5 than at pH 8, even though agonist affinity was lower at pH 6.5. This biophysical analysis was corroborated by functional studies; basal (agonist-independent) activation of Gαs by the β2AR was greater at pH 6.5 compared with pH 8.0. Taken together, these results provide evidence that protonation increases basal activity by destabilizing the inactive state of the receptor. In addition, we found that the pH sensitivity of β2AR activation is not abrogated by mutation of Asp130, which is homologous to the highly conserved acidic amino acids that link protonation to activation of rhodopsin (Glu134) and the α1B adrenergic receptor (Asp142).