Fluorescence Resonance Energy Transfer Analysis of α_2a-Adrenergic Receptor Activation Reveals Distinct Agonist-Specific Conformational Changes

Abstract

Several lines of evidence suggest that G-protein-coupled receptors can adopt different active conformations, but their direct demonstration in intact cells is still missing. Using a fluorescence resonance energy transfer (FRET)-based approach we studied conformational changes in ␣ 2A -adrenergic receptors in intact cells. The receptors were C-terminally labeled with cyan fluorescent protein and with fluorescein arsenical hairpin binder at different sites in the third intracellular loop: N-terminally close to transmembrane domain V (I3-N), in the middle of the loop (I3-M), or C-terminally close to transmembrane domain VI (I3-C). All constructs retained normal ligand binding and signaling properties. Changes in FRET between the labels were determined in intact cells in response to different agonists. The full agonist norepinephrine evoked similar FRET changes for all three constructs. The strong partial agonists clonidine and dopamine induced partial FRET changes for all constructs. However, the weak partial agonists octopamine and norphenephrine only induced detectable changes in the construct I3-C but no change in I3-M and I3-N. Dopamineinduced FRET-signals were Ϸ1.5-fold slower than those for norepinephrine in I3-C and I3-M but Ͼ3-fold slower in I3-N. Our data indicate that the different ligands induced conformational changes in the receptor that were sensed differently in different positions of the third intracellular loop. This agrees with X-ray receptor structures indicating larger agonist-induced movements at the cytoplasmic ends of transmembrane domain VI than V and suggests that partial agonism is linked to distinct conformational changes within a G-protein-coupled receptor.Stimulation of G-protein-coupled receptors (GPCRs) by an agonist leads to a conformational change and to a transition of the receptor into an active conformation, which can then couple to its G-protein. Conformational changes have been well established to occur within the transmembrane domains (TMs) III and VI (Gether, 2000;Hubbell et al., 2003). These changes are believed to be transmitted into the third intracellular loop. This loop seems to contain the key domains for coupling to G-proteins, particularly in its C terminus (adjacent to TMVI) but also in its N terminus (adjacent to TMV) regions (Wess, 1998).Whereas classic theory assumed that receptors simply switch between "off" and "on" states, more recent data indicate that agonists of different efficacy might induce different changes in receptor conformations (Kobilka and Deupi, 2007). To accommodate the growing body of evidence for multiple conformational states into theoretical considerations, different models have been proposed. These models propose either that each agonist might promote its own specific active receptor conformation, thus leading to an almost unlimited number of receptor conformations R n *, or suggest that there might be a limited number of active conformations into which different agonists might switch a receptor (Kenakin, 1995). The accumulating evid...

G protein-mediated stretch reception

“…In recent years it became obvious that AT 1 R and most probably also other GPCRs possess more than one active or inactive receptor conformation in analogy to the different states of ion channels. In line with this notion, different partial agonists were shown to induce several distinct active receptor conformations (116). In the performance of bioluminescence resonance energy transfer (BRET) experiments, it was demonstrated that membrane stretch favors an active AT 1 R conformation that is recognized by ␤-arrestin (62), an observation that was recently verified by Rakesh et al (81) in performing intermolecular fluorescence resonance energy transfer (FRET) experiments.…”

Spatial and Temporal Aspects of Signaling by G-Protein–Coupled Receptors

“…However, once this has happened, the protein states that follow resemble those formed in other GPCRs; in the case of rhodopsin, the socalled Meta states all contain the all-trans-retinal agonist bound by a deprotonated retinal Schiff base, but are distinguished by specific arrangements of crucial amino acids and their connecting hydrogen bonds. Corresponding R and R* conformational states can be delineated for other GPCRs and strongly suggest the presence of multiple active forms of GPCRs (Zürn et al, 2009;Nygaard et al, 2013;Manglik et al, 2015).…”

From Molecular Details of the Interplay between Transmembrane Helices of the Thyrotropin Receptor to General Aspects of Signal Transduction in Family A G-protein-coupled Receptors (GPCRs)

“…Different GPCR conformations are causally related to different signaling activity states (3,10,11). Furthermore, differences between activated conformations depend on particular interacting ligands, and they are probably involved in determination of G-protein subtype preferences (12)(13)(14)(15)(16)(17). The previously published GPCR structures of inactive receptor conformations (for reviews, see Refs.…”