Engineered Hyperphosphorylation of theβ₂-Adrenoceptor Prolongs Arrestin-3 Binding and Induces Arrestin Internalization

Abstract

G protein-coupled receptor phosphorylation plays a major role in receptor desensitization and arrestin binding. It is, however, unclear how distinct receptor phosphorylation patterns may influence arrestin binding and subsequent trafficking. Here we engineer phosphorylation sites into the C-terminal tail of the b 2 -adrenoceptor (b2AR) and demonstrate that this mutant, termed b2AR SSS , showed increased isoprenaline-stimulated phosphorylation and differences in arrestin-3 affinity and trafficking. By measuring arrestin-3 recruitment and the stability of arrestin-3 receptor complexes in real time using fluorescence resonance energy transfer and fluorescence recovery after photobleaching, we demonstrate that arrestin-3 dissociated quickly and almost completely from the b2AR, whereas the interaction with b2AR SSS was 2-to 4-fold prolonged. In contrast, arrestin-3 interaction with a b 2 -adrenoceptor fused to the carboxyl-terminal tail of the vasopressin type 2 receptor was nearly irreversible. Further analysis of arrestin-3 localization revealed that by engineering phosphorylation sites into the b 2 -adrenoceptor the receptor showed prolonged interaction with arrestin-3 and colocalization with arrestin in endosomes after internalization. This is in contrast to the wild-type receptor that interacts transiently with arrestin-3 at the plasma membrane. Furthermore, b2ARSSS internalized more efficiently than the wild-type receptor, whereas recycling was very similar for both receptors. Thus, we show how the interaction between arrestins and receptors can be increased with minimal receptor modification and that relatively modest increases in receptor-arrestin affinity are sufficient to alter arrestin trafficking.

Identification of key phosphorylation sites in PTH1R that determine arrestin3 binding and fine-tune receptor signaling

“…Thus, to study whether the removal of phospho-acceptor clusters in the PTH1R C-tail changes the temporal stability of activated arrestin–receptor complexes at the plasma membrane, we used an assay based on dual-color FRAP. This technique has previously been used to monitor the steady-state affinity of arrestin3 to agonist-bound receptors in single living cells [27]. In these experiments, HA-tagged PTH1R–YFP receptors were cross-linked at the plasma membrane and, therefore, the lateral mobility of arrestin–receptor complexes was reduced.…”

“…[ 32 P]Orthophosphate labeling, agonist incubation, receptor solubilization, immunoprecipitation and autoradiography were performed as described recently [27]. Briefly, HEK293 cells stably expressing HA–PTH1Rs were grown in six-well plates.…”

“…Individual FRET recordings were averaged and shown as absolute alterations in FRET normalized to baseline. Quantification of relative expression levels of fluorescently labeled proteins was performed as described recently [27]. …”

“…Fluorescence recovery after photobleaching (FRAP) was performed as described previously [27,28] with minor modifications. HEK293T cells transiently expressing arrestin3–CFP and N-terminally HA-tagged PTHRs labeled with YFP at the C-terminus were treated with a monoclonal anti-HA antibody (HA.11 16B12, BioLegend) followed by 30 min incubation with a polyclonal anti-mouse antibody at 37°C before being stimulated with 100 nM PTH(1–34) in 0.1% BSA/Tyrode's buffer.…”

Heterologous phosphorylation–induced formation of a stability lock permits regulation of inactive receptors by β-arrestins

“…We used a mutant β2AR with engineered additional phosphorylation sites (β2AR-SSS; G361S, E362S, Q363S), which shows class B-type binding (39). The interaction phenotype of the mutant receptor was reversed to class A by K2A-β-arrestin2 ( Figure 4c).…”