Background: G-Protein Coupled Receptors (GPCRs) are central mediators of signaling cascades in many biological systems. GPCRs internalization rates vary based on the presence/absence of its ligand and the type of the receptor. β2-Adrenergic Receptor (β2AR) is a prototypical GPCR, reaching its maximum in internalization within ~10 minutes after ligand treatment. One regulator of β2AR internalization is β-arrestin2. GPCR–β-arrestin binding is biphasic where the phosphorylated carboxyl terminus of GPCRs docks to the N-domain of β-arrestin first, and then the seven transmembrane core of the receptor engages with β-arrestin. However, it has been discovered that the core interaction is dispensable for receptor endocytosis. AKAP12 is a large scaffolding protein that was reported to be essential in agonist-induced internalization and resensitization of β2AR. Our central hypothesis is that higher expression levels of AKAP12 may enhance β-arrestin2 recruitment to the β2AR and subsequently increase β2AR internalization.
Methods: HEK293 cells were grouped into three groups: [1] Controls: endogenous AKAP12 expression, [2] Overexpression (OX): transiently overexpressing human AKAP12, and [3] Knockdown (KD): treated with human AKAP12 siRNA. β-arrestin2 recruitment to the receptor and β2AR internalization was evaluated using the Nanobit luciferase system. For each assay, we tested four β2AR-β-arrestin2 or Early Endosome-β2AR orientations. Orientation pairs with the highest luminescence signals were used for the experiments to obtain maximum sensitivity. At 48 hours post-transfection, Nanobit assays were performed by recording baseline luminescence for 10 minutes. Next, 10µM epinephrine was added and luminescence measurements were recorded immediately and then once every minute for 1 hour. Results are represented as Luminescence Fold Induction (LFI). Receptor trafficking to early endosomes post epinephrine treatment was further confirmed using the Proximity Ligation Assay (PLA).
Results: HEK cells, OX AKAP12 [9 fold higher] had a significantly lower β-arrestin2 recruitment to the β2AR (2.26±0.39, LFI) as compared to controls (7.47±1.12, LFI; p=0.0004). AKAP12 KD [AKAP12 ~80% silenced] had a significantly higher β-arrestin2 recruitment to the β2AR (10.35±0.56, LFI) as compared to both control and OX AKAP12 groups (p=0.035 and plt;0.0001), respectively. At 15 minutes post epinephrine addition, β2AR trafficking to early endosomes was significantly lower in the OX AKAP12 group (1.82±0.22, LFI), as compared to the control (2.47±0.09, LFI; p=0.01), while the KD AKAP12 group had a significantly higher internalization (2.98±0.14, LFI), as compared to control and OX AKAP12 groups (p=0.005 and p=0.0003), respectively. Our PLA data further confirmed that the OX AKAP12 group displayed significantly lower β2AR trafficking as defined by its decreased interaction with Early Endosome Antigen 1 (EEA1), as compared to the control (p= 0.0451).
Conclusions: Contrary to previously thought, higher AKAP12 expression levels may interfere with β2AR trafficking to early endosomes partially through inhibiting β-arrestin2 recruitment to the β2AR.
This study was supported by the National Heart, Lung, and Blood Institute of the National Institutes of Health (award number R15 HL141963 to B.K.M.); the American Heart Association (award number 18AIREA 33960175 to B.K.M.)
