AKAP12 Modulates β‐arrestin2 Recruitment to the β2‐Adrenergic Receptors
Authors: Qasim Hanan , Reyes‐Alcaraz Arfaxad , McConnell Bradley K. 
Published: May 2022

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 p<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.