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Visualizing differences in ligand-induced beta-arrestin-GFP interactions and trafficking between three recently
N A Evans1, D A Groarke, J Warrack
1SmithKline Beecham Pharmaceuticals, Harlow, Essex, UK. Nicholas_A_Evans@sbphrd.com
Abstract:
beta-Arrestin 1-GFP or beta-arrestin 2-GFP were coexpressed transiently with G protein-coupled receptor kinase 2 within cells stably expressing the orexin-1, apelin or melanin-concentrating hormone (MCH), receptors. In response to agonist ligands both the orexin-1 and apelin receptors were able to rapidly translocate both beta-arrestin 1-GFP and beta-arrestin 2-GFP from cytoplasm to the plasma membrane. For the MCH receptor this was only observed for beta-arrestin 2-GFP. beta-Arrestin 1-GFP translocated by the apelin receptor remained at the plasma membrane during prolonged exposure to ligand even though the receptor became internalized. By contrast, for the orexin-1 receptor, internalization of beta-arrestin 1-GFP within punctate vesicles could be observed for over 60 min in the continued presence of agonist. Co-internalization of the orexin-1 receptor was observed by monitoring the binding and trafficking of TAMRA-(5- and 6-carboxytetramethylrhodamine) labelled orexin-A. Subsequent addition of an orexin-1 receptor antagonist resulted in cessation of incorporation of beta-arrestin 1-GFP into vesicles at the plasma membrane and a gradual clearance of beta-arrestin 1-GFP from intracellular vesicles. For the melanin-concentrating hormone receptor the bulk of translocated beta-arrestin 2-GFP was maintained at concentrated foci close to, or at, the plasma membrane. These results demonstrate very distinct features of beta-arrestin-GFP interactions and trafficking for three G protein-coupled receptors for which the natural ligands have only recently been identified and which were thus previously considered as orphan receptors.
Insights
This study reveals distinct beta-arrestin trafficking patterns for orexin-1, apelin, and MCH receptors. These differences highlight unique cellular responses to newly identified G protein-coupled receptors.
Area of Science:
- Molecular and Cellular Biology
- Receptor Pharmacology
- G protein-coupled receptor (GPCR) signaling
Background:
- Beta-arrestins are key regulators of GPCR signaling and trafficking.
- Orexin-1, apelin, and melanin-concentrating hormone (MCH) receptors are recently identified GPCRs.
- Understanding beta-arrestin interactions with these receptors is crucial for elucidating their functions.
Purpose of the Study:
- To investigate the distinct trafficking behaviors of beta-arrestin 1 and beta-arrestin 2 in response to agonists for orexin-1, apelin, and MCH receptors.
- To characterize the dynamics of beta-arrestin-GPCR interactions during ligand stimulation and internalization.
Main Methods:
- Transient coexpression of beta-arrestin 1-GFP or beta-arrestin 2-GFP with G protein-coupled receptor kinase 2 in cells stably expressing orexin-1, apelin, or MCH receptors.
- Agonist stimulation to induce receptor activation and beta-arrestin translocation.
- Confocal microscopy to monitor beta-arrestin and receptor localization and trafficking, including co-internalization studies using fluorescently labeled ligands.
Main Results:
- Orexin-1 and apelin receptors rapidly translocated both beta-arrestin 1-GFP and beta-arrestin 2-GFP to the plasma membrane upon agonist stimulation.
- MCH receptor translocation was observed only for beta-arrestin 2-GFP.
- Distinct intracellular trafficking patterns were observed: beta-arrestin 1-GFP associated with the apelin receptor remained at the membrane during internalization, while with the orexin-1 receptor, it was internalized into punctate vesicles. Beta-arrestin 2-GFP largely remained at the MCH receptor plasma membrane foci.
Conclusions:
- Significant differences exist in beta-arrestin 1 and beta-arrestin 2 recruitment and trafficking dynamics among orexin-1, apelin, and MCH receptors.
- These distinct interactions provide insights into the specific signaling and regulatory mechanisms of these previously orphan GPCRs.
- The findings underscore the complexity of beta-arrestin-mediated GPCR regulation.