The impact of linker region between receptor and fluorescent protein on arrestin recruitment assays.

S. Al-Sabah¹, M. Bünemann², C. Krasel². ¹Pharmacology & Toxicology, Kuwait University, Kuwait, Kuwait, ²Institut für Pharmakologie und Klinische Pharmazie, Philipps-Universität Marburg, Marburg, Germany.

Introduction: The incretin hormones, glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), are secreted from the gut in response to nutrient ingestion. They potentiate glucose dependent insulin secretion through their specific receptors (GLP-1R and GIPR, respectively) expressed on pancreatic β-cells. Using fluorescence resonance energy transfer (FRET) between YFP-tagged receptors and CFP-tagged arrestin3, we have previously reported that GLP-1R interacted robustly with arrestin3 in response to GLP-1 whereas GIPR showed no interaction with arrestin3 in response to GIP (1). In subsequent experiments a GIPR construct was employed that used a modified version of YFP (SYFP2). This receptor was found to be able to recruit arrestin.

Method: Arrestin3 recruitment to GIPR was investigated by monitoring FRET between YFP-labelled GIPR and CFP labelled arrestin in transiently transfected HEK-293T cells. Cells were perfused with buffer or 1 μM GIP. Arrestin recruitment was observed as an increase in FRET. The original GIPR-YFP construct contained a 10 amino acid linker between the receptor and a XbaI restriction site upstream of the YFP. This linker was no present in the modified GIPR-SYFP2. However, this results in the introduction of a serine residue to the end of GIPR’s C-terminal tail which could potentially be a phosphorylation site. The serine/arginine (SR) coded by the XbaI site was then substituted with glycine/glycine (GG) by site-directed mutagenesis.

Results: Deletion of the 10 amino acid linker between GIPR and the XbaI restriction site results in a receptor that can recruit arrestin when stimulated with 1 μM GIP. However, substitution of SR with GG between the receptor and YFP abolishes arrestin recruitment (n=5).

Conclusion: These results highlight the importance of the linker between receptor and fluorescent protein in arrestin recruitment assays. The use of the commonly used XbaI restriction site may unintentionally introduce an additional phosphorylation site, potentially resulting in false positive results.

References:

1. Al-Sabah S et al., (2014) PLoS One 9: e106890.