Journal Article

Extracellular bimolecular fluorescence complementation for investigating membrane protein dimerization: a proof of concept using class B GPCRs

Alexander, Tyla I., Hay, Debbie L., Garelja, Michael L., Walker, Christopher S.

2024

Overview

Bimolecular fluorescence complementation (BiFC) methodology uses split fluorescent proteins to detect interactions between proteins in living cells. To date, BiFC has been used to investigate receptor dimerization by splitting the fluorescent protein between the intracellular portions of different receptor components. We reasoned that attaching these split proteins to the extracellular N-terminus instead may improve the flexibility of this methodology and reduce the likelihood of impaired intracellular signal transduction. As a proof-of-concept, we used receptors for calcitonin gene-related peptide, which comprise heterodimers of either the calcitonin or calcitonin receptor-like receptor in complex with an accessory protein (receptor activity-modifying protein 1). We created fusion constructs in which split mVenus fragments were attached to either the C-termini or N-termini of receptor subunits. The resulting constructs were transfected into Cos7 and HEK293S cells, where we measured cAMP production in response to ligand stimulation, cell surface expression of receptor complexes, and BiFC fluorescence. Additionally, we investigated ligand-dependent internalization in HEK293S cells. We found N-terminal fusions were better tolerated with regards to cAMP signaling and receptor internalization. N-terminal fusions also allowed reconstitution of functional fluorescent mVenus proteins; however, fluorescence yields were lower than with C-terminal fusion. Our results suggest that BiFC methodologies can be applied to the receptor N-terminus, thereby increasing the flexibility of this approach, and enabling further insights into receptor dimerization.

• Publisher: Portland Press Ltd The Biochemical Society,Portland Press Ltd
• Subject: Animals / Calcitonin / Calcitonin gene-related peptide / Calcitonin Receptor-Like Protein - genetics / Calcitonin Receptor-Like Protein - metabolism / Cell surface / Cell surface receptors / Chlorocebus aethiops / Complementation / COS Cells / Cyclic AMP / Cyclic AMP - metabolism / Dimerization / Flexibility / Fluorescence / Fusion protein / HEK293 Cells / Humans / Internalization / Intracellular / Investigations / Kinases / Ligands / Luminescent Proteins - genetics / Luminescent Proteins - metabolism / Molecular Interactions / N-Terminus / Peptides / Proof of Concept Study / Protein Multimerization / Protein structure / Proteins / Receptor Activity-Modifying Protein 1 - genetics / Receptor Activity-Modifying Protein 1 - metabolism / Receptors / Receptors, Calcitonin - genetics / Receptors, Calcitonin - metabolism / Recombinant Fusion Proteins - genetics / Recombinant Fusion Proteins - metabolism / Signal Transduction / Signaling / Therapeutics & Molecular Medicine