Extracellular bimolecular fluorescence complementation for investigating membrane protein dimerization: a proof of...

Area of Science:

• Molecular Biology
• Cell Biology
• Biochemistry

Background:

• Bimolecular fluorescence complementation (BiFC) is a method to detect protein interactions in living cells.
• Current BiFC approaches typically split fluorescent proteins intracellularly, potentially affecting signaling.
• Receptor dimerization is crucial for cellular communication and signal transduction.

Purpose of the Study:

• To investigate the feasibility of applying BiFC by fusing split fluorescent proteins to the extracellular N-termini of receptors.
• To compare the functional consequences of N-terminal versus C-terminal BiFC fusions in receptor signaling and localization.
• To assess the utility of N-terminal BiFC for studying calcitonin gene-related peptide (CGRP) receptor dimerization.

Main Methods:

• Constructed fusion proteins with split mVenus fragments at the N-termini or C-termini of CGRP receptor subunits.
• Transfected constructs into Cos7 and HEK293S cells.
• Assessed cAMP production, cell surface expression, BiFC fluorescence, and ligand-dependent internalization.

Main Results:

• N-terminal BiFC fusions were better tolerated, showing less impact on cAMP signaling and receptor internalization.
• Functional fluorescent mVenus proteins were reconstituted with N-terminal fusions, albeit with lower fluorescence intensity compared to C-terminal fusions.
• BiFC methodology can be successfully applied to receptor N-termini, demonstrating increased flexibility.

Conclusions:

• Applying BiFC to receptor N-termini enhances methodological flexibility and reduces potential interference with intracellular signaling pathways.
• N-terminal BiFC provides a viable alternative for studying receptor dimerization and dynamics.
• This approach offers new avenues for investigating complex receptor-ligand interactions.