Opsin Oligomerization in a Heterologous Cell System

Abstract

Using bioluminescence resonance energy transfer (BRET) we studied opsin oligomerization in heterologous expression systems and quantitatively assessed its oligomerization state. BRET² saturation and competition experiments were performed with live COS-7 cells expressing Rluc-and GFP²-tagged receptor constructs. BRET² saturation curves obtained were hyperbolic, and the calculated oligomerization state (N = 1 for dimers) suggested that opsin (N = 1.34 ± 0.25) forms higher oligomers. Very high BRET² values obtained for the opsin homo-dimer pair indicated a large energy transfer efficiency (E) and for cases where E ≫ 0.1 a modified saturation curve was proposed. The existence of homo-dimer complexes was additionally supported by competition assay results and was also observed in HEK-293 cells. Furthermore, evidence was provided for homo-and hetero-dimerization of family A (β₂-adrenergic) and B (gastric inhibitory polypeptide, GIP) receptors. In summary, these experiments demonstrate homo-and hetero-dimerization for opsin, β ₂-adrenergic, and GIP receptors.

Key Words: :

• Opsin
• GIPR
• β₂AR
• Oligomerization
• BRET.