Physical interactions among NADPH‐cytochrome P450 reductase, CYP1A2, and CYP2B4 in the endoplasmic reticulum

Abstract

In previous reports, our laboratory has shown that CYP1A2 and CYP2B4 form complexes in mixed reconstituted systems. Formation of the CYP1A2•CYP2B4 complex alters the function of these proteins, particularly in their ability to associate with NADPH‐P450 reductase (CPR). When in mixed reconstituted systems, the affinity of the CYP1A2 moiety of the complex for CPR was dramatically increased, demonstrating not only that CYP1A2 and CYP2B4 form complexes, but also that protein function is affected in the CYP1A2•CYP2B4 complex. To further explore the nature of these P450•P450 interactions, we used bioluminescence resonance energy transfer (BRET) to characterize the complexes in living cells. Transfection of GFP‐containing CYP1A2 and luc‐containing CYP2B4 into HEK293T cells led to a strong BRET signal, indicative of the CYP1A2•CYP2B4 complex. Next, we determined if the CYP1A2•CYP2B4 complex led to an alteration in CPR‐CYP binding by measuring the effect of unlabeled CYP1A2 on BRET for the CYP2B4‐GFP CPR‐luc pair. The results showed that CYP1A2 caused a dramatic disruption of the CYP2B4‐GFP•CPR‐luc complex. Conversely, CYP2B4 was unable to disrupt the CYP1A2‐GFP•CPR‐luc complex. These results are consistent with our model of CPR binding the CYP1A2•CYP2B4 complex and provide further evidence that functional P450•P450 interactions occur in living cells.