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CYP1A2 and CYP2B4 Form Complexes Associated with Altered Catalytic Activity

Our lab has shown that CYP1A2 activity increased and CYP2B4 activity decreased when both proteins were incorporated into the same vesicles at subsaturating NADPH‐cytochrome P450 reductase (CPR). These data, particularly the apparent activation of CYP1A2 by CYP2B4, are inconsistent with simple competition between the P450s for CPR. Previous work has also provided evidence for physical interactions between P450s. The goal of this study was to show that physical interactions can occur between CYP1A2 and CYP2B4 in the endoplasmic reticulum and to determine how these interactions might affect CPR binding and P450 activities. To achieve this we utilized bioluminescence resonance energy transfer (BRET). HEK293T cells were co‐transfected with two proteins—one with a GFP tag and the other with a Renilla luciferase (Rluc) tag—and energy transfer between these proteins was measured to determine whether they formed physical complexes. A complex was formed by CYP1A2‐Rluc and CYP2B4‐GFP, and this complex was stable in the presence of co‐transfected, untagged CPR. Physical interactions were also detected between each GFP‐tagged P450 and CPR‐Rluc. Whereas untagged CYP1A2 was able to disrupt the CYP2B4:CPR interaction, untagged CYP2B4 had no effect the CYP1A2:CPR complex. CYP1A2 can also form homomeric complexes that are sometimes associated with lower activity at subsaturating [CPR]. Here, we detected these complexes using BRET and showed that they were disrupted by the presence of either CPR or CYP2B4. These data are consistent with a model in which CYP1A2:CYP1A2 complexes are broken up by CYP2B4 to form a stable CYP1A2:CYP2B4 heteromer whose CYP1A2 moiety is preferentially bound by CPR. (Supported by R01 ES004344 and P42 ES013648)