β2-Chimaerin Is a High Affinity Receptor for the Phorbol Ester Tumor Promoters

Abstract β2-chimaerin, a member of the GTPase-activating proteins for the small GTP-binding protein p21Rac, possesses a single cysteine-rich domain with high homology to those implicated in phorbol ester and diacylglycerol binding in protein kinase C (PKC) isozymes. We have expressed β2-chimaerin in Sf9 insect cells using the baculovirus expression system and determined that, like PKCs, β2-chimaerin binds phorbol esters with high affinity in the presence of phosphatidylserine as a cofactor. Scatchard plot analysis using the radioligand [3H]phorbol 12,13-dibutyrate revealed a dissociation constant of 1.9 ± 0.2 nm for β2-chimaerin. Likewise, β2-chimaerin is a high affinity receptor for the bryostatins, a class of atypical PKC activators. A detailed comparison of structure-activity relations using several phorbol ester analogs revealed striking differences in binding recognition between β2-chimaerin and PKCα. Although the diacylglycerol 1-oleoyl-2-acetylglycerol binds with similar potency to both β2-chimaerin and PKCα, the mezerein analog thymeleatoxin has 56-fold less affinity for binding to β2-chimaerin. To establish whether β2-chimaerin responds to phorbol esters in cellular systems, we overexpressed β2-chimaerin in COS-7 cells and monitored its subcellular distribution after phorbol ester treatment. Interestingly, as described previously for PKC isozymes, β2-chimaerin translocates from cytosolic to particulate fractions as a consequence of phorbol ester treatment. Our results demonstrate that β2-chimaerin is a novel target for the phorbol ester tumor promoters. The expansion of the family of phorbol ester receptors strongly suggests a potential for the “non-kinase” receptors as cellular mediators of the phorbol ester responses.