beta 2-chimaerin is a novel target for diacylglycerol: Binding properties and changes in subcellular localization mediated by ligand binding to its C1 domain

The members of the chimaerin family of Rac-GTPase-activating proteins posse ss a single C1 domain with high homology to those present in protein kinase C (PKC) isozymes. This domain in PKCs is involved in phorbol ester and dia cylglycerol (DAG) binding. We previously have demonstrated that one of the chimaerin isoforms, beta 2-chimaerin, binds phorbol esters with high affini ty. In this study we analyzed the properties of beta 2-chimaerin as a DAG r eceptor by using a series of conformationally constrained cyclic DAG analog ues (DAG lactones) as probes. We identified analogs that bind to beta 2-chi maerin with more than 100-fold higher affinity than 1-oleoyl-2-acetylglycer ol. The potencies of these analogs approach those of the potent phorbol est er tumor promoters. The different DAG lactones show some selectivity for th is novel receptor compared with PKC alpha, Cellular studies revealed that t hese DAG analogs induce translocation of beta 2-chimaerin from cytosolic (s oluble) to particulate fractions. Using green fluorescent protein-fusion pr oteins for beta 2-chimaerin we determined that this novel receptor transloc ates to the perinuclear region after treatment with DAG lactones, Binding a nd translocation were prevented by mutation of the conserved Cys-246 in the C1 domain. The structural homology between the C1 domain of beta 2-chimaer in and the C1b domain of PKC delta also was confirmed by modeling analysis, Our results demonstrate that beta 2-chimaerin is a high affinity receptor for DAG through binding to its C1 domain and supports the emerging concept that multiple pathways transduce signaling through DAG and the phorbol este rs.