β 2-Chimaerin Is a Novel Target for Diacylglycerol: Binding Properties and Changes in Subcellular Localization Mediated by Ligand Binding to Its C1 Domain

• Published in: Proceedings of the National Academy of Sciences - PNAS 1999-10, Vol.96 (21), p.11854-11859
• Main Authors: Caloca, Maria Jose, Garcia-Bermejo, Maria Laura, Blumberg, Peter M., Lewin, Nancy E., Kremmer, Elisabeth, Mischak, Harald, Wang, Shaomeng, Nacro, Kassoum, Bienfait, Bruno, Marquez, Victor E., Kazanietz, Marcelo G.
• Format: Article
• Language: English
• Subjects: Amino acids; COS cells; Directed acyclic graphs; Drug interactions; Lactones; Ligands; Particulate matter; Phorbol esters; Phorbols; Receptors
• ISSN: 0027-8424
• DOI: 10.1073/pnas.96.21.11854

The members of the chimaerin family of Rac-GTPase-activating proteins possess 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 diacylglycerol (DAG) binding. We previously have demonstrated that one of the chimaerin isoforms, β 2-chimaerin binds phorbol esters with high affinity. In this study we analyzed the properties of β 2-chimaerin as a DAG receptor by using a series of conformationally constrained cyclic DAG analogues (DAG lactones) as probes. We identified analogs that bind to β 2-chimaerin with more than 100-fold higher affinity than 1-oleoyl-2-acetylglycerol. The potencies of these analogs approach those of the potent phorbol ester tumor promoters. The different DAG lactones show some selectivity for this novel receptor compared with PKCα . Cellular studies revealed that these DAG analogs induce translocation of β 2-chimaerin from cytosolic (soluble) to particulate fractions. Using green fluorescent protein-fusion proteins for β 2-chimaerin we determined that this novel receptor translocates to the perinuclear region after treatment with DAG lactones. Binding and translocation were prevented by mutation of the conserved Cys-246 in the C1 domain. The structural homology between the C1 domain of β 2-chimaerin and the C1b domain of PKCδ also was confirmed by modeling analysis. Our results demonstrate that β 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 esters.