CHARACTERIZATION OF THE CYSTEINE-RICH REGION OF THE CAENORHABDITIS-ELEGANS PROTEIN UNC-13 AS A HIGH-AFFINITY PHORBOL ESTER RECEPTOR - ANALYSIS OF LIGAND-BINDING INTERACTIONS, LIPID COFACTOR REQUIREMENTS, AND INHIBITOR SENSITIVITY

The Caenorhabditis elegans Unc-13 protein is a novel member of the pho rbol ester receptor family having a single cysteine-rich region with h igh homology to those present in protein kinase C (PKC) isozymes and t he chimaerins, We expressed the cysteine-rich region of Unc-13 in Esch erichia coli and quantitatively analyzed its interactions with phorbol esters and related analogs, its phospholipid requirements, and its in hibitor sensitivity, [H-3]Phorbol 12,13-dibutyrate [H-3]PDBu bound wit h high affinity to the cysteine-rich region of Unc-13 (K-d = 1.3 +/- 0 .2 nM). This affinity is similar to that of other single cysteine-rich regions from PKC isozymes as well as n-chimaerin, As also described f or PKC isozymes and n-chimaerin, Unc-13 bound diacylglycerol with an a ffinity about 2 orders of magnitude weaker than [H-3]PDBu. Structure a ctivity analysis revealed significant but modest differences between r ecombinant cysteine-rich regions of Unc-13 and PKC delta. In addition, Unc-13 required slightly higher concentrations of phospholipid for re constitution of [H-3]PDBu binding. Calphostin C, a compound described as a selective inhibitor of PKC, was also able to inhibit [H-3]PDBu bi nding to Unc-13, suggesting that this inhibitor is not able to disting uish between different classes of phorbol ester receptors, In conclusi on, although our results revealed some differences in ligand and lipid cofactor sensitivities, Unc-13 represents a high affinity cellular ta rget for the phorbol esters as well as for the lipid second messenger diacylglycerol, at least in C. elegans, The use of phorbol esters or s ome ''specific'' antagonists of PKC does not distinguish between cellu lar pathways involving different PKC isozymes or novel phorbol ester r eceptors such as n-chimaerin or Unc-13.