COMPARATIVE CHARACTERIZATION OF A WILD-TYPE AND TRANSMEMBRANE DOMAIN-DELETED FATTY-ACID AMIDE HYDROLASE - IDENTIFICATION OF THE TRANSMEMBRANE DOMAIN AS A SITE FOR OLIGOMERIZATION

Fatty acid amide hydrolase (FAAH) is an integral membrane protein resp onsible for the hydrolysis of a number of primary and secondary fatty acid amides, including the neuromodulatory compounds anandamide and ol eamide. Analysis of FAAH's primary sequence reveals the presence of a single predicted transmembrane domain at the extreme N-terminus of the enzyme. A mutant form of the rat FAAH protein lacking this N-terminal transmembrane domain (Delta TM-FAAH) was generated and, like wild typ e FAAH (WT-FAAH), was found to be tightly associated with membranes wh en expressed in COS-7 cells. Recombinant forms of WT- and Delta TM-FAA H expressed and purified from Escherichia coli exhibited essentially i dentical enzymatic properties which were also similar to those of the native enzyme from rat liver. Analysis of the oligomerization states o f WT- and Delta TM-FAAH by chemical cross-linking, sedimentation veloc ity analytical ultracentrifugation, and size exclusion chromatography indicated that both enzymes were oligomeric when membrane-bound and af ter solubilization. However, WT-FAAH consistently behaved as a larger oligomer than Delta TM-FAAH. Additionally, SDS-PAGE analysis of the re combinant proteins identified the presence of SDS-resistant oligomers for WT-FAAH, but not for Delta TM-FAAH. Self-association through FAAH' s transmembrane domain was further demonstrated by a FAAH transmembran e domain-GST fusion protein which formed SDS-resistant dimers and larg e oligomeric assemblies in solution.