Identification of two tamoxifen target proteins by photolabeling with 4-(2-morpholinoethoxy)benzophenone.
Our quest to identify target proteins involved in the activity of tamoxifen led to the design of photoaffinity ligand analogues of tamoxifen able to cross-link such proteins. A new tritiated photoprobe, 4-(2-morpholinoethoxy)benzophenone (MBoPE), was synthesized and used to identify proteins involved in tamoxifen binding in rat liver. MBoPE, which has structural features in common with the potential antagonist of the intracellular histamine receptor (N,N-diethyl-2-[(4-phenylmethyl)phenoxy]ethanamine HCl: DPPE) is unable to bind the estrogen receptor although it does compete with tamoxifen for an antiestrogen binding site (AEBS). This tritiated benzophenone derivative was obtained by metal-catalyzed halogen-tritium replacement reaction. Because of its high specific activity, four target proteins could be photolabeled, three of which were identified with M(r) of 60,000, 49,500, and 14,000, while the fourth at 27,500 was in too low an amount and could not be sequenced. The 49.5 kDa protein corresponded by mass spectrometry to the microsomal epoxide hydrolase already identified with an aryl azide photoprobe [Mesange, F., et al. (1998) Biochem. J. 334, 107-112]. The 60 and 14 kDa proteins were identified as the carboxylesterase (ES10) and the liver fatty acid binding protein (L-FABP), respectively. The inhibitory effect of tamoxifen on carboxylesterase activity and the competitive efficacy of oleic acid on [(3)H]tamoxifen binding suggest that both proteins are AEBS subunits. Moreover, treatment of hepatocytes with antisense mRNA directed against ES10 or L-FABP abolished both tamoxifen and MBoPE binding. On the basis of previous pharmacological arguments, the 27.5 kDa protein might correspond to the sigma I receptor. Altogether, these results confirm that the microsomal epoxide hydrolase is a target for tamoxifen and provide evidence of two new target proteins implicated in cell lipid metabolism.
INSERM U397 Institut Claudius Regaud 20-24 rue du pont Saint Pierre 31052 Toulouse Cedex, France.
Fabienne
F
Mésange
Mohamed
M
Sebbar
Joël
J
Capdevielle
Jean-Claude
JC
Guillemot
Pascual
P
Ferrara
Francis
F
Bayard
Marc
M
Poirot
Jean-Charles
JC
Faye
D000818:Animals; D001577:Benzophenones / Q000138:chemical synthesis; D001665:Binding Sites; D043182:Carboxylesterase; D002265:Carboxylic Ester Hydrolases / Q000032:analysis / Q000378:metabolism; D002352:Carrier Proteins / Q000032:analysis / Q000378:metabolism; D003432:Cross-Linking Reagents; D016503:Drug Delivery Systems; D004851:Epoxide Hydrolases / Q000032:analysis / Q000378:metabolism; D020847:Estrogen Receptor Modulators; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D022781:Hepatocytes / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D009025:Morpholines / Q000138:chemical synthesis; D009363:Neoplasm Proteins / Q000032:analysis*; D009419:Nerve Tissue Proteins; D019920:Photoaffinity Labels; D011869:Radioligand Assay; D051381:Rats; D011960:Receptors, Estrogen; D013629:Tamoxifen / Q000737:chemistry / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D025521:Tumor Suppressor Proteins
C476043:4-(2-morpholinoethoxy)benzophenone; D001577:Benzophenones; D002352:Carrier Proteins; D003432:Cross-Linking Reagents; D020847:Estrogen Receptor Modulators; C493319:FABP1 protein, human; C107608:FABP7 protein, human; C493320:Fabp1 protein, mouse; C493321:Fabp1 protein, rat; C490174:Fabp7 protein, rat; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D009025:Morpholines; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D019920:Photoaffinity Labels; D011960:Receptors, Estrogen; D025521:Tumor Suppressor Proteins; D013629:Tamoxifen; D002265:Carboxylic Ester Hydrolases; D043182:Carboxylesterase; D004851:Epoxide Hydrolases