While many physiological and biochemical activities have been described for the EETs (4
), the initiation step in the actions of EETs has not been defined. Several lines of evidence implicate a cell surface, high affinity, G-protein coupled receptor as the initial step (17
); however, further characterization and identification of this receptor is needed. Photoaffinity labeling has been a useful approach to receptor identification. These studies describe the characterization of the first EET photoaffinity probe, 20-I-14,15-EE8ZE-APSA, and its use to characterize the EET receptor. 20-I-14,15-EE8ZE-APSA relaxed the bovine coronary artery with a similar ED50
as 14,15-EET and displaced the EET antagonist radioligand 20-125
I-14,15-EE5ZE from its binding site on U937 cell membranes with an IC50
similar to 14,15-EET. Thus, 20-I-14,15-EE8ZE-APSA is high affinity agonist ligand for the EET binding site/receptor. Importantly, this photoactive ligand can be radiolabeled with high specific activity.
When incubated with U937 membranes and cross-linked with UV light, 20-125
I-14,15-EE8ZE-APSA radiolabeled a single protein band of 47 KDa. The radiolabeling of the 47 KDa protein was specific for the EET structure since EET agonists (8,9-EET, 11,12-EET, and 14,15-EET) and the structurally-related EET antagonist (14,15-EE5ZE). In contrast, the inactive EET analogs 14,15-thiirane and 8,9-DHET did not inhibit the radiolabeling. The IC50
’s of the EET analogs for inhibition of labeling by 20-125
I-14,15-EE8ZE-APSA suggest the 47 KDa protein is a high affinity receptor for 11,12-EET and 14,15-EET but has lower affinity for 8,9-EET and 14,15-EE5ZE. This affinity ranking order, 11,12-EET = 14,15-EET > 8,9-EET=14,15-EE5ZE and no binding for 8,9-DHET and 14,15-thiirane is same as was determined in radioligand binding assays and correlates with agonist activity (15
Epoxide hydrolase and cytochrome P450 inhibitors were screened for binding to the EET receptor using the 20-125
I-14,15-EE5ZE as the radioligand (30
). Miconazole and MSPPOH inhibited binding with Ki’s of 350 nM and 1558 nM, respectively. Ketoconazole (50 μM) inhibited binding by 50%. The concentrations of the drugs that inhibited binding of the EET radioligand differed from the concentrations that inhibited cytochrome P450 (30
). Thus, inhibition of EET binding was not associated with cytochrome P450 inhibition. Other cytochrome P450 inhibitors such as sulfaphenazole and proadifen and epoxide hydrolase inhibitors did not alter binding of the radioligand. In addition, miconazole and MSPPOH blocked 14,15-EET-induced relaxations of bovine coronary arteries. Proadifen did not alter 14,15-EET relaxations. These studies indicate that MSPPOH and miconazole, besides inhibiting cytochrome P450, act as EET antagonists like 14,15-EE5ZE. Miconazole, MSPPOH, ketoconazole and 14,15-EE5ZE inhibited 20-125
I-14,15-EE8ZE-APSA photolabeling of the 47 KDa protein. These data are consistent with our previous conclusion that these drugs are EET antagonists and suggests that the 47 KDa protein is the binding site for 20-125
I-14,15-EE5ZE and an EET receptor.
The initial characterization of the EET photoprobe used U937 cell membranes since this cell line has been a model system to study the EET receptor (26
). However, the photolabeling of the 47 KDa protein by 20-125
I-14,15-EE8ZE-APSA was also observed in membranes from rabbit, bovine and human vascular smooth muscle cells, bovine coronary arterial endothelial cells, bovine coronary artery, canine heart and rat kidney. As with U937 cells, this labeling was inhibited by EET agonists. Since EETs relax vascular smooth muscle (10
), inhibit adhesion molecule expression on endothelial cells (4
), alter renal tubular and vascular function (45
) and cause cardioprotection (8
), a protein involved in binding EETs and initiating EET actions would be expected in these cells and tissues.
Several studies indicate the utility of the EET photoprobe in characterizing the EET receptor. These photolabeling experiments reveal for the first time that the EET binding site/receptor has a molecular weight of 47 KDa. This information may be used to eliminate a number of proteins that may bind EETs. For example: CYP2J and CYP2C synthesize EETs, fatty acid binding proteins and PPARs bind EETs and soluble epoxide hydrolase converts EETs to DHETs (4
). However, these proteins cannot represent the EET binding site labeled by the photoprobe since the molecular weights of CYPs, PPARs and epoxide hydrolase exceed 47 KDa and fatty acid binding proteins are less than 47 KDa. By the same logic, BKCa
channel proteins, TRPV4 channels, EP2 receptor and the TP receptor can be eliminated (47
) despite suggestions that these proteins may function as EET receptors (24
The photoprobe made EET receptor screening simple and applicable on a relatively large scale. With small amounts of protein, candidate EET receptor proteins can be tested directly for photolabeling. Additionally, competition by specific receptor agonists or antagonists for photolabeling by 20-125
I-14,15-EE8ZE-APSA can be used to eliminate other known receptors. For example, AM251 (a CB1 receptor antagonist), 5-oxo-ETE (a 5-oxo-ETE receptor agonist), naloxone (an opioid antagonist) and U46619 (a thromboxane receptor agonist) failed to inhibit photolabeling thereby eliminating these receptors as possible EET receptors. Thus, while high concentrations of EETs may inhibit TP receptors or activate EP2 receptors (23
), the TP and EP receptors are not the high affinity EET receptor.
A large number of GPCRs have no known ligand so are termed orphan receptors (40
). Many of these GPCRs may bind lipid mediators (41
). For this reason, a group of 79 GPCRs that bind lipids, are related phylogenetically to lipid binding receptors or are express in EET-responding tissues were tested for 14,15-EET binding by photolabeling. None of the 79 GPCRs were photolabeled by 20-125
I-14,15-EE8ZE-APSA. When U937 membranes were treated in an identical manner at the same time, the photoprobe labeled a 47 KDa protein. Since the method is capable of labeling and detecting the endogenous EET receptor in U937 cells, it should certainly detect an overexpressed GPCR that binds 14,15-EET. Thus, it appears that none of the 79 GPCRs represent the high affinity EET receptor labeled by 20-I-14,15-EE8ZE-APSA. Of particular note, free fatty acid receptors and receptors that bind modified or oxidized fatty acids such as GPR2A, GPR18, GPR40, GPR41, GPR43, GPR84 and GPR120 were not labeled by the photoprobe (51
The photoprobe that is characterized herein is an analog of 14,15-EET and may only label an EET receptor or receptors with high affinity for 14,15-EET and possibly 11,12-EET such as the 47 KDa protein. The failure to detect receptor labeling with 20-125I-14,15-EE8ZE-APSA in HEK293T cells overexpressing orphan GPCRs does not exclude the possibility that these GPCRs are receptors for other EET regioisomers or low affinity EET receptors. Photoprobes for the other EET regioisomers may be needed to identify other EET receptors.
In summary, 20-I-14,15-EE8ZE-APSA is the first EET photoaffinity probe and has EET agonist activity. This photoprobe labeled a 47 KDa high affinity EET binding protein in U937 cells and vascular cells. Since photolabeling was inhibited by several EET agonist (8,9-EET, 11,12-EET, and 14,15-EET) and EET antagonists (14,15-EE5ZE, miconazole, ketoconazole and MS-PPOH), we propose this 47 KDa protein as a high affinity EET receptor. Used in a same way as an antibody, the photoprobe could be a molecular marker for the EET receptor in normal cells and tissues and in disease and improve our understanding of the expression and regulation of the EET receptor.