Identification of GPR39 as a putative receptor for 14,15-EET
We implemented a chemical biology approach to purify 14,15-EET-binding proteins by producing a modified form of 14,15-EET (EET-P, Fig. 1A and Supplementary Material) that covalently crosslinks to target proteins following exposure to ultraviolet (UV) light. The probe also incorporates a click chemistry moiety that allows subsequent fluorophore labeling or biotin-streptavidin affinity purification and identification of linked proteins by mass spectrometry (Fig. 1A). Despite these additional functional groups, we were able to demonstrate that EET-P mimics 14,15-EET’s previously reported ability to dilate mouse mesenteric arteries pre-constricted with thromboxane A2 agonist U46619 (13) (Fig. S1). We next used this probe to identify putative mVSMC 14,15-EET receptor(s) by treating mouse heart mVSMCs with EET-P (1 mM) in the presence or absence of 14,15-EET followed by a 5 min UV (365 nm) exposure. Cell lysates were reacted with biotin-azide for affinity purification and subsequent mass spectrometry analysis that yielded a number of intracellular and membrane-associated proteins that could be competitively displaced by 14,15-EET, including a known 14,15-EET metabolizing enzyme, epoxide hydrolase, an indication of probe specificity (Table S1; complete MS data has been deposited to the ProteomeXchange Consortium via the PRIDE database; dataset identifier PXD013952), A single GPCR was detected in the screen, GPR39, a 50 kDa orphan member of the ghrelin receptor family previously reported to be activated by zinc ions (Zn2+) (14). We also found that cultured VSMCs could be labeled with EET-P using a rhodamine azide in-cell click reaction. Importantly, this membrane labeling can be displaced by pretreatment with 14,15-EET (Fig. 1B). We further confirmed membrane-bound targets of EET-P by using rhodamine-azide labeling followed by membrane purification and SDS-PAGE analysis to detect EET-P-labeled proteins and observed a single band that was dose-dependently displaced by 14,15-EET and exhibited approximately the same molecular weight as GPR39 (Fig 1C). The GPR39 genomic locus encodes two isoforms, GPR39 1a (a full-length 7-transmembrane (7TM) isoform) and 1b (a truncated 5TM 1b isoform that lacks TM6 and 7) (15). Using immunocytochemistry and real-time quantitative PCR we confirmed expression of GPR39 1a, but not 1b in cultured mouse heart mVSMCs (Fig. 1D). We also determined that human embryonic kidney (HEK)-293 cells express only the 1b isoform (Fig. S2B). Probe binding specificity was confirmed by crosslinking EET-P to HEK-293 cells transiently transfected with HA-tagged human GPR39 1a (Fig. S2C-E). Western blots probed with HA-antisera (Fig. 1E) indicate that HA-tagged GRP39 1a co-purifies with EET-P (lane 1) and this band is absent in lysates of untransfected cells (lane 4). We further confirmed probe specificity by eliminating probe binding to HA-tagged GPR39 by pretreating GPR39-transfected HEK-293 cells with either 14,15-EET (5 mM, lane 2) or EETs antagonist 14,15-epoxyeicosa-5(Z)-enoic acid (16) (14,15-EEZE, 5 mM, lane 3) prior to EET-P exposure. Finally, dot-blot assay demonstrated dose-dependent saturable binding of 14,15-EET, but not 11,12-EET, to protein extracts from HEK-293 cells expressing GPR39 1a, but not control cells (Fig 1F).
Isoform-Specific Activation of GPR39 Signaling by 14,15-EET and 15-HETE
We transfected HEK-293 cells with either GPR39 1a or GPR39 1b to determine if 14,15-EET binding activates GPR39 signaling and if 14,15-EET binding is specific or whether related eicosanoids can also activate this receptor. Cells were subsequently stimulated with one of four regioisomers of EETs (5,6-EET; 8,9-EET; 11,12-EET; 14,15-EET), HETEs (11-HETE, 12-HETE, 15-HETE, 20-HETE), or vehicle. 14,15-EET and 15-HETE are the only regioisomers that significantly increase GPCR activation as monitored by ERK phosphorylation in cells transfected with GPR39 1a (Fig. 2A, 2B) but not untransfected cells (Fig. 2C-D), with both eicosanoids displaying a concentration-dependent activation of GPR39 (Fig. 2C and 2D). Zinc has been reported to be either a GPR39 agonist (14) or an allosteric modulator for synthetic ligands of the receptor (17). Therefore, we repeated the dose response-curves for 14,15-EET and 15-HETE in the presence of 1 µM zinc, which has no effect on ERK phosphorylation by itself. Zinc significantly potentiates the effects of both eicosanoids, consistent with its role as an allosteric modulator of GPR39. Dot-blot assays using lysates from GPR39 1a stably expressing HEK-293 cells demonstrate dose-dependent, saturable binding of 15-HETE, similar to 14,15-EET, that is not observed for 12-HETE (Fig S3A). Importantly, binding competition experiments indicate that 15-HETE and 14,15-EET can displace each other while 12-HETE (Fig S3B) and 11,12-EET (Fig S3C), respectively, fail to do so.
GPR39-Ligand Interaction Modeling Predicts Selectivity for 14,15-EET and 15-HETE
We next used a homology model to investigate GPR39 1a-ligand specificity in silico (Fig S4A and B) that predicted structural complementarity between 14,15-EET and GPR39, as indicated by a low docking energy (-8.7 kcal/mol). A fingerprint similarity search for other GPR39 ligands using 14,15-EET as a reference structure resulted in a list of long-chain fatty acids (Table S2) including 15-HETE, which had a predicted docking energy of -8.6 kcal/mol, similar to 14,15-EET. This likely reflects that both compounds have a polar group at the 15th carbon position and maintain double bonds in the cis configuration at the 5th, 8th and 11th positions (Fig. 2E) unlike the other EETs and HETEs regioisomers. A potential orthostatic pocket was identified using SiteMap V3.2 which revealed a hydrophilic major pocket and a hydrophobic minor pocket (Fig S4C) in GPR39 1a with the tail portion of these lipids extending into the minor binding pocket formed by transmembrane 1 (TM1), TM2, TM7 and the N-terminal loop (Fig. 2F, S4D-E). The major pocket accommodates the carboxylate moiety of 14,15-EET and 15-HETE by forming ionic interactions with positively charged residues from TM6 (Fig. 2F).
GPR39 Mediates Effects of 14,15-EET and 15-HETE on mVSMC Calcium Transients
We next enquired whether 14,15-EET and 15-HETE regulate the signaling of mVSMC via GPR39, and if their actions are modulated by zinc. Using live-cell fluorescent imaging to monitor calcium transients, we first evaluated the effect of Zn2+ alone in mVSMCs and found that it increases intracellular calcium with an EC50 of 7.8 mM (Fig 3A). We then established the dose-response relationship for 15-HETE in the presence and absence of 1, 4 and 10 mM zinc. We observed that zinc, at 4 or 10 mM, significantly potentiates the response of GPR39 to 15-HETE stimulation (Fig 3B; only 1 and 10 µM are shown). Application of 15-HETE alone elicits a calcium response in mVSMCs only at concentrations above 100 nM (Fig. 3B). However, in the presence of 10 µM (Fig. 3B) or 4 µM (Fig. 3C) zinc, 15-HETE elicits a significant calcium response in mVSMCs at low nanomolar concentrations (Fig. 3B), suggesting that zinc serves as a positive allosteric modulator for 15-HETE in mVSMCs. Importantly, GPR39 RNAi knockdown in mVSMCs attenuated the increase in mVSMC intracellular calcium produced by 15-HETE (Fig 3C, S5B), confirming a significant role for GPR39 in mediating the effect of 15-HETE on mVSMC calcium transients. 14,15-EET had no effect on intracellular calcium in mVSMCs at concentrations between 1 pM - 10 mM (data not shown), but it was observed to inhibit 15-HETE-dependent increases in intracellular calcium (Fig. 3D), consistent with GPR39 acting as a dual sensor for both 14,15-EET and 15-HETE. Taken together, our modeling data along with pERK, calcium imaging and binding assays indicate a competitive structure-activity relationship between 14,15-EET and 15-HETE.
GPR39 Localization and Function in Myocardial Microvessels
Consistent with a role in regulating coronary vascular resistance, immunofluorescence indicates that GPR39 expression is predominantly restricted to microvessels in the mouse heart (Fig 4A). Co-staining with α-smooth muscle actin (α-SMA, upper panel) and CD31 (middle panel) confirms expression in arteriolar smooth muscle cells, but not endothelial cells (Fig 4A). Antibody specificity was confirmed using heart tissue from GPR39 knockout (KO) mice, which lacked microvascular immunoreactivity observed in hearts from wild-type littermates (Fig 4A, lower panel). This microvascular pattern of expression was further confirmed using non-fluorescent immunostaining where we also observed GPR39 expression in both mouse and human heart within perivascular cells that were consistently adjacent to microvessels including capillaries (Fig. S6), suggestive of GPR39 expression in pericytes as well.
Role of GPR39 in Microvascular Regulation and Response to Myocardial Ischemia
We next determined the contribution of GPR39 activation by eicosanoids to microvascular tone regulation. This was accomplished using a mouse heart Langendorff preparation where changes in coronary perfusion pressure (CPP) are determined by microvascular resistance at a constant flow rate. Accordingly, 15-HETE (1 µM) increases CPP (Fig 4B), and this effect is inhibited by co-administration of 14,15-EET (1 µM) or abolished in GPR39-null hearts (Fig 4B, Fig S7). Importantly, the effects of two other vasoactive agents on CPP, angiotensin II (Ang II) and prostaglandin I2 (PGI2), were unaffected by GPR39 deletion. Interestingly, CPP response to sodium nitroprusside (SNP) was significantly attenuated in GPR39 KO. Finally, consistent with the established role of P450 eicosanoids in myocardial ischemia, GPR39 KO mice were protected from I/R injury in-vivo compared to their wild-type littermates (Fig 4C).