Heterodimerization with the prostacyclin receptor triggers thromboxane receptor relocation to lipid rafts

Abstract

Objective: Prostacyclin and thromboxane mediate opposing cardiovascular actions through receptors termed IP and TP, respectively. When dimerized with IP, the TP shifts to IP-like function. IP localizes to cholesterol-enriched membrane rafts, but TP and IPTP heterodimer localization is not defined. We examined these receptors' membrane localization and the role of rafts in receptor function.

Methods and results: Microdomain distribution of IP, TP, and IPTP heterodimers was examined in COS-7 cells by measuring energy transfer from renilla luciferase-fused receptors to fluorescently labeled rafts. IP raft association was confirmed. TP was raft excluded, but redistributed to rafts upon dimerization with IP. Signaling of the IP and IPTP heterodimer, but not TP alone, was suppressed after raft disruption by cholesterol depletion. Cholesterol enrichment also selectively suppressed IP and IPTP function. Native IP and IPTP signaling in smooth muscle cells and macrophages were similarly sensitive to cholesterol manipulation, whereas macrophages from hypercholesterolemic mice displayed suppressed IP and IPTP function.

Conclusions: IP and TP function within distinct microdomains. Raft incorporation of TP in the IPTP heterodimer likely facilitates its signaling shift. We speculate that changes in IP and IPTP signaling after perturbation of membrane cholesterol may contribute to cardiovascular disease associated with hypercholesterolemia.

Fig. 1. IP and TP membrane distribution in living cells

COS-7 cells were transfected with IPrLuc, TPrLuc, TPrLuc plus untagged IP, or β₂-ARrLuc and the L_o membrane phase labeled with increasing concentrations of DiIC16. Energy transfer (BRET) from rLuc to DiIC16 was quantified as a measure of the receptor’s localization to the L_o phase. Data are expressed as % of maximum BRET and are mean ± sem; n=3; **p<0.005 for either IPrLUC or TPrLuc-IP versus either TPrLUC or β₂-ARrLuc; ns = non significant.

Fig. 2. Effect of cholesterol depletion on receptor signaling

COS-7 cells were transfected with (A) IP alone, (B) IP plus TP, (C) TP alone or (D) β₂-AR and, 48hr later, subjected to no treatment (control) or cholesterol depletion (2-hydroxypropyl-β-cyclodextrin 20mM, 1hr). IP and IPTP signaling were determined as cAMP generation in response to cicaprost (IP agonist) or U46619 (TP agonist), respectively; TP signaling was determined as U46619-induced inositol phosphate (InsP) generation and β₂-AR signaling as metaproterenol (β₂-AR agonist) stimulated cAMP. In (A), (E) and (I) IP signaling is shown for IP-COS-7 cells, hAoSMC and RAW 246.7, respectively. In (B), (F) and (J) IPTP signaling is shown IPTP-COS-7 cells, hAoSMC and RAW 246.7, respectively. In (C) and (G) TP signaling is shown in TP-COS-7 cells and hAoSMC, respectively. In (D), (H) and (K) β₂-AR signaling is shown in β₂-AR-COS-7 cells hAoSMC and RAW 246.7 respectively. Results are % of control ± sem from n=3. * p<0.05, ** p<0.005, *** p<0.0005 compared to control.

Fig. 3. IP and TP dimerization in living cells

Receptor dimerization was examined by measuring BRET from (A) IPrLuc to IPYFP, (B) TPrLuc to IPYFP or (C) TPrLuc to TP YFP in transfected COS-7 subjected to cholesterol depletion (grey line), loading (solid black line), or no manipulation (dashed line). Data are the % of maximum in control cells vs. the ratio of YFP-receptor/rLuc-receptor and are mean ± sem of n=3.

Fig. 4. Effect of cholesterol enrichment on receptor signaling

COS-7 cells were transfected with (A) IP alone, (B) IP plus TP, (C) TP alone or (D) β₂-AR and, 48hr later, subjected to no treatment (control) or cholesterol loading (cholesterol-methyl-β-cyclodextrin complex 80μg/ml, 1hr). IP and IPTP signaling were determined as cAMP generation in response to cicaprost (IP agonist) or U46619 (TP agonist), respectively; TP signaling was determined as U46619-induced inositol phosphate (InsP) generation and β₂-AR signaling as metaproterenol (β₂-AR agonist) stimulated cAMP. In (A), (E) and (I) IP signaling is shown for IP-COS-7 cells, hAoSMC and RAW 246.7, respectively. In (B), (F) and (J) IPTP signaling is shown IPTP-COS-7 cells, hAoSMC and RAW 246.7, respectively. In (C) and (G) TP signaling is shown in TP-COS-7 cells and hAoSMC, respectively. In (D), (H) and (K) β₂-AR signaling is shown in β₂-AR-COS-7 cells hAoSMC and RAW 246.7 respectively. Results are % of control ± sem from n=3. * p<0.05, ** p<0.005 compared to control.

Fig. 5. IP- and IPTP-mediated signaling in mouse peritoneal macrophages

Peritoneal macrophages isolated from wild type (WT) or hypercholesterolemic low density lipoprotein receptor deficient (LDLR^−/−) mice (6–8 months of age) were stimulated with (A) cicaprost or (B) U46619 and cAMP generation quantified. Results are fold over basal ± sem; n=6–7; * p<0.05 compared to WT.