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, 154 (1), 283-95

Characterization, Neurosteroid Binding and Brain Distribution of Human Membrane Progesterone Receptors δ and {Epsilon} (mPRδ and mPR{epsilon}) and mPRδ Involvement in Neurosteroid Inhibition of Apoptosis

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Characterization, Neurosteroid Binding and Brain Distribution of Human Membrane Progesterone Receptors δ and {Epsilon} (mPRδ and mPR{epsilon}) and mPRδ Involvement in Neurosteroid Inhibition of Apoptosis

Yefei Pang et al. Endocrinology.

Abstract

Three members of the progestin and adipoQ receptor (PAQR) family, PAQR-7, PAQR-8, and PAQR-5 [membrane progesterone (P4) receptor (PR) (mPR)α, mPRβ, and mPRγ], function as plasma mPRs coupled to G proteins in mammalian cells, but the characteristics of two other members, PAQR6 and PAQR9 (mPRδ and mPRε), remain unclear, because they have only been investigated in yeast expression systems. Here, we show that recombinant human mPRδ and mPRε expressed in MDA-MB-231 breast cancer cells display specific, saturable, high-affinity [(3)H]-P4 binding on the plasma membranes of transfected cells with equilibrium dissociation constants (K(d)s) of 2.71 and 2.85 nm, respectively, and low affinity for R5020, characteristics typical of mPRs. P4 treatment increased cAMP production as well as [(35)S]-guanosine 5'-triphosphate (GTP)γS binding to transfected cell membranes, which was immunoprecipitated with a stimulatory G protein antibody, suggesting both mPRδ and mPRε activate a stimulatory G protein (Gs), unlike other mPRs, which activate an inhibitory G protein (Gi). All five mPR mRNAs were detected in different regions of the human brain, but mPRδ showed greatest expression in many regions, including the forebrain, hypothalamus, amygdala, corpus callosum, and spinal cord, whereas mPRε was abundant in the pituitary gland and hypothalamus. Allopregnanolone and other neurosteroids bound to mPRδ and other mPRs and acted as agonists, activating second messengers and decreased starvation-induced cell death and apoptosis in mPRδ-transfected cells and in hippocampal neuronal cells at low nanomolar concentrations. The results suggest that mPRδ and mPRε function as mPRs coupled to G proteins and are potential intermediaries of nonclassical antiapoptotic actions of neurosteroids in the central nervous system.

Figures

Fig. 1.
Fig. 1.
Expression, localization, and ligand binding characteristics of recombinant mPRδ in stably transfected MDA-MB-231 cells. A and B, Expression of mRNA (left), recombinant protein on cell membrane (right) (A) and immunocytochemical analysis (B) of mPRδ-transfected (mPRδ) cells. Mkr, Molecular weight marker; Vec, empty vector-transfected cells; P, peptide antigen. C, Single point-specific [3H]-P4 binding to plasma membranes from Vec and mPRδ cells. D, Representative saturation analysis and Scatchard plot of [3H]-P4 binding to mPRδ cell membranes. E and F, Representative competition curves of steroid binding to mPRδ cell membranes expressed as a percentage of maximum [3H]-P4 binding. T, Testosterone; E2, 17β-estradiol; Cort, cortisol; 02, Org OD-02-0; 13, Org OD-13-0; Mkr, DNA size markers. *, P < 0.001 compared with Vec, n = 6.
Fig. 2.
Fig. 2.
Expression, localization, and ligand binding characteristics of recombinant mPRϵ in stably transfected MDA-MB-231 cells. A and B, Expression of mRNA (left), recombinant protein on cell membrane (right) (A) and immunocytochemical analysis (B) of mPRϵ-transfected cells (mPRϵ). C, Single point-specific [3H]-P4 binding to plasma membranes from control (Vec) and mPRϵ cells. D, Representative saturation analysis and Scatchard plot of [3H]-P4 binding to mPRϵ cells. E and F, Representative competition curves of steroid binding to mPRϵ cell membranes expressed as a percentage of maximum [3H]-P4 binding. Mkr, DNA size markers; P, peptide antigen; T, testosterone; E2, 17β-estradiol; Cort, cortisol; 02, Org OD-02-0; 13, Org OD-13-0; 5020, R5020. *, P < 0.001 compared with Vec, n = 6.
Fig. 3.
Fig. 3.
G protein activation and signal transduction of recombinant mPRδ and mPRϵ. A and D, [35S]-GTPγS binding to plasma membranes of mPRδ-transfected (A) and mPRϵ-transfected (D) (mPRδ and mPRϵ) cells. Vec, Control vector-transfected cells; Veh, vehicle control. *, P < 0.05 compared with Veh, n = 6. B and E, Immunoprecipitation of activated G protein on plasma membranes of mPRδ (B) and mPRϵ (E) cells. IgG, Control IgG; Gs, anti-Gαs-subunit antibody; Gi, anti-Gαi-subunit antibody. *, P < 0.05 compared with IgG, n = 6. Western blot analyses show immunoprecipitated G proteins probed with mPRδ (B) and mPRϵ (E) antibodies. C and F, Effects of 15-min progestin treatments on cAMP concentrations in mPRδ (C) and mPRϵ (F) cells. *, P < 0.05 compared with Veh. n = 6.
Fig. 4.
Fig. 4.
Relative mRNA expression levels of mPR subtypes and PR in different regions of human brain determined by QPCR. A, relative expression of mPRδ and mPRϵ mRNAs in selected brain regions. B, Expression levels of mPRδ, mPRϵ, mPRα, mPRβ, mPRγ, and nPR mRNAs in different human brain regions shown with same arbitrary units as in graph A, Expression scale (arbitrary units): −, ≤1; +, >1 to ≤5; ++, >5 to ≤10; +++: >10 to ≤30; ++++, >30 to ≤60; +++++, >60 to ≤150; ++++++, >150. F. L., Frontal lobe; T. L., temporal lobe; O. L., occipital lobe; P. L., parietal lobe; Pa. G., paracentral gyrus; Po. G., postcentral gyrus; O. B., olfactory bulb; Thal, thalamus; C. C., corpus callosum; Hypo, hypothalamus; Amyg, amygdala; Hippo, hippocampus; Caud, caudate; Puta, putamen; S. N., substantia nigra; P. G., pituitary gland; C. G., cerebellum gray; C. W., cerebellum white; C. V., cerebellum vermis; N. A., nucleus accumbens; Pons, pons; Medu., medulla; S. C., spinal cord; C. P., choroid plexus.
Fig. 5.
Fig. 5.
Representative competition curves of neurosteroid binding to cell membranes of mPRα-transfected (A), mPRβ-transfected (B), mPRδ-transfected (C), and mPRϵ-transfected (D) cells expressed as a percentage of maximum [3H]-P4 binding. DHEA, Dehydroepiandrosterone; Pregna, pregnanolone; Preg, pregnenolone; Allo, allopregnanolone.
Fig. 6.
Fig. 6.
Effects of P4 and allopregnanolone (Allo) on second messengers and cell death in mPRδ-transfected (mPRδ), vector (Vec), and hippocampal neuronal cells. A, Effects of 15-min treatments with P4 or Allo and forskolin (Fors) on cAMP concentrations in mPRδ cells. *, P < 0.05; **, P < 0.01 compared with respective vehicle (Veh) control. n = 6. B, Representative Western blot analyses of effects of 20-min treatments with P4 or Allo on activation of ERK 1/2 (left) and Akt (right) in mPRδ and Vec cells. EGF, Epidermal growth factor control; P-ERK, phosphorylated ERK; P-Akt, phosphorylated Akt. C, Effects of P4 and Allo on cell death of Vec and mPRδ cells. *, P < 0.01 compared with Veh control, n = 6. D, Antiapoptotic effects of P4 and Allo in mPRδ or Vec cells (detected with TUNEL assay). E, RT-PCR of mPRδ and mPRϵ mRNAs and antiapoptotoic effects of P4, Allo, and R5020 in rat hippocampal neuronal (H19-7) cells. **, P < 0.001, compared with Veh control, n = 6.

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