Characterization, Neurosteroid Binding and Brain Distribution of Human Membrane Progesterone Receptors δ and ϵ (mPRδ and mPRϵ) and mPRδ Involvement in Neurosteroid Inhibition of Apoptosis

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 [3H]-P4 binding on the plasma membranes of transfected cells with equilibrium dissociation constants (Kds) 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 [35S]-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.