Although nonclassical estrogen actions initiated at the cell surface have been described in many tissues, the identities of the membrane estrogen receptors (mERs) mediating these actions remain unclear. Here we show that GPR30, an orphan receptor unrelated to nuclear estrogen receptors, has all the binding and signaling characteristics of a mER. A high-affinity (dissociation constant 2.7 nm), limited capacity, displaceable, single binding site specific for estrogens was detected in plasma membranes of SKBR3 breast cancer cells that express GPR30 but lack nuclear estrogen receptors. Progesterone-induced increases and small interfering RNA-induced decreases in GPR30 expression in SKBR3 cells were accompanied by parallel changes in specific estradiol-17β (E2) binding. Plasma membranes of human embryonic kidney 293 cells transfected with GPR30, but not those of untransfected cells, and human placental tissues that express GPR30 also displayed high-affinity, specific estrogen binding typical of mERs. E2 treatment of transfected cell membranes caused activation of a stimulatory G protein that is directly coupled to the receptor, indicating GPR30 is a G protein-coupled receptor (GPCR), and also increased adenylyl cyclase activity. The finding that the antiestrogens tamoxifen and ICI 182,780, and an environmental estrogen, ortho,para-dichlorodiphenyldichloroethylene (o,p′-DDE), have high binding affinities to the receptor and mimic the actions of E2 has important implications for both the development and treatment of estrogen-dependent breast cancer. GPR30 is structurally unrelated to the recently discovered family of GPCR-like membrane progestin receptors. The identification of a second distinct class of GPCR-like steroid membrane receptors suggests a widespread role for GPCRs in nonclassical steroid hormone actions.

The structures of membrane receptors mediating rapid, nongenomic actions of steroids have not been identified. We describe the cloning of a cDNA from spotted seatrout ovaries encoding a protein that satisfies the following seven criteria for its designation as a steroid membrane receptor: plausible structure, tissue specificity, cellular distribution, steroid binding, signal transduction, hormonal regulation, and biological relevance. For plausible structure, computer modeling predicts that the protein has seven transmembrane domains, typical of G protein-coupled receptors. The mRNA (4.0 kb) is only detected in the brain and reproductive tissues on Northern blots. Antisera only detect the protein (40 kDa) in plasma membranes of reproductive tissues. The recombinant protein produced in an Escherichia coli expression system has a high affinity ( K _d = 30 nM), saturable, displaceable, single binding site specific for progestins. Progestins alter signal transduction pathways, activating mitogen-activated protein kinase and inhibiting adenylyl cyclase, in a transfected mammalian cell line. Inhibition of adenylyl cyclase is pertussis toxin sensitive, suggesting the receptor may be coupled to an inhibitory G protein. Progestins and gonadotropin up-regulate both mRNA and protein levels in seatrout ovaries. Changes in receptor abundance in response to hormones and at various stages of oocyte development, its probable coupling to an inhibitory G protein and inhibition of progestin induction of oocyte maturation upon microinjection of antisense oligonucleotides are consistent with the identity of the receptor as an intermediary in oocyte maturation. These characteristics suggest the fish protein is a membrane progestin receptor mediating a “nonclassical” action of progestins to induce oocyte maturation in fish.

Recently we discovered a previously uncharacterized gene with the characteristics of a membrane progestin receptor (mPR) in a fish model, spotted seatrout. Here, we report the identification, cloning, and characteristics of other members of this hitherto unknown family of putative mPRs from several vertebrate species, including human, mouse, pig,Xenopus, zebrafish, andFugu, with highly conserved nucleotide and deduced amino acid sequences and similar structures to the spotted seatrout mPR. The 13 vertebrate genes identified seem to belong to an unknown gene family. Phylogenetic analysis indicates these cDNAs comprise three distinct groups (named α, β, and γ) within this gene family. Structural analyses of the translated cDNAs suggest they encode membrane proteins with seven transmembrane domains. The transcript sizes of the human α, β, and γ putative mPR mRNAs varied from 2.8 to 5.8 kb and showed distinct distributions in reproductive, neural, kidney and intestinal tissues, respectively. Recombinant human α, γ, and mouse β proteins produced in anEscherichia coliexpression system demonstrated high affinity (K_d= 20–30 nM) saturable binding for progesterone. Further analysis of binding to the γ-subtype revealed binding was specific for progestins and was displaceable, with rapid rates of association and dissociation (t_1/2= 2–8 min). These results suggest this is a new family of steroid receptors unrelated to nuclear steroid receptors, but instead having characteristics of G protein-coupled receptors.

A novel progestin receptor (mPR) with seven-transmembrane domains was recently discovered in spotted seatrout and homologous genes were identified in other vertebrates. We show that cDNAs for the mPR α subtypes from spotted seatrout (st-mPRα) and humans (hu-mPRα) encode progestin receptors that display many functional characteristics of G protein-coupled receptors. Flow cytometry and immunocytochemical staining of whole MDA-MB-231 cells stably transfected with the mPRαs using antibodies directed against their N-terminal regions show the receptors are localized on the plasma membrane and suggest the N-terminal domain is extracellular. Both recombinant st-mPRα and hu-mPRα display high affinity (Kd 4.2–7.8 nm), limited capacity (Bmax 0.03–0.32 nm), and displaceable membrane binding specific for progestins. Progestins activate a pertussis toxin-sensitive inhibitory G protein (G_i) to down-regulate membrane-bound adenylyl cyclase activity in both st-mPRα- and hu-mPRα-transfected cells. Coimmunoprecipitation experiments demonstrate the receptors are directly coupled to the G_i protein. Similar to G protein-coupled receptors, dissociation of the receptor/G protein complex results in a decrease in ligand binding to the mPRαs and mutation of the C-terminal, and third intracellular loop of st-mPRα causes loss of ligand-dependent G protein activation. Phylogenetic analysis indicates the mPRs are members of a progesterone and adipoQ receptor (PAQR) subfamily that is only present in chordates, whereas other PAQRs also occur in invertebrates and plants. Progesterone and adipoQ receptors are related to the hemolysin3 family and have origins in the Eubacteria. Thus, mPRs arose from Eubacteria independently from members of the GPCR superfamily, which arose from Archeabacteria, suggesting convergent evolution of seven-transmembrane hormone receptors coupled to G proteins.

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 (K_ds) 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.