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-17beta (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 mitogenactivated 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. Many physiological effects of steroid hormones are too rapid to be mediated by the classical genomic mechanism of steroid action involving activation of nuclear steroid receptors (1-3). Rapid, nongenomic steroid actions initiated on the cell surface have been described in a wide variety of animal, tissue, and cell models (4-8). Steroid membrane receptors characterized in plasma membrane fractions of many target tissues are the likely intermediaries of these nonclassical steroid actions (6, 9-11). For example, extensive studies in our laboratory have demonstrated that a progestin membrane receptor characterized in spotted seatrout (Cynoscion nebulosus) ovaries mediates the induction of oocyte meiotic maturation by the maturationinducing steroid, 17,20␤,21-trihydroxy-4-pregnen-3-one (20␤-S), in this species (12-15). However, detailed knowledge of the molecular structures and mechanisms of action of steroid membrane receptors has eluded investigators, despite intensive research efforts in many laboratories over several decades to purify and sequence the receptor proteins (16)(17)(18)(19)(20). The minute quantities of receptor proteins present in target tissues and major losses of binding activity during solubilizatio...

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, and Fugu, 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 ␣, ␤, and ␥) 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 ␣, ␤, and ␥ 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 ␣, ␥, and mouse ␤ proteins produced in an Escherichia coli expression system demonstrated high affinity (Kd ‫؍‬ 20 -30 nM) saturable binding for progesterone. Further analysis of binding to the ␥-subtype revealed binding was specific for progestins and was displaceable, with rapid rates of association and dissociation (t1/2 ‫؍‬ 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 lthough the existence of specific receptors on the surface of target cells mediating rapid nongenomic actions of steroids was recognized 20 years ago (1, 2), efforts to determine the structures of steroid membrane receptors have been unsuccessful until now (3-5). In the accompanying paper in this issue of PNAS (6), we described a gene discovered in a teleost species, spotted seatrout, whose protein fulfils the criteria for its designation as a steroid membrane receptor, including structural plausibility, specific tissue and plasma membrane localization, steroid binding characteristic of steroid and progestin receptors, coupling to second messenger pathways, regulation by steroid hormones, and biological relevance. Evidence was obtained that this progestin membrane receptor (mPR) is the intermediary in progestin induction of oocyte meiotic maturation in teleost fishes and activates an inhibitory G protein (G i/o ), which suggests it may be a G protein-coupled receptor (GPCR).The aims of this study were to search for related cDNA fragments of other vertebrates in the genomic databases, clone and sequence their full-length cDNAs, and partially characterize these genes and their recombinant proteins. This paper reports the identification of 13 additional vertebrate genes closely related to the spotted seatrout mPR. Structural and phylogenetic evidence is presented that these vertebrate genes encode for membrane proteins with seven or occasionally six transmembrane domains that can be classified into three subty...

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