Twenty years of the G protein-coupled estrogen receptor GPER: Historical and personal perspectives

Abstract

Estrogens play a critical role in many aspects of physiology, particularly female reproductive function, but also in pathophysiology, and are associated with protection from numerous diseases in premenopausal women. Steroids and the effects of estrogen have been known for ∼90 years, with the first evidence for a receptor for estrogen presented ∼50 years ago. The original ancestral steroid receptor, extending back into evolution more than 500 million years, was likely an estrogen receptor, whereas G protein-coupled receptors (GPCRs) trace their origins back into history more than one billion years. The classical estrogen receptors (ERα and ERβ) are ligand-activated transcription factors that confer estrogen sensitivity upon many genes. It was soon apparent that these, or novel receptors may also be responsible for the "rapid"/"non-genomic" membrane-associated effects of estrogen. The identification of an orphan GPCR (GPR30, published in 1996) opened a new field of research with the description in 2000 that GPR30 expression is required for rapid estrogen signaling. In 2005-2006, the field was greatly stimulated by two studies that described the binding of estrogen to GPR30-expressing cell membranes, followed by the identification of a GPR30-selective agonist (that lacked binding and activity towards ERα and ERβ). Renamed GPER (G protein-coupled estrogen receptor) by IUPHAR in 2007, the total number of articles in PubMed related to this receptor recently surpassed 1000. In this article, the authors present personal perspectives on how they became involved in the discovery and/or advancement of GPER research. These areas include non-genomic effects on vascular tone, receptor cloning, molecular and cellular biology, signal transduction mechanisms and pharmacology of GPER, highlighting the roles of GPER and GPER-selective compounds in diseases such as obesity, diabetes, and cancer and the obligatory role of GPER in propagating cardiovascular aging, arterial hypertension and heart failure through the stimulation of Nox expression.

Keywords: Adolf Butenand; Adolf Windaus; Charles-Édouard Brown-Séquard; Clara Szego; Edward Doisy; Estrogen; Ferdinand Mainzer; GPCR; History; IUPHAR; Non-genomic; Pathology; Pathophysiology; Physiology; Rudolf Chrobak.

Figure 1. Biologist Clara M. Szego, PhD

Szego was born in Budapest, Hungary, on March 23, 1916. She was a pioneer in the study of estrogen biology (beginning in the 1940s) and in the rapid responses to estrogen (in the 1960s and 1970s), making seminal contributions in both fields. In 1956, Dr. Szego became a Guggenheim fellow while working at the University of California, Los Angeles, and in 1957, she was honored as one of ten Women of the Year by the Los Angeles Times. The photograph was taken in 1957 and provided by the Los Angeles Times.

Figure 2. Rapid non-genomic effects of estrogen in a human coronary artery ring (Barton)

Shown on the left is an organ bath allowing ex vivo study of vascular tone of an intact human coronary artery (4 mm in diameter) obtained during cardiac transplantation (suspended between to steel hooks in physiological salt solution at 37°C). The right panel shows an original tracing of the first ever recorded response to 17β-estradiol of a human coronary artery obtained from a female patient. The cardiac transplantation and experiment took place on May 1, 1992. The left anterior descending (LAD) artery was obtained from a 22 year-old women requiring a heart transplant after developing dilated cardiomyopathy (DCM) and severe heart failure as a complication of a viral infection. Upper panel: Coronary artery tone was recorded after exposure to a contractile substance (prostaglandin F₂). Once a stable plateau was reached, a single concentration of the non-selective estrogen receptor agonist 17β-estradiol (E2, 3 μmol/L) was added to the bath, causing immediate and complete relaxation of the pre-contracted artery. By contrast, the solvent control ethanol (ETOH, lower panel) had only marginal effects on arterial tone. Within 30 min, E2 also increased coronary artery cAMP content [57]. At the time of the experiment, only a single ER protein was known; neither ER nor GPER had been cloned, and it was later shown that the rapid dilator response [70] and cAMP increase [58, 202] involve GPER. Left panel: original organ chamber showing a human coronary artery suspended from a Hugo Sachs force transducer; photograph taken by the author (M.B.) at the Division of Cardiology of Hannover Medical School in 1991. Right panel reproduced from reference [60].

Figure 3. The first cloning of a cDNA for a receptor that would later be called GPR30 and eventually GPER (Lolait)

The cloning of the human GPR30 (GPER) cDNA was first published in 1996 [44] as detailed in the text. In fact, this cDNA (designated clone 47-2) was isolated over 4 years earlier, as evidenced in a note from the first (Christer Owman) to senior (Stephen Lolait) author indicating that the clone was isolated prior to mid-Feb 1992. The note details the DNA concentrations of sets of oligonucleotides (48 base pairs) specific for a number of cDNA clones encoding some orphan GPCRs isolated in the lab around the same time. The GPER clone 47-2 and oligonucleotides Lym 1 and Lym 2 (in red) are detailed in the publication. Clone 21-9 encoded a GPCR that was later deorphanised by others as the leukotriene B4 receptor (LTB4R) [203].

Figure 4. The first image of GPR30 expressed in COS7 cells (Prossnitz)

In order to localize the site(s) of GPR30 expression, the cDNA for GPR30 was fused in frame at the 3′ (C-terminal) end to GFP (green fluorescent protein) or mRFP (monomeric red fluorescent protein [204], shown in red) and subcloned in pcDNA3. COS7 cells were transfected with the plasmid construct and imaged by confocal microscopy, using TO-PRO-3 as a nuclear stain (blue) as in [66]. This first image to test the new plasmid construct came as a great surprise as GPCRs (see localization of β2-adrenergic receptor in Fig. 1A of [66]) are usually expressed predominantly at the plasma membrane (with some expression often detectable in the Golgi apparatus, as a result of accumulation during processing and transport to the plasma membrane). On the contrary, however, the fluorescent protein fusions of GPR30 showed a predominantly intracellular localization, being expressed in the endoplasmic reticulum and Golgi apparatus with essentially no detectable receptor at the plasma membrane. Although at first we suspected the C-terminal fusion of fluorescent proteins might be impeding processing and transport, staining of endogenously expressed GPR30 with newly generated antibodies would reveal a similar expression pattern. Overtime, it would become clear that although most cells exhibit significant localization of GPER intracellularly, GPER does traffic to the plasma membrane (to varying extents, likely dependent on cell type) [82, 83, 105], from where it is constitutively internalized (in an apparently ligand-independent manner) leading to retrograde transport to intracellular membrane compartments, including the endoplasmic reticulum [88, 205, 206]. It is perhaps not entirely surprising that GPER can function intracellularly [99], as estrogen is freely membrane permeable and must “find its way” to the nucleus to activate the classical estrogen receptors ERα and ERβ.