L. M. Fredrik Leeb-Lundberg

Deletion of the G protein-coupled receptor 30 impairs glucose tolerance, reduces bone growth, increases blood pressure, and eliminates estradiol-stimulated insulin release in female mice.

In vitro studies suggest that the G protein-coupled receptor (GPR) 30 is a functional estrogen receptor. However, the physiological role of GPR30 in vivo is unknown, and it remains to be determined whether GPR30 is an estrogen receptor also in vivo. To this end, we studied the effects of disrupting the GPR30 gene in female and male mice. Female GPR30((-/-)) mice had hyperglycemia and impaired glucose tolerance, reduced body growth, increased blood pressure, and reduced serum IGF-I levels. The reduced growth correlated with a proportional decrease in skeletal development. The elevated blood pressure was associated with an increased vascular resistance manifested as an increased media to lumen ratio of the resistance arteries. The hyperglycemia and impaired glucose tolerance in vivo were associated with decreased insulin expression and release in vivo and in vitro in isolated pancreatic islets. GPR30 is expressed in islets, and GPR30 deletion abolished estradiol-stimulated insulin release both in vivo in ovariectomized adult mice and in vitro in isolated islets. Our findings show that GPR30 is important for several metabolic functions in female mice, including estradiol-stimulated insulin release.

Bradykinin sequesters B2 bradykinin receptors and the receptor-coupled Galpha subunits Galphaq and Galphai in caveolae in DDT1 MF-2 smooth muscle cells

In this report, we show that the vasoactive peptide agonist bradykinin (BK) when bound to B2 BK receptors on DDT1 MF-2 smooth muscle cells promotes the recruitment and sequestration of the occupied receptors and the receptor-coupled G-protein α subunits Gαq and Gαi in caveolae. Association of ligand receptor complexes and Gα subunits with caveolae was indicated by their co-enrichment on density gradients with caveolin, a marker protein for caveolae. Caveolin and Gα subunits were monitored by immunoblotting, whereas receptors were monitored as ligand receptor complexes formed by labeling receptors with the agonist BK or the antagonist NPC17731 prior to cell disruption and caveolae enrichment. These complexes were detected with radioligand and by immunoblotting with BK antibodies. A direct interaction of Gα subunits with caveolin was also indicated by their co-immunoprecipitation. Immunoelectron microscopy revealed that the enriched caveolin, Gα subunits, and BK receptor complexes were present in structures of 0.1–0.2 μm. At 4 °C, BK and NPC17731 receptor complexes were detected in caveolae, and both complexes were sensitive to acid washing prior to cell disruption and caveolae enrichment. Elevation of the temperature to 37 °C increased the amount of BK receptor complexes in caveolae with a maximal response at 10 min (continuous labeling) or 20 min (single-round labeling), and the complexes became acid-resistant. These conditions also increased the amount of Gαq and Gαi in caveolae with a maximal response at 5–10 min. In contrast, the NPC17731 receptor complexes remained acid-sensitive and dissociated at this temperature, and antagonists did not increase the amount of Gα subunits in caveolae. These results show that some agonists that act through G-protein-coupled receptors promote the association of their receptors and receptor-coupled Gα subunits with caveolae.

PI 3-kinase regulation of dopamine uptake

The magnitude and duration of dopamine (DA) signaling is defined by the amount of vesicular release, DA receptor sensitivity, and the efficiency of DA clearance, which is largely determined by the DA transporter (DAT). DAT uptake capacity is determined by the number of functional transporters on the cell surface as well as by their turnover rate. Here we show that inhibition of phosphatidylinositol (PI) 3‐kinase with LY294002 induces internalization of the human DAT (hDAT), thereby reducing transport capacity. Acute treatment with LY294002 reduced the maximal rate of [3H]DA uptake in rat striatal synaptosomes and in human embryonic kidney (HEK) 293 cells stably expressing the hDAT (hDAT cells). In addition, LY294002 caused a significant redistribution of the hDAT from the plasma membrane to the cytosol. Conversely, insulin, which activates PI 3‐kinase, increased [3H]DA uptake and blocked the amphetamine‐induced hDAT intracellular accumulation, as did transient expression of constitutively active PI 3‐kinase. The LY294002‐induced reduction in [3H]DA uptake and hDAT cell surface expression was inhibited by expression of a dominant negative mutant of dynamin I, indicating that dynamin‐dependent trafficking can modulate transport capacity. These data implicate DAT trafficking in the hormonal regulation of dopaminergic signaling, and suggest that a state of chronic hypoinsulinemia, such as in diabetes, may alter synaptic DA signaling by reducing the available cell surface DATs.

GPR30/GPER1: searching for a role in estrogen physiology

Estrogens are sex hormones that are central to health and disease in both genders. These hormones have long been recognized to act in complex ways, both through relatively slow genomic mechanisms and via fast non-genomic mechanisms. Several recent in vitro studies suggest that GPR30, or G protein-coupled estrogen receptor 1 (GPER1), is a functional membrane estrogen receptor involved in non-genomic estrogen signaling. However, this function is not universally accepted. Studies concerning the role of GPER1 in vivo are now beginning to appear but with divergent results. In this review we discuss current knowledge on the physiological role of GPER1 in the nervous system as well as in reproduction, metabolism, bone, and in the cardiovascular and immune systems.

G Protein-Coupled Estrogen Receptor 1 (GPER1)/GPR30 Localizes in the Plasma Membrane and Trafficks Intracellularly on Cytokeratin Intermediate Filaments.

G protein-coupled receptor 30 [G protein-coupled estrogen receptor 1 (GPER1)], has been introduced as a membrane estrogen receptor and a candidate cancer biomarker and therapeutic target. However, several questions surround the subcellular localization and signaling of this receptor. In native cells, including mouse myoblast C2C12 cells, Madin-Darby canine kidney epithelial cells, and human ductal breast epithelial tumor T47-D cells, G-1, a GPER1 agonist, and 17β-estradiol stimulated GPER1-dependent cAMP production, a defined plasma membrane (PM) event, and recruitment of β-arrestin2 to the PM. Staining of fixed and live cells showed that GPER1 was localized both in the PM and on intracellular structures. One such intracellular structure was identified as cytokeratin (CK) intermediate filaments, including those composed of CK7 and CK8, but apparently not endoplasmic reticulum, Golgi, or microtubules. Reciprocal coimmunoprecipitation of GPER1 and CKs confirmed an association of these proteins. Live staining also showed that the PM receptors constitutively internalize apparently to reach CK filaments. Receptor localization was supported using FLAG- and hemagglutinin-tagged GPER1. We conclude that GPER1-mediated stimulation of cAMP production and β-arrestin2 recruitment occur in the PM. Furthermore, the PM receptors constitutively internalize and localize intracellularly on CK. This is the first observation that a G protein-coupled receptor is capable of associating with intermediate filaments, which may be important for GPER1 regulation in epithelial cells and the relationship of this receptor to cancer.

Kinins promote B2 receptor endocytosis and delay constitutive B1 receptor endocytosis.

Upon sustained insult, kinins are released and many kinin responses, such as inflammatory pain, adapt from a B2 receptor (B2R) type in the acute phase to a B1 receptor (B1R) type in the chronic phase. In this study, we show that kinins modulate receptor endocytosis to rapidly decrease B2R and increase B1R on the cell surface. B2Rs, which require agonist for activity, are stable plasma membrane components without agonist but recruit β-arrestin 2, internalize in a clathrin-dependent manner, and recycle rapidly upon agonist treatment. In contrast, B1Rs, which are inducible and constitutively active, constitutively internalize without agonist via a clathrin-dependent pathway, do not recruit β-arrestin 2, bind G protein-coupled receptor sorting protein, and target lysosomes for degradation. Agonist delays B1R endocytosis, thus transiently stabilizing the receptor. Most of the receptor trafficking phenotypes are transplantable from one receptor to the other through exchange of the C-terminal receptor tails, indicating that the tails contain epitopes that are important for the binding of protein partners that participate in the endocytic and postendocytic receptor choices. It is noteworthy that the agonist delay of B1R endocytosis is not transplanted to the B2R via the B1R tail, suggesting that this property of the B1R requires another domain. These events provide a rapid kinin-dependent mechanism for 1) regulating the constitutive B1R activity and 2) shifting the balance of accessible receptors in favor of B1R.

The human B1 bradykinin receptor exhibits high ligand-independent, constitutive activity: Roles of residues in the fourth intracellular and third transmembrane domains

The B1 bradykinin (BK) receptor (B1R) is a seven-transmembrane domain, G protein-coupled receptor that is induced by injury and important in inflammation and nociception. Here, we show that the human B1R exhibits a high level of ligand-independent, constitutive activity. Constitutive activity was identified by the increase in basal cellular phosphoinositide hydrolysis as a function of the density of the receptors in transiently transfected HEK293 cells. Several B1R peptide antagonists were neutral antagonists or very weakly efficacious inverse agonists. Constitutive B1R activity was further increased by alanine mutation of Asn121 in the third transmembrane domain of the receptor (B1A121). This mutant resembled the agonist-preferred receptor state since it also exhibited increased agonist affinity and decreased agonist responsiveness. A dramatic loss of constitutive activity occurred when the fourth intracellular C-terminal domain (IC-IV) of the human B2 BK receptor subtype (B2R), which exhibits minimal constitutive activity, was substituted in either B1R or B1A121 to make B1(B2ICIV) and B1(B2ICIV)A121, respectively. Activity was partially recovered by subsequent alanine mutation of a cluster of two serines and two threonines in IC-IV of either B1(B2ICIV) or B1(B2ICIV)A121, a cluster that is important for B2R desensitization. The ligand-independent, constitutive activity of B1R therefore depends on epitopes in both transmembrane and intracellular domains. We propose that the activity is primarily due to the lack of critical epitopes in IC-IV that regulate such activity.

Requirement for direct cross-talk between B1 and B2 kinin receptors for the proliferation of androgen-insensitive prostate cancer PC3 cells.

Stimulation of endogenous kinin receptors promotes growth of androgen-independent prostate cancer PC3 cells via activation of the mitogenic extracellular-signal-regulated kinase (ERK) pathway. In the present study, we show that kinin-mediated mitogenic signalling and prostate-cell growth involves two subtypes of bradykinin (BK) receptors, B1R and B2R. Specific stimulation of either B1R or B2R by their respective agonists des-Arg(9)-BK and Lys-BK promoted ERK activation and cell growth, whereas selective blockade with specific antagonists des-Arg(9)-[Leu(8)]BK and Hoe 140 respectively obliterated this effect, indicating the presence of both receptor subtypes. However, blockade of B1R also inhibited B2R-mediated ERK activation and cell growth, and, similarly, antagonism of B2R inhibited the B1R-mediated response. Furthermore, both B1R and B2R agonists promoted internalization of B1R, whereas both receptor antagonists blocked this effect. The B1R ligands des-Arg(9)-BK and des-Arg(9)-[Leu(8)]BK had no effect on the binding of BK to B2R, as demonstrated by radioligand competitive binding studies. However, blockade of either B1R or B2R impaired the ability of the reciprocal receptor to produce inositol phosphates, suggesting that the interaction between B1R and B2R is proximal to activation of phospholipase C. These results provide evidence for the existence of B1R-B2R complexes in prostate cancer PC3 cells and demonstrate that antagonism of one receptor interferes with the signalling ability of the other, possibly at the level of receptor-Galpha(q) protein coupling. Selective inhibition of B1R, which is up-regulated in injured and cancerous tissue, may be beneficial for the treatment of advanced prostate cancer.

The GPER1 Agonist G-1 Attenuates Endothelial Cell Proliferation by Inhibiting DNA Synthesis and Accumulating Cells in the S and G2 Phases of the Cell Cycle

G protein-coupled receptor 30 (GPR30) or G protein-coupled estrogen receptor 1 (GPER1) is expressed in the vasculature, but the importance of vascular GPER1 remains to be clarified. Here we investigate effects of the GPER1 agonist G-1 on endothelial cell proliferation using mouse microvascular endothelial bEnd.3 cells. The bEnd.3 cells express mRNA for GPER1. The bEnd.3 cells expressed both ERα and ERβ immunoreactivities. Treatment with G-1 reduced DNA synthesis and cell number with IC50 values of about 2 µM. GPER1 siRNA prevented G-1-induced attenuation of DNA synthesis. G-1 accumulated cells in S and G2 phases of the cell cycle, suggesting that G-1 blocks transition between G2 and M. G-1 had no effect on DNA synthesis in COS-7 cells only weakly expressing GPER1 mRNA. 17β-Estradiol had no effect on DNA synthesis in physiological concentrations (nM). The ER blocker ICI182780 reduced DNA synthesis with similar potency as G-1. Treatment with the ERK/MAP kinase inhibitor PD98059 had no effect on G-1-induced attenuation of DNA synthesis. G-1- induced antiproliferation was observed not only in bEnd.3 cells but also in human umbilical vein endothelial cells and HMEC-1 endothelial cells. We conclude that the GPER1 agonist G-1 attenuates endothelial cell proliferation via inhibition of DNA synthesis and by accumulation of cells in S and G2.

G protein-coupled oestrogen receptor 1 (GPER1)/GPR30: a new player in cardiovascular and metabolic oestrogenic signalling

Oestrogens are important sex hormones central to health and disease in both genders that have protective effects on the cardiovascular and metabolic systems. These hormones act in complex ways via both genomic and non‐genomic mechanisms. The genomic mechanisms are relatively well characterized, whereas the non‐genomic ones are only beginning to be explored. Two oestrogen receptors (ER), ERα and ERβ, have been described that act as nuclear transcription factors but can also associate with the plasma membrane and influence cytosolic signalling. ERα has been shown to mediate both anti‐atherogenic effects and pro‐survival effects in pancreatic β‐cells. In recent years, a third membrane‐bound ER has emerged, G protein‐coupled receptor 30 or G protein‐coupled oestrogen receptor 1 (GPER1), which mediates oestrogenic responses in cardiovascular and metabolic regulation. Both GPER1 knock‐out models and pharmacological agents are now available to study GPER1 function. These tools have revealed that GPER1 activation may have several beneficial effects in the cardiovascular system including vasorelaxation, inhibition of smooth muscle cell proliferation, and protection of the myocardium against ischaemia/reperfusion injury, and in the metabolic system including stimulation of insulin release and protection against pancreatic β‐cell apoptosis. Thus, GPER1 is emerging as a candidate therapeutic target in both cardiovascular and metabolic disease.