Adaptor protein-2 interaction with arrestin regulates GPCR recycling and apoptosis.

G protein-coupled receptors (GPCRs) are integral to cellular function in nearly all physiologic and many pathologic processes. GPCR signaling represents an intricate balance between receptor activation, inactivation (desensitization, internalization and degradation) and resensitization (recycling and de novo synthesis). Complex formation between phosphorylated GPCRs, arrestins and an ever-increasing number of effector molecules is known to regulate cellular function. Previous studies have demonstrated that, although N-formyl peptide receptor (FPR) internalization occurs in the absence of arrestins, FPR recycling is arrestin-dependent. Furthermore, FPR stimulation in the absence of arrestins leads to receptor accumulation in perinuclear endosomes and apoptosis. In this study, we show that the interaction of GPCR-bound arrestin with adaptor protein-2 (AP-2) is a critical anti-apoptotic event. In addition, AP-2 associates with the receptor-arrestin complex in perinuclear endosomes and is required for proper post-endocytic GPCR trafficking. Finally, we observed that depletion of endogenous AP-2 results in the initiation of apoptosis upon stimulation of multiple GPCRs, including P2Y purinergic receptors and CXCR2, but not CXCR4. We propose a model in which the abnormal accumulation of internalized GPCR-arrestin complexes in recycling endosomes, resulting from defective arrestin-AP-2 interactions, leads to the specific initiation of aberrant signaling pathways and apoptosis.

Calmodulin modulates Akt activity in human breast cancer cell lines.

Growth factor-induced activation of Akt occurs in the majority of human breast cancer cell lines resulting in a variety of cellular outcomes, including suppression of apoptosis and enhanced survival. We demonstrate that epidermal growth factor (EGF)-initiated activation of Akt is mediated by the ubiquitous calcium sensing molecule, calmodulin, in the majority of human breast cancer cell lines. Specifically, in estrogen receptor (ER)-negative, but not ER-positive, breast cancer cells, Akt activation is abolished by treatment with the calmodulin antagonist, W-7. Suppression of calmodulin expression by siRNAs against all three calmodulin genes in c-Myc-overexpressing mouse mammary carcinoma cells results in significant inhibition of EGF-induced Akt activation. Additionally, transient expression of constitutively active Akt (Myr-Akt) can overcome W-7-mediated suppression of Akt activation. These results confirm the involvement of calmodulin in the Akt pathway. The calmodulin independence of EGF-initiated Akt signaling in some cells was not explained by calmodulin expression level. Additionally, it was not explained by ER status or activation, since removal of estrogen and ablation of the ER did not convert the ER-positive, W-7 insensitive, MCF-7 cell line to calmodulin dependent signaling. However, forced overexpression of either epidermal growth factor receptor (EGFR) or ErbB2 did partially restore calmodulin dependent EGF-stimulated Akt activation. This is consistent with observation that W-7 sensitive cells tend to be estrogen independent and express high levels of EGFR family members. In an attempt to address how calmodulin is regulating Akt activity, we looked at localization of fluorescently tagged Akt and calmodulin in MCF-7 and SK-BR-3 cells. We found that both Akt and calmodulin translocate to the membrane after EGF-stimulation, and this translocation to the same sub-cellular compartment is inhibited by the calmodulin inhibitor W-7. Thus, calmodulin may be regulating Akt activity by modulating its sub-cellular location and is a novel target in the poor prognosis, ER-negative subset of breast cancers.

A Selective Ligand for Estrogen Receptor Proteins Discriminates Rapid and Genomic Signaling.

Estrogen exerts extensive and diverse effects throughout the body of women. In addition to the classical nuclear estrogen receptors (ERα and ERß), the G protein-coupled estrogen receptor GPER is an important mediator of estrogen action. Existing ER-targeted therapeutic agents act as GPER agonists. Here, we report the identification of a small molecule, named AB-1, with the previously unidentified activity of high selectivity for binding classical ERs over GPER. AB-1 also possesses a unique functional activity profile as an agonist of transcriptional activity but an antagonist of rapid signaling through ERα. Our results define a class of small molecules that discriminate between the classical ERs and GPER, as well as between modes of signaling within the classical ERs. Such an activity profile, if developed into an ER antagonist, could represent an opportunity for the development of first-in-class nuclear hormone receptor-targeted therapeutics for breast cancer exhibiting reduced acquired and de novo resistance.

GPR30: a novel indicator of poor survival for endometrial carcinoma.

OBJECTIVE: This study was undertaken to evaluate the relationship between GPR30, classical steroidal receptor expression, and clinical outcome in patients with endometrial carcinoma. STUDY DESIGN: Immunohistochemistry was used to investigate the expression of GPR30, estrogen, progesterone, epidermal growth factor receptors and Ki-67 in 47 consecutive consenting patients with endometrial carcinoma diagnosed between 1997 and 2001. Results were correlated with clinical and pathologic predictors of adverse outcome and survival. RESULTS: GPR30 correlated positively with epidermal growth factor receptor (P = .005), but negatively with progesterone (P = .05) receptor expression. GPR30 overexpression occurred more frequently in tumors with deep myometrial invasion, high-grade, biologically aggressive histologic subtypes, and advanced stage. In patients with GPR30 overexpression, survival was significantly poorer (65.2% vs 100%, P = .005). CONCLUSION: GPR30 represents an alternative estrogen-responsive receptor that is overexpressed in tumors where estrogen and progesterone receptors are downregulated, and in high-risk endometrial cancer patients with lower survival rates.

Altered Ca2+ homeostasis in polymorphonuclear leukocytes from chronic myeloid leukaemia patients.

BACKGROUND: In polymorphonuclear leukocytes (PMNL), mobilization of calcium ions is one of the early events triggered by binding of chemoattractant to its receptors. Besides chemotaxis, a variety of other functional responses are dependent on calcium ion mobilization. PMNL from chronic myeloid leukaemia (CML) patients that were morphologically indistinguishable from normal PMNL were found to be defective in various functions stimulated by a chemoattractant - fMLP. To study the mechanism underlying defective functions in CML PMNL, we studied calcium mobilization in CML PMNL in response to two different classical chemoattractants, fMLP and C5a. RESULTS: Release of calcium estimated by flow cytometry and spectrofluorimetry using fluo-3 as an indicator showed that the [Ca2+]i levels were lower in CML PMNL as compared to those in normal PMNL. But, both normal and CML PMNL showed maximum [Ca2+]i in response to fMLP and C5a at 10 sec and 30 sec, respectively. Spectrofluorimetric analysis of the total calcium release in chemoattractant treated PMNL indicated more and faster efflux of [Ca2+]i in CML PMNL as compared to normal PMNL. CONCLUSION: Fine-tuning of Ca2+ homeostasis was altered in CML PMNL. The altered Ca2+ homeostasis may contribute to the defective functions of CML PMNL.

Synthetic estrogen derivatives demonstrate the functionality of intracellular GPR30.

Estrogen mediates its effects through multiple cellular receptors. In addition to the classical nuclear estrogen receptors (ERalpha and ERbeta), estrogen also signals through the seven-transmembrane G-protein-coupled receptor (GPCR) GPR30. Although estrogen is a cell-permeable ligand, it is often assumed that all GPCRs function solely as cell surface receptors. Our previous results showed that GPR30 appeared to be expressed predominantly in the endoplasmic reticulum. A critical question that arises is whether this localization represents the site of functional receptor. To address this question, we synthesized a collection of cell-permeable and cell-impermeable estrogen derivatives. We hypothesized that if functional GPR30 were expressed at the cell surface, both permeable and impermeable derivatives would show activity. However, if functional GPR30 were predominantly intracellular, like ERalpha, only the permeable ligands should show activity. Cell permeability was assessed using cells expressing ERalpha as a model intracellular estrogen-binding receptor. Our results reveal that despite exhibiting similar binding affinities for GPR30, only the cell-permeable ligands are capable of stimulating rapid calcium mobilization and phosphoinositide 3-kinase (PI3K) activation. We conclude that GPR30 expressed intracellularly is capable of initiating cellular signaling and that there is insufficient GPR30 expressed on the cell surface to initiate signaling in response to impermeable ligands in the cell lines examined. To our knowledge, this is the first definitive demonstration of a functional intracellular transmembrane estrogen receptor.

Linkage effects on binding affinity and activation of GPR30 and estrogen receptors ERalpha/beta with tridentate pyridin-2-yl hydrazine tricarbonyl-Re/(99m)Tc(I) chelates.

We describe a new structural class of neutral tridentate pyridin-2-yl hydrazine chelates for labeling with tricarbonyl Re/99mTc(I) under aqueous conditions and investigate the receptor binding of synthetic estradiol derivatives with the novel G-protein-coupled receptor GPR30 and estrogen receptors ERalpha/beta. The steroid linkage affected the affinity and selectivity of estrogen binding with these receptors. Fluorescence assays based on calcium signaling demonstrate that membrane-permeable chelates 2 and 3 interact with the receptors in whole cells. These results suggest that in vitro assays will facilitate the development of targeted imaging agents for intracellular receptors and the feasibility of targeting GPR30 and ERalpha/beta for diagnostic tumor imaging.

Virtual and biomolecular screening converge on a selective agonist for GPR30.

Estrogen is a hormone critical in the development, normal physiology and pathophysiology of numerous human tissues. The effects of estrogen have traditionally been solely ascribed to estrogen receptor alpha (ERalpha) and more recently ERbeta, members of the soluble, nuclear ligand-activated family of transcription factors. We have recently shown that the seven-transmembrane G protein-coupled receptor GPR30 binds estrogen with high affinity and resides in the endoplasmic reticulum, where it activates multiple intracellular signaling pathways. To differentiate between the functions of ERalpha or ERbeta and GPR30, we used a combination of virtual and biomolecular screening to isolate compounds that selectively bind to GPR30. Here we describe the identification of the first GPR30-specific agonist, G-1 (1), capable of activating GPR30 in a complex environment of classical and new estrogen receptors. The development of compounds specific to estrogen receptor family members provides the opportunity to increase our understanding of these receptors and their contribution to estrogen biology.

A transmembrane intracellular estrogen receptor mediates rapid cell signaling.

The steroid hormone estrogen regulates many functionally unrelated processes in numerous tissues. Although it is traditionally thought to control transcriptional activation through the classical nuclear estrogen receptors, it also initiates many rapid nongenomic signaling events. We found that of all G protein-coupled receptors characterized to date, GPR30 is uniquely localized to the endoplasmic reticulum, where it specifically binds estrogen and fluorescent estrogen derivatives. Activating GPR30 by estrogen resulted in intracellular calcium mobilization and synthesis of phosphatidylinositol 3,4,5-trisphosphate in the nucleus. Thus, GPR30 represents an intracellular transmembrane estrogen receptor that may contribute to normal estrogen physiology as well as pathophysiology.

Arrestins block G protein-coupled receptor-mediated apoptosis.

G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptors (GPCRs) activate numerous cellular signals through the combined actions of G proteins, GPCR kinases, and arrestins. Although arrestins have traditionally been thought of as mediating GPCR desensitization, they have now been shown to play important roles in the internalization, trafficking, and signaling of many GPCRs. We demonstrate that in cells devoid of arrestins, the stimulation of numerous GPCRs including the N-formyl peptide receptor (FPR) initiates rapid cell rounding, annexin V positivity, and caspase activation followed by cell death. The apoptotic response is initiated by G protein signaling and involves activation of phosphoinositide 3-kinase, mitogen-activated protein kinases, and c-Src resulting in cytochrome c release from mitochondria and ultimately caspase 9 and caspase 3 activation. Reconstitution with either arrestin-2 or arrestin-3 is completely sufficient to prevent FPR-mediated apoptosis. Surprisingly, a non-desensitizing and non-internalizing mutant of the FPR is unable to initiate apoptosis, indicating that receptor phosphorylation and internalization, but not solely chronic activation due to a lack of desensitization, are critical determinants for the induction of apoptosis by the FPR. We further demonstrate that this response is not unique to the FPR with numerous additional GPCRs, including the V2 vasopressin, angiotensin II (type 1A), and CXCR2 receptors, capable of initiating apoptosis upon stimulation, whereas GPCRs such as the beta(2)-adrenergic receptor and CXCR4 are not capable of initiating apoptotic signaling. These data demonstrate for the first time that arrestins play a critical and completely unexpected role in the suppression GPCR-mediated apoptosis, which we show is a common consequence of GPCR-mediated cellular activation in the absence of arrestins.

N-formyl peptide receptors internalize but do not recycle in the absence of arrestins.

Arrestins mediate phosphorylation-dependent desensitization, internalization, and initiation of signaling cascades for the majority of G protein-coupled receptors (GPCRs). Many GPCRs undergo agonist-mediated internalization through arrestin-dependent mechanisms, wherein arrestin serves as an adapter between the receptor and endocytic proteins. To understand the role of arrestins in N-formyl peptide receptor (FPR) trafficking, we stably expressed the FPR in a mouse embryonic fibroblast cell line (MEF) that lacked endogenous arrestin 2 and arrestin 3 (arrestin-deficient). We compared FPR internalization and recycling kinetics in these cells to congenic wild type MEF cell lines. Internalization of the FPR was not altered in the absence of arrestins. Since the FPR remains associated with arrestins following internalization, we investigated whether the rate of FPR recycling was altered in arrestin-deficient cells. While the FPR was able to recycle in the wild type cells, receptor recycling was largely absent in the arrestin double knockout cells. Reconstitution of the arrestin-deficient line with either arrestin 2 or arrestin 3 restored receptor recycling. Confocal fluorescence microscopy studies demonstrated that in arrestin-deficient cells the FPR may become trapped in the perinuclear recycling compartment. These observations indicate that, although the FPR can internalize in the absence of arrestins, recycling of internalized receptors to the cell surface is prevented. Our results suggest a novel role for arrestins in the post-endocytic trafficking of GPCRs.