Robert H. Oakley

Association of beta-arrestin with G protein-coupled receptors during clathrin-mediated endocytosis dictates the profile of receptor resensitization.

Resensitization of G protein-coupled receptors (GPCRs) following agonist-mediated desensitization is a necessary step for maintaining physiological responsiveness. However, the molecular mechanisms governing the nature of GPCR resensitization are poorly understood. Here, we examine the role of β-arrestin in the resensitization of the β2 adrenergic receptor (β2AR), known to recycle and resensitize rapidly, and the vasopressin V2 receptor (V2R), known to recycle and resensitize slowly. Upon agonist activation, both receptors recruit β-arrestin to the plasma membrane and internalize in a β-arrestin- and clathrin-dependent manner. However, whereas β-arrestin dissociates from the β2AR at the plasma membrane, it internalizes with the V2R into endosomes. The differential trafficking of β-arrestin and the ability of these two receptors to dephosphorylate, recycle, and resensitize is completely reversed when the carboxyl-terminal tails of these two receptors are switched. Moreover, the ability of β-arrestin to remain associated with desensitized GPCRs during clathrin-mediated endocytosis is mediated by a specific cluster of phosphorylated serine residues in the receptor carboxyl-terminal tail. These results demonstrate that the interaction of β-arrestin with a specific motif in the GPCR carboxyl-terminal tail dictates the rate of receptor dephosphorylation, recycling, and resensitization, and thus provide direct evidence for a novel mechanism by which β-arrestins regulate the reestablishment of GPCR responsiveness.

Proinflammatory cytokines regulate human glucocorticoid receptor gene expression and lead to the accumulation of the dominant negative beta isoform: a mechanism for the generation of glucocorticoid resistance.

Inflammatory responses in many cell types are coordinately regulated by the opposing actions of NF-κB and the glucocorticoid receptor (GR). The human glucocorticoid receptor (hGR) gene encodes two protein isoforms: a cytoplasmic alpha form (GRα), which binds hormone, translocates to the nucleus, and regulates gene transcription, and a nuclear localized beta isoform (GRβ), which does not bind known ligands and attenuates GRα action. We report here the identification of a tumor necrosis factor (TNF)-responsive NF-κB DNA binding site 5′ to the hGR promoter that leads to a 1.5-fold increase in GRα mRNA and a 2.0-fold increase in GRβ mRNA in HeLaS3 cells, which endogenously express both GR isoforms. However, TNF-α treatment disproportionately increased the steady-state levels of the GRβ protein isoform over GRα, making GRβ the predominant endogenous receptor isoform. Similar results were observed following treatment of human CEMC7 lymphoid cells with TNF-α or IL-1. The increase in GRβ protein expression correlated with the development of glucocorticoid resistance.

The Interaction of β-Arrestin with the AP-2 Adaptor Is Required for the Clustering of β2-Adrenergic Receptor into Clathrin-coated Pits

β-Arrestins are cytosolic proteins that regulate the signaling and the internalization of G protein-coupled receptors (GPCRs). Although termination of receptor coupling requires β-arrestin binding to agonist-activated receptors, GPCR endocytosis involves the coordinate interactions between receptor-β-arrestin complexes and other endocytic proteins such as adaptor protein 2 (AP-2) and clathrin. Clathrin interacts with a conserved motif in the β-arrestin C-terminal tail; however, the specific molecular determinants in β-arrestin that bind AP-2 have not been identified. Moreover, the respective contributions of the interactions of β-arrestin with AP-2 and clathrin toward the targeting of GPCRs to clathrin-coated vesicles have not been established. Here, we identify specific arginine residues (Arg394 and Arg396) in the β-arrestin 2 C terminus that mediate β-arrestin binding to AP-2 and show, in vitro, that these domains in β-arrestin 1 and 2 interact equally well with AP-2 independently of clathrin binding. We demonstrate in HEK 293 cells by fluorescence microscopy that β2-adrenergic receptor-β-arrestin complexes lacking the β-arrestin-clathrin binding motif are still targeted to clathrin-coated pits. In marked contrast, receptor-β-arrestin complexes lacking the β-arrestin/AP-2 interactions are not effectively compartmentalized in punctated areas of the plasma membrane. These results reveal that the binding of a receptor-β-arrestin complex to AP-2, not to clathrin, is necessary for the initial targeting of β2-adrenergic receptor to clathrin-coated pits.

The biology of the glucocorticoid receptor: new signaling mechanisms in health and disease.

Glucocorticoids are primary stress hormones necessary for life that regulate numerous physiologic processes in an effort to maintain homeostasis. Synthetic derivatives of these hormones have been mainstays in the clinic for treating inflammatory diseases, autoimmune disorders, and hematologic cancers. The physiologic and pharmacologic actions of glucocorticoids are mediated by the glucocorticoid receptor (GR), a member of the nuclear receptor superfamily of ligand-dependent transcription factors. Ligand-occupied GR induces or represses the transcription of thousands of genes through direct binding to DNA response elements, physically associating with other transcription factors, or both. The traditional view that glucocorticoids act through a single GR protein has changed dramatically with the discovery of a large cohort of receptor isoforms with unique expression, gene-regulatory, and functional profiles. These GR subtypes are derived from a single gene by means of alternative splicing and alternative translation initiation mechanisms. Posttranslational modification of these GR isoforms further expands the diversity of glucocorticoid responses. Here we discuss the origin and molecular properties of the GR isoforms and their contribution to the specificity and sensitivity of glucocorticoid signaling in healthy and diseased tissues.

Expression and Subcellular Distribution of the β-Isoform of the Human Glucocorticoid Receptor1

Alternative splicing of the human glucocorticoid receptor (hGR) primary transcript produces two highly homologous protein isoforms, termed hGRα and hGRβ, that differ at their carboxy-termini. In contrast to the well characterized hGRα isoform, which modulates gene expression in a hormone-dependent fashion, the biological significance of hGRβ has only recently begun to emerge. We and others have shown that the hGRβ messenger RNA transcript is widely expressed in human tissues and that the hGRβ protein functions as a dominant negative inhibitor of hGRα in transfected cells. Unfortunately, these initial studies did not determine whether the hGRβ protein was made in vivo. Such analyses are hindered because available anti-hGR antibodies cannot discriminate between the similarly sized hGRα and hGRβ proteins. Therefore, to investigate the expression of the hGRβ protein, we have produced an antipeptide, hGRβ-specific antibody termed BShGR. This antibody was made against the unique 15-amino acid peptide at the car...

Role of CRF Receptor Signaling in Stress Vulnerability, Anxiety, and Depression

Markers of hyperactive central corticotropin releasing factor (CRF) systems and CRF‐related single nucleotide polymorphisms (SNPs) have been identified in patients with anxiety and depressive disorders. Designing more effective antagonists may now be guided by data showing that small molecules bind to transmembrane domains. Specifically, CRF1 receptor antagonists have been developed as novel anxiolytic and antidepressant treatments. Because CRF1 receptors become rapidly desensitized by G protein‐coupled receptor kinase (GRK) and β‐arrestin mechanisms in the presence of high agonist concentrations, neuronal hypersecretion of synaptic CRF alone may be insufficient to account for excessive central CRF neurotransmission in stress‐induced affective pathophysiology. In addition to desensitizing receptor function, GRK phosphorylation and β‐arrestin binding can shift a G protein‐coupled receptor (GPCR) to signal selectively via the extracellular signal‐regulated kinase/mitogen‐activated protein kinase (ERK‐MAPK) or Akt pathways independent of G proteins. Also, Epac‐dependent CRF1 receptor signaling via the ERK‐MAPK pathway has been found to potentiate brain‐derived neurotrophic factor (BDNF)‐stimulated TrkB signaling. Thus, genetic or acquired abnormalities in GRK and β‐arrestin function may be involved in the pathophysiology of stress‐induced anxiety and depression.

A β-Arrestin Binding Determinant Common to the Second Intracellular Loops of Rhodopsin Family G Protein-coupled Receptors

β-Arrestins have been shown to inhibit competitively G proteindependent signaling and to mediate endocytosis for many of the hundreds of nonvisual rhodopsin family G protein-coupled receptors (GPCR). An open question of fundamental importance concerning the regulation of signal transduction of several hundred rhodopsin-like GPCRs is how these receptors of limited sequence homology, when considered in toto, can all recruit and activate the two highly conserved β-arrestin proteins as part of their signaling/desensitization process. Although the serine and threonine residues that form GPCR kinase phosphorylation sites are common β-arrestin-associated receptor determinants regulating receptor desensitization and internalization, the agonist-activated conformation of a GPCR probably reveals the most fundamental determinant mediating the GPCR and arrestin interaction. Here we identified a β-arrestin binding determinant common to the rhodopsin family GPCRs formed from the proximal 10 residues of the second intracellular loop. We demonstrated by both gain and loss of function studies for the serotonin 2C, β2-adrenergic, α2a-adrenergic, and neuropeptide Y type 2 receptorsthat the highly conserved amino acids, proline and alanine, naturally occurring in rhodopsin family receptors six residues distal to the highly conserved second loop DRY motif regulate β-arrestin binding and β-arrestin-mediated internalization. In particular, as demonstrated for the β2 AR, this occurs independently of changes in GPCR kinase phosphorylation. These results suggest that a GPCR conformation directed by the second intracellular loop, likely using the loop itself as a binding patch, may function as a switch for transitioning β-arrestin from its inactive form to its active receptor-binding state.

Dual Role for Glucocorticoids in Cardiomyocyte Hypertrophy and Apoptosis

Glucocorticoids and their synthetic derivatives are known to alter cardiac function in vivo; however, the nature of these effects and whether glucocorticoids act directly on cardiomyocytes are poorly understood. To explore the role of glucocorticoid signaling in the heart, we used rat embryonic H9C2 cardiomyocytes and primary cardiomyocytes as model systems. Dexamethasone (100 nm) treatment of cardiomyocytes caused a significant increase in cell size and up-regulated the expression of cardiac hypertrophic markers, including atrial natriuretic factor, β-myosin heavy chain, and skeletal muscle α-actin. In contrast, serum deprivation and TNFα exposure triggered cardiomyocyte apoptosis, and these apoptotic effects were inhibited by dexamethasone. Both the hypertrophic and anti-apoptotic actions of glucocorticoids were abolished by the glucocorticoid receptor (GR) antagonist RU486 and by short hairpin RNA-mediated GR depletion. Blocking the activity of the mineralocorticoid receptor had no effect on these glucocorticoid-dependent cardiomyocyte responses. Aldosterone (1 μm) activation of GR also promoted cardiomyocyte hypertrophy and cell survival. To elucidate the mechanism of the dual glucocorticoid actions, a genome-wide microarray was performed on H9C2 cardiomyocytes treated with vehicle or dexamethasone in the absence or presence of serum. Serum dramatically influenced the transcriptome regulated by GR, revealing potential glucocorticoid signaling mediators in both cardiomyocyte hypertrophy and apoptosis. These studies reveal a direct and dynamic role for glucocorticoids and GR signaling in the modulation of cardiomyocyte function.

Quantitative cell-based high-content screening for vasopressin receptor agonists using transfluor technology.

The authors demonstrate the use of a simple, universal G-protein-coupled receptor (GPCR) assay to screen for agonists for a specific GPCR. Cells stably expressing a green fluorescent protein (GFP)-labeled β-arrestin fusion protein and the vasopressin V2 receptor (V2R) were used in a high-content screening (HCS) assay to screen a small peptide library for V2R agonists. Cells were treated with the peptides at a final concentration of 500 nM for 30min. Agonist stimulation causes V2R internalization into endosomes. GFP-β-arrestin remains associated with the V2R in endosomes, resulting in a fluorescent pattern of intracellular spots. Assay plates were automatically imaged and quantitatively analyzed using an HCS imaging platformand a fast turnkey image analysis application optimized for detection of receptor activation and intracellular spots. Hits were further evaluated to determine their potency. The combination of unique biology, automated high-content analysis, and a powerful means of validating hits results in better leads.

Immunocytochemical analysis of the glucocorticoid receptor alpha isoform (GRα) using a GRα-specific antibody

Abstract The alpha isoform of the glucocorticoid receptor (GRα) binds glucocorticoids and functions as a ligand-dependent transcription factor. Although GRα is expressed in almost all tissues and cells, its subcellular distribution is controversial. Many studies have reported that GRα translocates from the cytoplasm to the nucleus in a hormone-dependent manner whereas others have concluded that GRα is constitutively located in the nucleus. These conflicting data may result from the use of antibodies that do not discriminate GRα from a splice variant of the GR gene termed GRβ. Using a GRβ-specific antibody, we have recently demonstrated that GRβ resides in the nucleus of cells independent of glucocorticoid treatment. In the following study we have generated a novel GRα-specific antibody (AShGR) in order to assess, unambiguously, the subcellular distribution of GRα. AShGR recognizes recombinant GRα on Western blots and in immunoprecipitation experiments but does not cross-react with recombinant GRβ. Endogenous GRα is detected by AShGR in a variety of human cell lines including HeLa S 3 , CEM-C7, HEK-293, MCF-7, Hep G2, and secondary lung epithelial cells. In addition, AShGR detects endogenous rat and mouse GRα. Immunocytochemistry was performed with AShGR on COS-1 cells transfected with human GRα and on HTC rat hepatoma cells expressing endogenous GRα. In both systems, GRα was found in the cytoplasm of cells in the absence of hormone and in the nucleus after hormone treatment. These studies mark the first time a GRα-specific antibody has been employed to examine the expression and subcellular distribution of endogenous GRα.