The contribution of classical (beta1/2-) and atypical beta-adrenoceptors to the stimulation of human white adipocyte lipolysis and right atrial appendage contraction by novel beta3-adrenoceptor agonists of differing selectivities

The role of beta3- and other putative atypical beta-adrenoceptors in human white adipocytes and right atrial appendage has been investigated using CGP 12177 and novel phenylethanolamine and aryloxypropanolamine beta3-adrenoceptor (beta3AR) agonists with varying intrinsic activities and selectivities for human cloned betaAR subtypes. The ability to demonstrate beta1/2AR antagonist-insensitive (beta3 or other atypical betaAR-mediated) responses to CGP 12177 was critically dependent on the albumin batch used to prepare and incubate the adipocytes. Four aryloxypropanolamine selective beta3AR agonists (SB-226552, SB-229432, SB-236923, SB-246982) consistently elicited beta1/2AR antagonist-insensitive lipolysis. However, a phenylethanolamine (SB-220646) that was a selective full beta3AR agonist elicited full lipolytic and inotropic responses that were sensitive to beta1/2AR antagonism, despite it having very low efficacies at cloned beta1- and beta2ARs. A component of the response to another phenylethanolamine selective beta3AR agonist (SB-215691) was insensitive to beta1/2AR antagonism in some experiments. Because no [corrected] novel aryloxypropanolamine had a beta1/2AR antagonist-insensitive inotropic effect, these results establish more firmly that beta3ARs mediate lipolysis in human white adipocytes, and suggest that putative 'beta4ARs' mediate inotropic responses to CGP 12177. The results also illustrate the difficulty of predicting from studies on cloned betaARs which betaARs will mediate responses to agonists in tissues that have a high number of beta1- and beta2ARs or a low number of beta3ARs.     

Desensitization of the beta-adrenergic response in human brown adipocytes

Activation of adenylyl cyclase by beta-adrenergic receptors (betaARs) plays a major role in adipose tissue homeostasis. The increase in cAMP promotes lipolysis in white adipose tissue, activates both thermogenesis and lipolysis in brown adipose tissue (BAT), and induces BAT hypertrophy. Previous studies indicated that among the three betaAR subtypes present in adipose tissue, beta3AR could be a potential target for antiobesity treatments in humans. We studied immortalized human brown adipocytes (PAZ6 adipocytes) as a model of beta-adrenergic response in human BAT. PAZ6 adipocytes and freshly isolated mature human brown adipocytes display the same proportions of betaAR subtypes, with beta3AR being the most abundant (approximately 80% of the total). However, beta3AR was poorly coupled to the adenylyl cyclase pathway in PAZ6 cells, contributing to only 10% of the isoproterenol-induced accumulation of cAMP, whereas 20% and 70% of the signal depended on beta1- and beta2-subtypes, respectively. Upon isoproterenol stimulation, beta1- and beta2AR down-regulated with a half-life of about 3 h and the beta3AR with a half-life of 30-40 h. Long term stimulation with both saturating (micromolar) and nonsaturating (nanomolar) concentrations of beta-adrenergic agonists caused a complete desensitization of the beta-adrenergic response at the adenylyl cyclase level and loss of stimulated protein kinase A activity and CREB phosphorylation. These results suggest that cAMP-dependent processes will be desensitized upon permanent treatment with beta3AR agonists. Further studies should establish whether the beta3AR is coupled to other signaling pathways in human brown adipocytes and whether these may contribute to BAT hypertrophy and/or thermogenesis.     

Reciprocal in vivo regulation of myocardial G protein-coupled receptor kinase expression by beta-adrenergic receptor stimulation and blockade

BACKGROUND: Impaired myocardial beta-adrenergic receptor (betaAR) signaling, including desensitization and functional uncoupling, is a characteristic of congestive heart failure. A contributing mechanism for this impairment may involve enhanced myocardial beta-adrenergic receptor kinase (betaARK1) activity because levels of this betaAR-desensitizing G protein-coupled receptor kinase (GRK) are increased in heart failure. An hypothesis has emerged that increased sympathetic nervous system activity associated with heart failure might be the initial stimulus for betaAR signaling alterations, including desensitization. We have chronically treated mice with drugs that either activate or antagonize betaARs to study the dynamic relationship between betaAR activation and myocardial levels of betaARK1. METHODS AND RESULTS: Long-term in vivo stimulation of betaARs results in the impairment of cardiac +betaAR signaling and increases the level of expression (mRNA and protein) and activity of +betaARK1 but not that of GRK5, a second GRK abundantly expressed in the myocardium. Long-term beta-blocker treatment, including the use of carvedilol, improves myocardial betaAR signaling and reduces betaARK1 levels in a specific and dose-dependent manner. Identical results were obtained in vitro in cultured cells, demonstrating that the regulation of GRK expression is directly linked to betaAR signaling. CONCLUSIONS: This report demonstrates, for the first time, that betaAR stimulation can significantly increase the expression of betaARK1 , whereas beta-blockade decreases expression. This reciprocal regulation of betaARK1 documents a novel mechanism of ligand-induced betaAR regulation and provides important insights into the potential mechanisms responsible for the effectiveness of beta-blockers, such as carvedilol, in the treatment of heart failure.     

The isoflavone genistein inhibits copper and peroxyl radical mediated low density lipoprotein oxidation in vitro

OBJECTIVES: Hormone-independent and cytotoxic drug-resistant tumor growth in osteoblastic metastases defines poor survival in patients with advanced prostate cancer. Therefore, we analyzed the ability of human osteoblast-like cells (MG-63 cells) and MG-63 conditioned media (MG-63 CM) to protect PC-3 human prostate cancer cells from adriamycin cytotoxicity in vitro. METHODS: Adriamycin cytotoxicity was assessed in MG-63 osteoblast-like and PC-3 prostate cancer monolayer and three-dimensional collagen coculture systems using the DNA content and trypan blue exclusion assays, analysis of indexes of cell cycle by flow cytometry, determination of DNA fragmentation on simple agarose gel and terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) assay, and immunocytochemistry. RESULTS: Adriamycin (100 nM) arrested both the PC-3 and MG-63 cells at the G2/M phase in the cell cycle but induced apoptosis only in PC-3 cells, as assessed by flow cytometry, trypan blue exclusion, and agarose gel. Optimal doses of MG-63 CM (50 microg/mL), insulin-like growth factor I (50 ng/mL), and transforming growth factor-beta-1 (25 ng/mL), as determined by DNA content assay, partially neutralized the adriamycin cytotoxicity of PC-3 cells detected by flow cytometry and trypan blue exclusion. In addition, MG-63 cells rescued PC-3 cells from adriamycin apoptosis in the three-dimensional type I collagen gel coculture system, as analyzed by TUNEL assay. CONCLUSIONS: These data suggest that osteoblast-like cells and osteoblast-derived growth factors can optimize survival of metastatic prostate cancer cells, thereby helping to develop cytotoxic drug-resistant growth in vitro.     

Endogenous lesions, S-phase-independent spontaneous mutations, and evolutionary strategies for base excision repair

The mitogen-activated protein (MAP) kinases (p44mapk and p42mapk), also known as extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2), are activated in response to a variety of extracellular signals, including growth factors, hormones and, neurotransmitters. We have investigated MAP kinase signal transduction pathways in normal human osteoblastic cells. Normal human bone marrow stromal (HBMS), osteoblastic (HOB), and human (TE85, MG-63, SaOS-2), rat (ROS 17/2.8, UMR-106) and mouse (MC3T3-E1) osteoblastic cell lines contained immunodetectable p44mapk/ERK1 and p42mapk/ERK2. MAP kinase activity was measured by 'in-gel' assay using myelin basic protein as the substrate. Mainly ERK2 was rapidly activated (within 10 min) by bFGF, IGF-I and PDGF-BB in normal HOB, HBMS and human osteosarcoma cells, whereas both ERK1 and ERK2 were activated by growth factors in rat osteoblast-like cell lines, ROS 17/2.8 and UMR-106. The ERK1 activation was greater than the ERK2 in ROS 17/2.8 cells. Furthermore, ERK2 was also activated by bFGF and PDGF-BB in the mouse osteoblastic cell line, MC3T3-E1. This is the first demonstration of inter-species differences in the activation of MAP kinases in osteoblastic cells. Cyclic AMP derivatives or cAMP generating agents such as PTH and forskolin inhibited ERK2 activation by bFGF and PDGF-BB suggesting a 'cross-talk' between the two different signalling pathways activated by receptor tyrosine kinases and cAMP-dependent protein kinase. The accumulated results also suggest that the MAP kinases may be involved in mediating mitogenic and other biological actions of bFGF, IGF-I and PDGF-BB in normal human osteoblastic and bone marrow stromal cells.     

Integrin alpha 2 beta 1 is a positive regulator of collagenase (MMP-1) and collagen alpha 1(I) gene expression

A classical model for studying the effects of extracellular matrix is to culture cells inside a three-dimensional collagen gel. When surrounded by fibrillar collagen, many cell types decrease the production of type I collagen, and the expression of interstitial collagenase (matrix metalloproteinase-1; MMP-1) is simultaneously induced. To study the role of the collagen-binding integrins alpha 1 beta 1 and alpha 2 beta 1 in this process, we used three different osteogenic cell lines with distinct patterns of putative collagen receptors: HOS cells, which express only alpha 1 beta 1 integrin, MG-63 cells, which express only alpha 2 beta 1 integrin, and KHOS-240 cells, which express both. Inside collagen gels, alpha 1 (I) collagen mRNA levels were decreased in HOS and KHOS-240 cells but not in MG-63 cells. In contrast, MMP-1 expression was induced in KHOS-240 and MG-63 cells but not in HOS cells. Transfection of MG-63 cells with alpha 2 integrin cDNA in an antisense orientation reduced the expression level of alpha 2 integrin. These cell clones showed induction and reduction of mRNA levels for MMP-1, respectively. HOS cells normally lacking alpha 2 beta 1 integrin were forced to express it, and this prevented the down-regulation in the levels of alpha 1 (I) collagen mRNA when cells were grown inside collagen gels. The data indicate that the level of MMP-1 expression is regulated by the collagen receptor alpha 2 beta 1 integrin. The down-regulation of collagen alpha 1 (I) is mediated by another receptor. Integrin alpha 2 beta 1 may compete with it and thus be a positive regulator of collagen synthesis.     

Cloning of a novel retinoic acid-inducible mRNA from human osteoblast-like cells

Retinoids have long been known to influence skeletal development and bone remodeling. Cells of the osteoblastic lineage play a key role in these processes. In this study we have used the differential display PCR technique to identify retinoic acid (RA)-induced mRNAs in human osteoblast-like cells. We report the cloning and sequencing of one such mRNA, AT-RA 6, which was specifically induced by all-trans RA both in normal human osteoblast-like cells and in MG-63 osteosarcoma cells. Maximal expression was found after 60 min, suggesting that this may be an early response gene. Expression was found in all tissues examined. No homology to known mRNA sequences was detected.     

An inverted cAMP response element mediates the cAMP induction of the ovine beta 1-adrenergic receptor gene

We identified an inverted, functional cAMP response element (CRE) located at--1599 bp relative to the translation start site within the ovine beta 1-adrenergic receptor (beta 1 AR) gene promoter. In transfection studies with SK-N-MC cells, a 40-bp oligonucleotide containing the potential CRE, beta 1 AR-CRE, conferred a 3- to 4-fold increase in luciferase activity mediated by cAMP. The induction was mimicked by co-transfecting the cells with a vector overexpressing the alpha-catalytic subunit of the cAMP-dependent protein kinase (PKA) without treatment, and was blocked by overexpressing a PKA inhibitor (PKI). In electrophoretic mobility shift assays, a discrete binding pattern was shown in cell nuclear extract probed with the 40 bp beta 1 AR-CRE. The binding was shown to be specific and supershifted by addition of a CRE binding protein (CREB-1) antibody. These data demonstrate that cAMP mediates the induction of beta 1 AR gene expression by interacting with an inverted CRE within the promoter region. This is the first reported functional CRE among all beta 1 AR genes.     

3-Pyridylethanolamines: potent and selective human beta 3 adrenergic receptor agonists

The 3-pyridylethanolamine L-757,793 is a potent beta 3 AR agonist (EC50 6.3 nM, 70% activation) with 1,300- and 500-fold selectivity over binding to the beta 1 and beta 2 ARs, respectively. L-757,793 stimulated lipolysis in rhesus monkeys (ED50 0.2 mg/kg) with a maximum response equivalent to that elicited by isoproterenol.     

Detection of differential gene expression in human osteoblastic cells by non-radioactive RNA arbitrarily primed PCR

The aim of the present study was to detect differentially expressed genes in the human osteoblast-like osteosarcoma cell line SaOS-2 using non-radioactive RNA fingerprinting (RNA arbitrarily primed polymerase chain reaction, RAP-PCR). RNA was isolated at different time points from SaOS-2 cells grown with and without dexamethasone (DEX). By RAP-PCR we detected changes in band patterns of cells treated with DEX compared with untreated cells. PCR fragments further characterized and sequences from three of these gave perfect matches to the coding sequences of the human nucleophosmin gene B23, cDNA clone 4_c6 from P1 H25 and the human TRA1 gene, respectively. differential regulation of these genes in DEX-stimulated SaOS-2 cells could be demonstrated by RT-PCR.     

Ketamine inhibits the proinflammatory cytokine-induced reduction of cardiac intracellular cAMP accumulation

The proinflammatory cytokines, including tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma), are increased in heart failure and sepsis, clinical conditions for which the IV anesthetic ketamine is useful. The proinflammatory cytokines cause beta-adrenergic receptor (betaAR) hypofunction secondary to reduced function of the enzyme adenylylcyclase (AC). In this study, we evaluated the effect of ketamine alone, TNF-alpha and IFN-gamma, and ketamine plus TNF-alpha and IFN-gamma, on isoproterenol (ISO, a betaAR agonist) and forskolin (FSK, an activator of AC)-induced intracellular accumulation of cAMP. An in vitro culture of a rat heart cell line (H9c2) was labeled with [3H]adenine to produce [3H]ATP, and we measured the intracellular accumulation of [3H]cAMP after stimulation with ISO or FSK to convert the [3H]ATP to [3H]cAMP. Pretreatment with either cytokine alone did not significantly affect ISO or FSK-induced intracellular cAMP accumulation, whereas the combination of TNF-alpha and IFN-gamma caused a significant (P < 0.05 compared with untreated cells) reduction. Pretreatment with ketamine caused a significant (P < 0.05 compared with untreated cells) increase in ISO or FSK-induced cAMP accumulation. Pretreatment of the H9c2 cells with ketamine, plus the combination of TNF-alpha and IFN-gamma, inhibited the reduction of ISO or FSK-induced intracellular cAMP accumulation caused by the proinflammatory cytokines alone. These results demonstrate that the combination of the proinflammatory cytokines TNF-alpha and IFN-gamma reduce poststimulation (ISO or FSK) intracellular cAMP accumulation. This action of the proinflammatory cytokines is consistent with the observation of betaAR hyporesponsiveness to betaAR agonist therapy in sepsis and heart failure. Ketamine augments the poststimulation cAMP accumulation in H9c2 cells while inhibiting the cytokine-induced reduction of cAMP accumulation. This may partly explain the improvement in cardiac function after ketamine use in clinical conditions known to have increased systemic levels of proinflammatory cytokines, such as sepsis and heart failure. IMPLICATIONS: Tumor necrosis factor-alpha and interferon-gamma reduced poststimulation intracellular cAMP levels, whereas ketamine inhibits this action of the proinflammatory cytokines. Because cAMP is the second messenger for the beta-adrenergic receptor, this may be a mechanism for improved blood pressure and cardiac output in sepsis and heart failure after ketamine use.     

Regulation of endothelial permeability by beta-adrenoceptor agonists: contribution of beta 1- and beta 2-adrenoceptors

The barrier function of cultured, macrovascular endothelial cells derived from bovine aorta was analyzed using confluent monolayers of cells and measuring the exchange of fluorescein dextrans of different molecular masses. The effects of beta-adrenoceptor agonists with different selectivity for beta 1- and beta 2-adrenoceptors (AR) were investigated. Formoterol, a novel high-affinity agonist for beta 2-AR recently introduced in the treatment of bronchial asthma, showed a significant reduction of cell permeability with subnanomolar concentrations, whereas the catecholamines (-)-isoproterenol and (-)-norepinephrine only showed significant effects with micromolar concentrations. In order to elucidate if this difference in potential to regulate cell permeability is related to appropriate changes in the selectivity and affinity of the agonists for beta 2 AR, we investigated the beta AR-coupled adenylate cyclase (AC) in membranes from endothelial cells and compared AC stimulation with the binding of agonists to the receptors using [125I](-)-iodopindolol as radioligand. beta-Adrenoceptors revealed to be closely coupled to AC as assessed by a similar magnitude of effects by receptor agonists in comparison to GTP analogues and direct stimulants of AC activity. AC activity was increased by formoterol in parallel to its receptor occupancy of beta 2AR with nanomolar concentrations which were 50-fold higher than those used for the regulation of cell permeability indicating the existence of spare receptors. In contrast to formoterol, the catecholamines (-)-isoproterenol and (-)-norepinephrine stimulated AC activity through both beta 1AR and beta 2AR. From the overproportional high contribution of beta 1AR to AC stimulation (42%) in comparison to its low fraction (13%) in receptor binding we calculated that beta 1AR is 3-4-fold more effectively coupled to AC than beta 2 AR.     

Increase of vascular endothelial growth factor mRNA expression by 1,25-dihydroxyvitamin D3 in human osteoblast-like cells

Vascular endothelial growth factor (VEGF), a secreted endothelial cell-specific mitogen, is produced in endocrine organs and regulated by trophic hormones. Because angiogenesis and osteogenesis are closely regulated, we studied whether human osteoblast-like cells produce VEGF, and if so, what factors regulate VEGF mRNA expression. Human osteoblast-like cells (HObLC) derived from trabecular bone explants were cultured in alpha-MEM supplemented with 10% fetal calf serum. Northern blot analysis revealed that HObLC expressed VEGF mRNA, as did several human osteosarcoma cells. 1,25-(OH)2D3 increased the steady-state levels of VEGF mRNA in a time- and concentration-dependent manner in HObLC and one of the osteosarcoma cell lines, SaOS-2, accompanied by an increase in the concentration of immunoreactive VEGF in the conditioned medium. PTH and IGF-I also increased the level of VEGF mRNA in HObLC and SaOS-2 cells. Furthermore, 12-O-tetradecanoylphorbol ester stimulated VEGF mRNA in a time-and concentration-dependent manner. The VEGF mRNA expression induced by 1,25-(OH)2D3 was completely inhibited by H-7, but only partially by staurosporine. We have demonstrated that PTH, IGF-I, and most potently 1,25-(OH)2D3 stimulate the mRNA expression and secretion of VEGF in human osteoblast-like cells, suggesting that one of the anabolic effects of 1,25-(OH)2D3 on skeletal tissue may be mediated by VEGF produced by osteoblasts.     

Chromatographic and immunological identification of GnRH (gonadotropin-releasing hormone) variants. Occurrence of mammalian and a salmon-like GnRH in the forebrain of an eutherian mammal: Hydrochaeris hydrochaeris (Mammalia, Rodentia)

Phosphorylation of the beta 2-adrenergic receptor (beta 2AR) is the initial event that underlies rapid agonist-promoted desensitisation. However, the role of phosphorylation in mediating long-term beta 2AR desensitisation is not known. To investigate this possibility, we performed intact cell phosphorylation studies with COS-7 cells transiently expressing an epitope tagged wild-type beta 2AR and found that receptor phosphorylation in cells treated with 1 microM isoproterenol for 24 h was approximately 4-fold over the basal state. This finding suggested that persistent phosphorylation of the receptor might contribute to functional long-term desensitisation which we further explored with mutated beta 2AR lacking the determinants of phosphorylation by the beta AR kinase (beta ARK), PKA or both. In CHW cells expressing the WT beta 2AR, pretreatment with 1 microM isoproterenol for 24 h reduced the isoproterenol-stimulated cAMP response by 82 +/- 5%. Substitution of the PKA sites with alanines had no effect on the extent of desensitisation (77 +/- 6%, P = NS compared to WT). In contrast, desensitisation was only 49 +/- 4% (P < 0.001 compared to WT) when the beta ARK sites were similarly substituted. Removal of both the beta ARK and PKA sites impaired desensitisation to the same extent as the beta ARK mutant. The extent of receptor loss (downregulation) was the same among all of the cell lines used and therefore could not account for the observed differences in desensitisation. Cellular beta ARK activity, assessed by a rhodopsin phosphorylation assay, was equivalent in all cell lines and was unaffected by agonist treatment. PKA activity, however, was dynamically regulated, increasing 4-fold over basal levels after 15 min of isoproterenol and returning to near basal levels after 24 h. The lower level of PKA activity after long-term agonist exposure may therefore have contributed to the apparent lack of effect of removing PKA sites. Nonetheless, long-term desensitisation was clearly attenuated with beta 2AR lacking beta ARK phosphorylation sites. These findings show that in addition to its role in regulating short-term desensitisation, beta ARK-mediated phosphorylation is an important mechanism underlying long-term desensitisation of the beta 2AR as well.     

Cytokine-induced inhibition of bone matrix proteins is not mediated by prostaglandins

Interleukin-1 (IL-1) and tumor necrosis factor (TNF), two pleiotropic cytokines produced in inflammatory processes, inhibit bone matrix biosynthesis and stimulate prostanoid formation in osteoblasts. In the present study, the importance of prostaglandin formation in IL-1 and TNF-induced inhibition of osteocalcin and type I collagen formation has been examined. In the human osteoblastic cell line MG-63, IL-1 alpha (10-1000 pg/ml), IL-1 beta (3-300 pg/ml) and TNF-alpha (1-30 ng/ml) stimulated prostaglandin E2 (PGE2) formation and inhibited 1,25(OH)2-vitamin D3-induced osteocalcin biosynthesis as well as basal production of type I collagen. Addition of PGE2 or increasing the endogenous formation of PGE2 by treating the cells with arachidonic acid, bradykinin, Lys-bradykinin or des-Arg9-bradykinin, did not affect osteocalcin and type I collagen formation in unstimulated or 1,25(OH)2-vitamin D3-stimulated osteoblasts. Four non-steroidal antiinflammatory drugs, indomethacin, flurbiprofen, naproxen and meclofenamic acid, inhibited basal, IL-1 beta- and TNF-alpha-stimulated PGE2 formation in the MG-63 cells without affecting IL-1 beta- or TNF-alpha-induced inhibition of osteocalcin and type I collagen formation. In isolated, non-transformed, human osteoblast-like cells, IL-1 beta and TNF-alpha stimulated PGE2 formation and concomitantly inhibited 1,25(OH)2-vitamin D3-stimulated osteocalcin biosynthesis, without affecting type I collagen formation. In these cells, indomethacin and flurbiprofen abolished the effects of IL-1 beta and TNF-alpha on prostaglandin formation without affecting the inhibitory effects of the cytokines on osteocalcin biosynthesis. These data show that IL-1 and TNF inhibit osteocalcin and type I collagen formation in osteoblasts independently of prostaglandin biosynthesis and that non-steroidal antiinflammatory drugs do not affect the effects of IL-1 and TNF on bone matrix biosynthesis.     

A collagen peptide motif activates tyrosine kinase-dependent calcium signalling pathways in human osteoblast-like cells

A collagen peptide motif (DGEA) which is a putative alpha 2 beta 1 integrin binding site was examined for its ability to activate Ca2+ signalling pathways in the human osteoblast-like cell line SaOS-2. We show that these cells express both alpha 2 beta 1 integrin subunits (by immunocytochemistry) and that an anti-beta 1 monoclonal antibody (DF5) mobilizes Ca2+ in these cells. DGEA elevated intracellular Ca2+ in fura-2-loaded cells, in a concentration- and sequence-dependent fashion, with an EC50 of 250 microM. The tyrosine kinase inhibitor herbimycin A reduced the number of cells responding to DGEA and to transforming growth factor alpha. Thrombin also stimulated a rise in intracellular Ca2+, but the number of cells responding was not reduced by herbimycin A. The DGEA response was dependent on extracellular Ca2+, but was not due to Ca2+ influx, since it was blocked by thapsigargin and not by lanthanum. Using three different anti-alpha 2 monoclonal antibodies, we were unable to show that the DGEA-induced Ca2+ signal was mediated by the alpha 2 beta 1 integrin. In summary, the DGEA collagen motif does appear to activate receptor-mediated Ca2+ signalling events in SaOS-2 cells, in a divalent cation-dependent manner, but we were unable to demonstrate a role for alpha 2 beta 1 integrin in this response.     

Visualization of agonist-induced sequestration and down-regulation of a green fluorescent protein-tagged beta2-adrenergic receptor

To date, the visualization of beta2-adrenergic receptor (beta2AR) trafficking has been largely limited to immunocytochemical analyses of acute internalization events of epitope-tagged receptors in various transfection systems. The development of a beta2AR conjugated with green fluorescent protein (beta2AR-GFP) provides the opportunity for a more extensive optical analysis of beta2AR sequestration, down-regulation, and recycling in cells. Here we demonstrate that stable expression of beta2AR-GFP in HeLa cells enables a detailed temporal and spatial analysis of these events. Time-dependent colocalization of beta2AR-GFP with rhodamine-labeled transferrin and rhodamine-labeled dextran following agonist exposure demonstrates receptor distribution to early endosomes (sequestration) and lysosomes (down-regulation), respectively. The observed temporal distribution of beta2AR-GFP was consistent with measures of receptor sequestration and down-regulation generated by radioligand-receptor binding assays. Cells stimulated with different beta-agonists revealed time courses of beta2AR-GFP redistribution reflective of the intrinsic activity of each agonist.