Prolonged Agonist Stimulation Research Articles

Therapeutic potentials of nonpeptidic V2R agonists for partial cNDI-causing V2R mutants.

Loss-of-function mutations in the type 2 vasopressin receptor (V2R) are a major cause of congenital nephrogenic diabetes insipidus (cNDI). In the context of partial cNDI, the response to desmopressin (dDAVP) is partially, but not entirely, diminished. For those with the partial cNDI, restoration of V2R function would offer a prospective therapeutic approach. In this study, we revealed that OPC-51803 (OPC5) and its structurally related V2R agonists could functionally restore V2R mutants causing partial cNDI by inducing prolonged signal activation. The OPC5-related agonists exhibited functional selectivity by inducing signaling through the Gs-cAMP pathway while not recruiting β-arrestin1/2. We found that six cNDI-related V2R partial mutants (V882.53M, Y1283.41S, L1614.47P, T2736.37M, S3298.47R and S3338.51del) displayed varying degrees of plasma membrane expression levels and exhibited moderately impaired signaling function. Several OPC5-related agonists induced higher cAMP responses than AVP at V2R mutants after prolonged agonist stimulation, suggesting their potential effectiveness in compensating impaired V2R-mediated function. Furthermore, docking analysis revealed that the differential interaction of agonists with L3127.40 caused altered coordination of TM7, potentially contributing to the functional selectivity of signaling. These findings suggest that nonpeptide V2R agonists could hold promise as potential drug candidates for addressing partial cNDI.

Effect of docosahexaenoic acid, phorbol myristate acetate, and insulin on the interaction of the FFA4 (short isoform) receptor with Rab proteins

Human embryonic kidney (HEK) 293 cells were co-transfected with plasmids for the expression of mCherry fluorescent protein-tagged FFA4 receptors and the enhanced green fluorescent protein-tagged Rab proteins involved in retrograde transport and recycling, to study their possible interaction through Förster Resonance Energy Transfer (FRET), under the action of agents that induce FFA4 receptor phosphorylation and internalization through different processes, i.e., the agonist, docosahexaenoic acid, the protein kinase C activator phorbol myristate acetate, and insulin. Data indicate that FFA4 receptor internalization varied depending on the agent that induced the process. Agonist activation (docosahexaenoic acid) induced an association with early endosomes (as suggested by interaction with Rab5) and rapid recycling to the plasma membrane (as indicated by receptor interaction with Rab4). More prolonged agonist stimulation also appears to allow the FFA4 receptors to interact with late endosomes (interaction with Rab9), slow recycling (interaction with Rab 11), and target to degradation (Rab7). Phorbol myristate acetate, triggered a rapid association with early endosomes (Rab5), slow recycling to the plasma membrane (Rab11), and some receptor degradation (Rab7). Insulin-induced FFA4 receptor internalization appears to be associated with interaction with early endosomes (Rab5) and late endosomes (Rab9) and fast and slow recycling to the plasma membrane (Rab4, Rab11). Additionally, we observed that agonist- and PMA-induced FFA4 internalization was markedly reduced by paroxetine, which suggests a possible role of G protein-coupled receptor kinase 2.

Agonist-induced down-regulation of AMPA receptors in oligodendrocyte progenitors

Prolonged exposure of oligodendrocyte progenitor cultures to non-toxic concentrations of glutamate receptor agonists for 24 h decreased cellular proliferation mediated by α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. Since prolonged agonist stimulation can regulate the expression of various families of receptors, we examined this possibility. Pretreatment of progenitor cultures with 100 μM kainic acid (KA) for 1–24 h caused a time-dependent decrease in AMPA receptor activity, determined by agonist-induced 45Ca2+ uptake. The maximum effect (70–80% decrease), observed in the 24 h-pretreated cells, was accompanied by a significant reduction in AMPA receptor subunits, as determined by Western blotting. GluR2/3 and GluR4 subunits were the most affected. Receptor down-regulation and 45Ca2+ uptake were only partially reversible upon KA removal. Furthermore, 24 h co-treatment of cultures with CNQX blocked the KA-induced decreases in calcium uptake. To address whether calpain, a calcium-activated protease, was implicated in the regulation of the AMPA receptor subunits, cultures were treated with the specific inhibitor PD150606 alone or in combination with KA for 24 h. Calpain inhibition significantly increased GluR1 in both conditions and partly reversed downregulation of GluR4 by KA. Collectively, these results indicate that calpain is not involved in the agonist-induced down-regulation of AMPA receptors subunits 2/3 in oligodendrocyte progenitors, while it downregulates GluR1 and GluR4.

MARCH2 promotes endocytosis and lysosomal sorting of carvedilol-bound β2-adrenergic receptors

Lysosomal degradation of ubiquitinated β(2)-adrenergic receptors (β(2)ARs) serves as a major mechanism of long-term desensitization in response to prolonged agonist stimulation. Surprisingly, the βAR antagonist carvedilol also induced ubiquitination and lysosomal trafficking of both endogenously expressed β(2)ARs in vascular smooth muscle cells (VSMCs) and overexpressed Flag-β(2)ARs in HEK-293 cells. Carvedilol prevented β(2)AR recycling, blocked recruitment of Nedd4 E3 ligase, and promoted the dissociation of the deubiquitinases USP20 and USP33. Using proteomics approaches (liquid chromatography-tandem mass spectrometry), we identified that the E3 ligase MARCH2 interacted with carvedilol-bound β(2)AR. The association of MARCH2 with internalized β(2)ARs was stabilized by carvedilol and did not involve β-arrestin. Small interfering RNA-mediated down-regulation of MARCH2 ablated carvedilol-induced ubiquitination, endocytosis, and degradation of endogenous β(2)ARs in VSMCs. These findings strongly suggest that specific ligands recruit distinct E3 ligase machineries to activated cell surface receptors and direct their intracellular itinerary. In response to β blocker therapy with carvedilol, MARCH2 E3 ligase activity regulates cell surface β(2)AR expression and, consequently, its signaling.

Prolonged Stimulation of μ-Opioid Receptors Produces β-Arrestin-2-Mediated Heterologous Desensitization of α2-Adrenoceptor Function in Locus Ceruleus Neurons

Prolonged agonist stimulation of the μ-opioid receptor (MOR) initiates receptor regulatory events that rapidly attenuate receptor-mediated signaling (homologous desensitization). Emerging evidence suggests that persistent MOR stimulation can also reduce responsiveness of effectors to other G-protein-coupled receptors, termed heterologous desensitization. However, the mechanisms by which heterologous desensitization is triggered by MOR stimulation are unclear. This study used whole-cell patch-clamp recordings of ligand activated G-protein-activated inwardly rectifying potassium channel currents in mouse brain slices containing locus ceruleus (LC) neurons to determine the effects of prolonged stimulation of MOR on α(2)-adrenoceptor (α(2)-AR) function. The results show distinct and sequential development of homologous and heterologous desensitization during persistent stimulation of MOR in LC neurons with Met(5)-enkephalin (ME). ME stimulation of MOR promoted rapid homologous desensitization that reached a steady state after 5 min and partially recovered over 30 min. Longer stimulation of MOR (10 min) induced heterologous desensitization of α(2)-AR function that exhibited slower recovery than homologous desensitization. Heterologous (but not homologous) desensitization required β-arrestin-2 (βarr-2) because it was nearly abolished in βarr-2-knockout (ko) mice. Heterologous (but not homologous) desensitization was also prevented by inhibition of ERK1/2 and c-Src signaling in wild-type (wt) mouse LC neurons. Heterologous desensitization may be physiologically relevant during exposure to high doses of opioids because α(2)-AR-mediated slow inhibitory postsynaptic currents were depressed in wt but not βarr-2 ko LC neurons after prolonged exposure to opioids. Together, these findings demonstrate a novel mechanism by which βarr-2 can regulate postsynaptic responsiveness to neurotransmitter release.

Many ways to dilate the P2X7 receptor pore.

The P2X7 receptor is associated with two different membrane permeabilities: a small cation conductance which opens within milliseconds, followed by the appearance of a second channel carrying higher molecular weight compounds (including organic dyes) after prolonged agonist stimulation. This activation profile has also been found in cells expressing P2X2 and P2X4 receptors; however, the P2X7 receptor-dependent pathway has the unique ability to activate pro-inflammatory signalling in macrophages. In this issue of the BJP, Marques-da-Silva et al. demonstrate that colchicine is a potent inhibitor of both P2X7 and P2X2 receptor-dependent dye uptake, without affecting the ion channels. Colchicine also blocked the pro-inflammatory signalling downstream of P2X7 receptor activation, both in vitro and in vivo. This report suggests that the dye uptake associated with activation of P2X7 receptors is distinct from the P2X7 receptor ion channel and could be a therapeutic target for the treatment of chronic inflammation.

Modulation of doxorubicin-induced cardiac dysfunction in dominant-negative p38α mitogen-activated protein kinase mice

Doxorubicin (Dox) is a widely used antitumor drug, but its application is limited because of its cardiotoxic side effects. Increased expression of p38α mitogen-activated protein kinase (MAPK) promotes cardiomyocyte apoptosis and is associated with cardiac dysfunction induced by prolonged agonist stimulation. However, the role of p38α MAPK is not clear in Dox-induced cardiac injury. Cardiac dysfunction was induced by a single injection of Dox into wild-type (WT) mice and transgenic mice with cardiac-specific expression of a dominant-negative mutant form of p38α MAPK (TG). Left ventricular (LV) fractional shortening and ejection fraction were higher and the expression levels of phospho-p38 MAPK and phospho-MAPK-activated mitogen kinase 2 were significantly suppressed in TG mouse heart compared to WT mice after Dox injection. Production of LV proinflammatory cytokines, cardiomyocyte DNA damage, myocardial apoptosis, caspase-3-positive cells, and phospho-p53 expression were decreased in TG mice after Dox injection. Moreover, LV expression of NADPH oxidase subunits and reactive oxygen species was significantly less in TG mice compared to WT mice after Dox injection. These findings suggest that p38α MAPK may play a role in the regulation of cardiac function, oxidative stress, and inflammatory and apoptotic mediators in the heart after Dox administration.

Interactions of the G protein-coupled receptor-associated sorting proteins (GASP) 1 and 2 with the novel cannabinoid receptor GPR55

GPR55 was recently found to be a novel cannabinoid receptor and a member of the G protein-coupled receptor (GPCR) subfamily A. GPR55 is activated by different cannabinoid ligands as well as by the lipid ligand lysophosphatidylinositol (LPI). Generally, the activity of GPCRs is coordinated by receptor signaling, receptor desensitization and receptor resensitization. The latter two mechanisms are typically associated with the sorting of the GPCRs between degradation or recycling pathways and are highly regulated. Several sorting proteins have recently been identified, for example the G protein-coupled receptor-associated sorting protein-1 (GASP-1). GASP-1 was originally found to target delta opioid receptors (DORs) to lysosomes and hence a degradative pathway. This study shows that GPR55 can internalize after ligand activation and is subsequently targeted to intracellular vesicles of the lysosomal compartments. This result and the close similarity of GPR55 to the cannabinoid receptor CB1 – which is targeted to lysosomes via the GASP-1 protein – suggested that GASP may be involved in targeting GPR55 to lysosomes. In fact, here we show that the C-terminus of GPR55 binds GASP-1, cGASP-1 (the C-terminal part of GASP-1) and GASP-2 (the closest homologue to GASP-1) in vitro. Both GASP-1 and cGASP-1 show a high affinity to the C-terminus of GPR55 in vitro, with a ~2-fold higher affinity for GASP-1 when compared to the DOR. Interestingly, the GASP-2 isoform has the highest affinity for GPR55 with ~85% when compared to GASP-1 and cGASP-1 (~80%). This study provides first evidence that the novel cannabinoid receptor GPR55 is targeted to lysosomes after prolonged agonist stimulation and this mechanism is likely regulated by members of the newly discovered G protein-coupled receptor-associated sorting proteins, i.e GASP-1 and GASP-2.

ARNO/cytohesin-2 regulates desensitization of the A2A adenosine receptor in rat pheochromocytoma cells

The A2A adenosine receptor is a G protein-coupled receptor which desensitizes upon prolonged agonist stimulation. In order to understand the biological function of its unusually long C-terminus, we screened a human brain library for proteins capable of binding to the last 120 amino acids of the A2A receptor. We identified a guanine nucleotide exchange factor for the small G protein ARF6 (ARNO/cytohesin-2) as a binding partner. In this study, we investigated the impact of ARNO on A2A receptor signaling in rat pheochromocytoma (PC12) cells. These cells express the A2A receptors endogenously. We created cell lines with inducible expression of ARNO or its catalytic inactive mutant E156K. Neither wild type ARNO nor the mutant had an effect on receptor expression, signaling via adenylyl cyclase after activation or long-term de- and resensitization kinetics. In order to investigate effects of ARNO on A2A receptor short-term de- and resensitization we employed a FRET-based sensor to measure changes in cAMP in real time. Cells were transfected with plasmids encoding the regulatory and catalytic subunit of protein kinase A (PKA) fused to CFP and YFP, respectively. Accumulation of cAMP results in the dissociation of the PKA subunits, which can be measured in single cells as a loss of FRET. The presence of dominant negative ARNO accelerated the recovery of A2A receptor after stimulation and led to a pronounced signaling response when cells were re-challenged with agonist. While membrane recruitment of ARNO was not affected by the mutation, we observed a difference in the recovery of the A2A receptor after agonist treatment. Our results indicate that the interaction with ARNO/cytohesin-2 stabilizes short-term desensitization of the A2A receptor to prevent excessive stimulation.

The time-course of agonist-induced solubilization of trimeric Gqα/G11α proteins resolved by two-dimensional electrophoresis

Prolonged agonist stimulation results in specific transfer of activated Galpha subunits of G(q)alpha/G(11)alpha family from particulate membrane fraction to soluble (cytosol) cell fraction isolated as 250,000 x g supernatant. In this study, we have used 2D electrophoresis for more defined resolution of Galpha subunits of G(q)alpha/G(11)alpha family and followed the time course of solubilization effect. The small signal of soluble G proteins was already detected in control, hormone-unexposed cells. Hormone stimulation resulted in a slow but continuous increase of both intensity and number of immunoreactive signals/spots of these G proteins (10, 30, 60, 120 and 240 min). At longer times of agonist exposure (>2 hours), a marked increase of G(q)alpha/G(11)alpha proteins was detected. The maximal level of soluble G(q)alpha/G(11)alpha proteins was reached after 16 hours of continuous agonist exposure. At this time interval, eight individual immunoreactive signals of G(q)alpha/G(11)alpha proteins could be resolved. The relative proportion among these spots was 15:42:10:11:7:7:2:5. Solubilization of this class of Galpha proteins was thus observed after prolonged agonist stimulation only, induced by ultra high concentration of hormone and in cells expressing a large number of GPCRs. Our data therefore rather indicate tight/persisting binding of G(q)alpha/G(11)alpha proteins to the membrane.

P90 RSK-1 associates with and inhibits neuronal nitric oxide synthase.

Evidence is presented that RSK1 (ribosomal S6 kinase 1), a downstream target of MAPK (mitogen-activated protein kinase), directly phosphorylates nNOS (neuronal nitric oxide synthase) on Ser847 in response to mitogens. The phosphorylation thus increases greatly following EGF (epidermal growth factor) treatment of rat pituitary tumour GH3 cells and is reduced by exposure to the MEK (MAPK/extracellular-signal-regulated kinase kinase) inhibitor PD98059. Furthermore, it is significantly enhanced by expression of wild-type RSK1 and antagonized by kinase-inactive RSK1 or specific reduction of endogenous RSK1. EGF treatment of HEK-293 (human embryonic kidney) cells, expressing RSK1 and nNOS, led to inhibition of NOS enzyme activity, associated with an increase in phosphorylation of nNOS at Ser847, as is also the case in an in vitro assay. In addition, these phenomena were significantly blocked by treatment with the RSK inhibitor Ro31-8220. Cells expressing mutant nNOS (S847A) proved resistant to phosphorylation and decrease of NOS activity. Within minutes of adding EGF to transfected cells, RSK1 associated with nNOS and subsequently dissociated following more prolonged agonist stimulation. EGF-induced formation of the nNOS-RSK1 complex was significantly decreased by PD98059 treatment. Treatment with EGF further revealed phosphorylation of nNOS on Ser847 in rat hippocampal neurons and cerebellar granule cells. This EGF-induced phosphorylation was partially blocked by PD98059 and Ro31-8220. Together, these data provide substantial evidence that RSK1 associates with and phosphorylates nNOS on Ser847 following mitogen stimulation and suggest a novel role for RSK1 in the regulation of nitric oxide function in brain.

Seven Transmembrane Receptor Core Signaling Complexes Are Assembled Prior to Plasma Membrane Trafficking

Much is known about beta2-adrenergic receptor trafficking and internalization following prolonged agonist stimulation. However, less is known about outward trafficking of the beta2-adrenergic receptor to the plasma membrane or the role that trafficking might play in the assembly of receptor signaling complexes, important for targeting, specificity, and rapidity of subsequent signaling events. Here, by using a combination of bioluminescence resonance energy transfer, bimolecular fluorescence complementation, and confocal microscopy, we evaluated the steps in the formation of the core receptor-G protein heterotrimer complex. By using dominant negative Rab and Sar GTPase constructs, we demonstrate that receptor dimers and receptor-G betagamma complexes initially associate in the endoplasmic reticulum, whereas G alpha subunits are added to the complex during endoplasmic reticulum-Golgi transit. We also observed that G protein heterotrimers adopt different trafficking itineraries when expressed alone or with stoichiometric co-expression with receptor. Furthermore, deliberate mistargeting of specific components of these complexes leads to diversion of other members from their normal subcellular localization, confirming the role of these early interactions in targeting and formation of specific signaling complexes.

Knock-out Mice Reveal the Contributions of P2Y and P2X Receptors to Nucleotide-induced Ca2+ Signaling in Macrophages

Immune cell function is modulated by changes in extracellular nucleotide levels. Here we used reverse transcription-PCR analyses, single cell Ca2+ imaging, and knock-out mice to define the receptors mediating nucleotide-induced Ca2+ signaling in resident peritoneal macrophages. In Ca2+-free buffer, the potent (K0.5<1 microm) stimulatory effect of UTP (or ATP) on endoplasmic reticulum (ER) Ca2+ release was abolished in cells isolated from P2Y2/P2Y4 double knock-out mice. Moreover, P2Y4(0/-), but not P2Y2-/-, macrophages responded to UTP. In P2Y2-/- macrophages, we could elicit Ca2+ responses to "pure" P2X receptor activation by applying ATP in buffer containing Ca2+. Purified UDP and ADP were ineffective agonists, although modest UDP-induced Ca2+ responses could be elicited in macrophages after "activation" with lipopolysaccharide and interferon-gamma. Notably, in Ca2+-free buffer, UTP-induced Ca2+ transients decayed within 1 min, and there was no response to repeated agonist challenge. Measurements of ER [Ca2+] with mag-fluo-4 showed that ER Ca2+ stores were depleted under these conditions. When extracellular Ca2+ was available, ER Ca2+ stores refilled, but Ca2+ increased to only approximately 40% of the initial value upon repeated UTP challenge. This apparent receptor desensitization persisted in GRK2+/- and GRK6-/- macrophages and after inhibition of candidate kinases protein kinase C and calmodulin-dependent kinase II. Initial challenge with UTP also reduced Ca2+ mobilization by complement component C5a (and vice versa). In conclusion, homologous receptor desensitization is not the major mechanism that rapidly dampens Ca2+ signaling mediated by P2Y2, the sole Gq-coupled receptor for UTP or ATP in macrophages. UDP responsiveness (P2Y6 receptor expression) increases following macrophage activation.

Novel Function for Receptor Activity-modifying Proteins (RAMPs) in Post-endocytic Receptor Trafficking

RAMPs (1-3) are single transmembrane accessory proteins crucial for plasma membrane expression, which also determine receptor phenotype of various G-protein-coupled receptors. For example, adrenomedullin receptors are comprised of RAMP2 or RAMP3 (AM1R and AM2R, respectively) and calcitonin receptor-like receptor (CRLR), while a CRLR heterodimer with RAMP1 yields a calcitonin gene-related peptide receptor. The major aim of this study was to determine the role of RAMPs in receptor trafficking. We hypothesized that a PDZ type I domain present in the C terminus of RAMP3, but not in RAMP1 or RAMP2, leads to protein-protein interactions that determine receptor trafficking. Employing adenylate cyclase assays, radioligand binding, and immunofluorescence microscopy, we observed that in HEK293 cells the CRLR-RAMP complex undergoes agonist-stimulated desensitization and internalization and fails to resensitize (i.e. degradation of the receptor complex). Co-expression of N-ethylmaleimide-sensitive factor (NSF) with the CRLR-RAMP3 complex, but not CRLR-RAMP1 or CRLR-RAMP2 complex, altered receptor trafficking to a recycling pathway. Mutational analysis of RAMP3, by deletion and point mutations, indicated that the PDZ motif of RAMP3 interacts with NSF to cause the change in trafficking. The role of RAMP3 and NSF in AM2R recycling was confirmed in rat mesangial cells, where RNA interference with RAMP3 and pharmacological inhibition of NSF both resulted in a lack of receptor resensitization/recycling after agonist-stimulated desensitization. These findings provide the first functional difference between the AM1R and AM2R at the level of post-endocytic receptor trafficking. These results indicate a novel function for RAMP3 in the post-endocytic sorting of the AM-R and suggest a broader regulatory role for RAMPs in receptor trafficking.

Prolonged Agonist Stimulation Does Not Alter the Protein Composition of Membrane Domains in Spite of Dramatic Changes Induced in a Specific Signaling Cascade

Protein composition of membrane domains prepared by three different procedures (mechanical homogenization, alkaline treatment with 1 M Na2CO3 [pH 11.0], or extraction with nonionic detergent Triton X-100), and isolated from the bulk of plasma membranes by flotation on equilibrium sucrose density gradients, was analyzed by two-dimensional (2D) electrophoresis and compared in preparations from control (quiescent) and agonist-stimulated human embryonic kidney cells (HEK)293 or S49 cells. HEK293 cells (clone e2m11) stably expressing high levels of thyrotropin-releasing hormone receptor and G11alpha protein were stimulated by thyrotropin-releasing hormone and S49 lymphoma cells by the beta-adrenergic receptor agonist isoprenaline. Whereas sustained exposure (16 h) of both cell lines to the appropriate hormones led to substantial cellular redistribution and downregulation of the cognate G proteins (G(q)alpha/G11alpha and G(s)alpha, respectively), the distribution and levels of nonstimulated G(i) proteins remained unchanged. The 2D electrophoretic analysis of membrane domains distinguished approx 150-170 major proteins in these structures and none of these proteins was significantly altered by prolonged agonist stimulation. Furthermore, specific immunochemical determination of a number of plasma membrane markers, including transmembrane and glycosyl-phosphatidylinositol-anchored peripheral proteins, confirmed that their detergent-extractability/solubility was not influenced by hormone treatment. Collectively, our present data indicate that sustained hormone stimulation of target cells does not alter the basic protein composition of membrane domain/raft compartments of the plasma membrane in spite of marked changes proceeding in a given signaling cascade.

Alterations of beta-Adrenergic Receptor Signaling in Cardiac Hypertrophy and Heart Failure: beta-Adrenergic Receptor Desensitization in Cardiac Disease

β-adrenergic receptors (βAR) belong to the large family of G protein-coupled receptors that form the interface between the sympathetic nervous and cardiovascular systems. G protein-coupled receptors undergo adaptation to repeated or prolonged agonist stimulation, which is termed desensitization. Significant βAR desensitization occurs with the development of cardiac hypertrophy and heart failure, and uncoupling of βARs and defects in this pathway might be primary elements underlying the transition from compensated to uncompensated cardiac failure. Decreasing the level of myocardial βARK1 in established heart failure is a novel approach to improving impaired βAR receptor function, and potentially alter the pathogenesis of this disease. (Korean Circulation J 2005;35:485-492)

Resistance of the Human β1-Adrenergic Receptor to Agonist-induced Ubiquitination: A MECHANISM FOR IMPAIRED RECEPTOR DEGRADATION

Down-regulation is a classic response of most G protein-coupled receptors to prolonged agonist stimulation. We recently showed that when expressed in baby hamster kidney cells, the human beta1-but not the beta2-adrenergic receptor (AR) is totally resistant to agonist-mediated down-regulation, whereas both have similar rates of basal degradation (Liang, W., Austin, S., Hoang, Q., and Fishman, P. H. (2003) J. Biol. Chem. 278, 39773-39781). To identify the underlying mechanism(s) for this resistance, we investigated the role of proteasomes, lysosomes, and ubiquitination in the degradation of beta1AR expressed in baby hamster kidney and human embryonic kidney 293 cells. Both lysosomal and proteasomal inhibitors reduced beta1AR degradation in agonist-stimulated cells but were less effective on basal degradation. To determine whether beta1AR trafficked to lysosomes we used confocal fluorescence microscopy. We observed some colocalization of beta1AR and lysosomal markers in agonist-treated cells but much less than that of beta2AR even in cells co-transfected with arrestin-2, which increases beta1AR internalization. Ubiquitination of beta2AR readily occurred in agonist-stimulated cells, whereas ubiquitination of beta1AR was not detectable even under conditions optimal for that of beta2AR. Moreover, in cells expressing betaAR chimeras in which the C termini have been switched, the chimeric beta1AR with beta2AR C-tail underwent ubiquitination and down-regulation, but the chimeric beta2AR with beta1AR C-tail did not. Our results demonstrate for the first time that beta1AR and beta2AR differ in the ability to be ubiquitinated. Because ubiquitin serves as a signal for sorting membrane receptors to lysosomes, the lack of agonist-mediated ubiquitination of beta1AR may prevent its extensive trafficking to lysosomes and, thus, account for its resistance to down-regulation.

Receptor Trafficking via the Perinuclear Recycling Compartment Accompanied by Cell Division Is Necessary for Permanent Neurotensin Cell Sensitization and Leads to Chronic Mitogen-activated Protein Kinase Activation

Most G protein-coupled receptors are internalized after interaction with their respective ligand, a process that subsequently contributes to cell desensitization, receptor endocytosis, trafficking, and finally cell resensitization. Although cellular mechanisms leading to cell desensitization have been widely studied, those responsible for cell resensitization are still poorly understood. We examined here the traffic of the high affinity neurotensin receptor (NT1 receptor) following prolonged exposure to high agonist concentration. Fluorescence and confocal microscopy of Chinese hamster ovary, human neuroblastoma (CHP 212), and murine neuroblastoma (N1E-115) cells expressing green fluorescent protein-tagged NT1 receptor revealed that under prolonged treatment with saturating concentrations of neurotensin (NT) agonist, NT1 receptor and NT transiently accumulated in the perinuclear recycling compartment (PNRC). During this cellular event, cell surface receptors remained markedly depleted as detected by both confocal microscopy and (125)I-NT binding assays. In dividing cells, we observed that following prolonged NT agonist stimulation, NT1 receptors were removed from the PNRC, accumulated in dispersed vesicles inside the cytoplasm, and subsequently reappeared at the cell surface. This NT binding recovery allowed for constant cell sensitization and led to a chronic activation of mitogen-activated protein kinases p42 and p44. Under these conditions, the constant activation of NT1 receptor generates an oncogenic regulation. These observations support the potent role for neuropeptides, such as NT, in cancer progression.

A lysosomal tetraspanin associated with retinal degeneration identified via a genome-wide screen.

The Drosophila visual system has provided a model to study phototransduction and retinal degeneration. To identify new candidate proteins that contribute to these processes, we conducted a genome-wide screen for genes expressed predominately in the eye, using DNA microarrays. This screen appeared to be comprehensive as it led to the identification of all 22 eye-enriched genes previously shown to function in phototransduction or implicated in retinal degeneration. In addition, we identified 93 eye-enriched genes whose roles have not been previously defined. One of the eye-enriched genes encoded a member of a large family of transmembrane proteins, referred to as tetraspanins. We created a null mutation in the eye-enriched tetraspanin, Sunglasses (Sun), which resulted in light-induced retinal degeneration. We found that the Sun protein was distributed primarily in lysosomes, and functioned in a long-known but poorly understood phenomenon of light-induced degradation of rhodopsin. We propose that lysosomal tetraspanins in mammalian cells may also function in the downregulation of rhodopsin and other G-protein-coupled receptors, in response to intense or prolonged agonist stimulation.

Resolution of Gsα and Gqα/G11α proteins in membrane domains by two-dimensional electrophoresis: the effect of long-term agonist stimulation.

Low-density membrane-domain fractions were prepared from S49 lymphoma cells and clone e2m11 of HEK293 cells expressing a large number of thyrotropin-releasing hormone receptor (TRH-R) and G(11)alpha by flotation on sucrose density gradients. The intact cell structure was broken by detergent-extraction, alkaline-treatment or drastic homogenization. Three types of low-density membranes were resolved by two-dimensional electrophoresis and analyzed for G(s)alpha (S49) or G(q)alpha/G11) (e2m11) content. Four individual immunoblot signals of Gsalpha protein were identified in S49 lymphoma cells indicating complete resolution of the long G(s)alpha L+/-ser and short G(s)alpha S+/-ser variants of G(s)alpha. All these were diminished by prolonged agonist (isoprenaline) stimulation. In e2m11-HEK cells, five different immunoblot signals were detected indicating post-translational modification of G proteins of G(q)alpha/G(11)alpha family. The two major spots corresponding to exogenously (over)expressed G(11)alpha and endogenous G(q)alpha were reduced; the minor spots diminished by hormonal stimulation. Parallel analysis by silver staining of the total protein content indicated that no major changes in protein composition occurred under these conditions. Our data thus indicate that agonist-stimulation of target cells results in down-regulation of all different members of G(s) and G(q)/G(11) families. This agonist-specific effect may be demonstrated in crude membrane as well as domain/raft preparations and it is not accompanied by changes in overall protein composition.