Agonist-induced Receptor Internalization Research Articles

Amphetamine pretreatment blunts dopamine-induced D2/D3-receptor occupancy by an arrestin-mediated mechanism: A PET study in internalization compromised mice

While all reversible receptor-targeting radioligands for positron emission tomography (PET) can be displaced by competition with an antagonist at the receptor, many radiotracers show limited occupancies using agonists even at high doses. [11C]Raclopride, a D2/D3 receptor radiotracer with rapid kinetics, can identify the direction of changes in the neurotransmitter dopamine, but quantitative interpretation of the relationship between dopamine levels and radiotracer binding has proven elusive. Agonist-induced receptor desensitization and internalization, a homeostatic mechanism to downregulate neurotransmitter-mediated function, can shift radioligand-receptor binding affinity and confound PET interpretations of receptor occupancy. In this study, we compared occupancies induced by amphetamine (AMP) in drug-naive wild-type (WT) and internalization-compromised β-arrestin-2 knockout (KO) mice using a within-scan drug infusion to modulate the kinetics of [11C]raclopride. We additionally performed studies at 3 h following AMP pretreatment, with the hypothesis that receptor internalization should markedly attenuate occupancy on the second challenge, because dopamine cannot access internalized receptors. Without prior AMP treatment, WT mice exhibited somewhat larger binding potential than KO mice but similar AMP-induced occupancy. At 3 h after AMP treatment, WT mice exhibited binding potentials that were 15 % lower than KO mice. At this time point, occupancy was preserved in KO mice but suppressed by 60 % in WT animals, consistent with a model in which most receptors contributing to binding potential in WT animals were not functional. These results demonstrate that arrestin-mediated receptor desensitization and internalization produce large effects in PET [11C]raclopride occupancy studies using agonist challenges.

Effects of Electron Radiation on Serotonin Signaling and Reactivity of Rat Gastric Smooth Muscle.

Ionizing radiation in radiotherapy can disrupt cellular functions based on radiation type, energy, and dose. However, investigations on the effects of accelerated electrons, particularly on serotonin mediation, are limited. This study aimed to investigate changes in serotonin signal transduction (targeting 5-HT2A and 5-HT2B receptors) in gastric smooth muscle (SM) samples isolated from rats irradiated with accelerated electrons (linear accelerator Siemens Primus S/N 3561) and their effects on serotonin-induced reactions. The radiation effects were examined in samples prepared five days after the procedure. The contractile activity of smooth muscle samples was measured using an isometric method. The expression of 5-HT2A and 5-HT2B receptors was determined by immunohistochemical assay. Increased contractile reactivity to exogenous serotonin (1.10-8-1.10-4 mol/L) was observed in irradiated samples compared to controls. The expression of 5-HT2A and 5-HT2B receptors was significantly increased in the irradiated tissue. By selecting appropriate time intervals between equimolar (1.10-6 mol/L) sequential serotonin exposures, a process of desensitization associated with agonist-induced internalization was established in control samples, which was absent in irradiated samples. In conclusion, irradiation with accelerated electrons affects the agonist-induced receptor internalization of 5-HT2A and 5-HT2B receptors and increases their expression in rat gastric SM, which alters their contractile reactivity to exogenous serotonin.

The use of fluorescence correlation spectroscopy to monitor cell surface β2-adrenoceptors at low expression levels in human embryonic stem cell-derived cardiomyocytes and fibroblasts.

The importance of cell phenotype in determining the molecular mechanisms underlying β2 -adrenoceptor (β2AR) function has been noted previously when comparing responses in primary cells and recombinant model cell lines. Here, we have generated haplotype-specific SNAP-tagged β2AR human embryonic stem (ES) cell lines and applied fluorescence correlation spectroscopy (FCS) to study cell surface receptors in progenitor cells and in differentiated fibroblasts and cardiomyocytes. FCS was able to quantify SNAP-tagged β2AR number and diffusion in both ES-derived cardiomyocytes and CRISPR/Cas9 genome-edited HEK293T cells, where the expression level was too low to detect using standard confocal microscopy. These studies demonstrate the power of FCS in investigating cell surface β2ARs at the very low expression levels often seen in endogenously expressing cells. Furthermore, the use of ES cell technology in combination with FCS allowed us to demonstrate that cell surface β2ARs internalize in response to formoterol-stimulation in ES progenitor cells but not following their differentiation into ES-derived fibroblasts. This indicates that the process of agonist-induced receptor internalization is strongly influenced by cell phenotype and this may have important implications for drug treatment with long-acting β2AR agonists.

Investigation of G protein‐coupled receptor kinase 2 (GRK2) regulation of D2 dopamine receptor signaling: effects of GRK2 functional domains and pharmacologic and genetic inhibition of GRK2 activity

The D2 dopamine (DA) receptor (D2R) signals through two major pathways, namely activation of G proteins and recruitment of β‐arrestin. Agonist binding stabilizes a receptor state that activates G proteins and facilitates receptor phosphorylation by one or more members of the G protein‐coupled receptor kinase (GRK) family, and this phosphorylation is known to enhance β‐arrestin recruitment and initiate the process of internalization for many GPCRs. Previously, we found that only GRK2 and GRK3 were involved in D2R phosphorylation in response to agonist stimulation (Namkung et al., JBC: 284: 15038, 2009). In this study, we further investigated the influence of GRK2 on receptor signaling, β‐arrestin recruitment, and internalization of the D2R. Overexpression of GRK2 was found to increase DA‐stimulated β‐arrestin recruitment and receptor internalization, whereas it suppressed D2R‐mediated inhibition of cAMP accumulation. In contrast, a catalytically inactive GRK2 mutant (GRK2‐K220R) did not affect any of these functional outputs suggesting that GRK2 kinase activity is required to influence D2R signaling. A disabling mutation of the GRK2 RGS homology (RH) domain (GRK2‐D110A) behaved similarly as WT GRK2, suggesting that this domain is not required for GRK2’s regulatory effects on the D2R. Finally, overexpression of a GRK2 construct containing a mutation in the pleckstrin homology (PH) domain (GRK2‐R587Q), which negates GRK2 interaction with Gβγ subunits, increased DA‐stimulated β‐arrestin recruitment and receptor internalization (more than observed with WT GRK2), but did not affect D2R regulation of cAMP accumulation, indicating that this GRK2 domain is involved in regulating some, but not all, aspects of D2R signaling. To study D2R regulation in the complete absence of GRK2, we utilized a cell line in which GRK2 expression was knocked out using CRISPR‐Cas9 technology. DA‐stimulated β‐arrestin recruitment and receptor internalization were decreased, but not eliminated, in the GRK2 KO cells suggesting that, while GRK2 may be facilitatory, it is not essential for these processes. In parallel studies we characterized the effects of Compound 101, a dual GRK2/3 inhibitor. Compound 101 was found to negate GRK2 interactions with the D2R and attenuate, but not eliminate, β‐arrestin recruitment and receptor internalization. In contrast, in the GRK2 KO cells, Compound 101 had no effect on the residual DA‐stimulated β‐arrestin recruitment. In conclusion, our data suggest that different GRK2 domains affect specific regulatory pathways of the D2R and that both agonist‐induced β‐arrestin recruitment and receptor internalization can occur in the absence of GRK2 interactions with the D2R.Support or Funding InformationNINDS Intramural Research Program

Anaplastic Lymphoma Kinase Regulates Internalization of the Dopamine D2 Receptor.

The dopamine D2 receptor (D2R) is a G protein-coupled receptor (GPCR) expressed in regions of the brain that control motor function, cognition, and motivation. As a result, D2R is involved in the pathophysiology of disorders such as schizophrenia and drug addiction. Understanding the signaling pathways activated by D2R is crucial to finding new therapeutic targets for these disorders. D2R stimulation by its agonist, dopamine, causes desensitization and internalization of the receptor. A previous study found that inhibitors of the receptor tyrosine kinase anaplastic lymphoma kinase (ALK) blocked D2R desensitization in neurons in the ventral tegmental area of the brain. In the present study, using a cell-based system, we investigated whether ALK regulates D2R internalization. The ALK inhibitor alectinib completely inhibited dopamine-induced D2R internalization. Since GPCRs can transactivate receptor tyrosine kinases, we also examined if D2R stimulation activated ALK signaling. ALK phosphorylation increased by almost 2-fold after dopamine treatment and ALK coimmunoprecipitated with D2R. To identify the signaling pathways downstream of ALK that might regulate D2R internalization, we used pharmacological inhibitors of proteins activated by ALK signaling. Protein kinase Cγ was activated by dopamine in an ALK-dependent manner, and a protein kinase C inhibitor completely blocked dopamine-induced D2R internalization. Taken together, these results identify ALK as a receptor tyrosine kinase transactivated by D2R that promotes its internalization, possibly through activation of protein kinase C. ALK inhibitors could be useful in enhancing D2R signaling. SIGNIFICANCE STATEMENT: Receptor internalization is a mechanism by which receptors are desensitized. In this study we found that agonist-induced internalization of the dopamine D2 receptor is regulated by the receptor tyrosine kinase ALK. ALK was also transactivated by and associated with dopamine D2 receptor. Dopamine activated protein kinase C in an ALK-dependent manner and a PKC inhibitor blocked dopamine D2 receptor internalization. These results indicate that ALK regulates dopamine D2 receptor trafficking, which has implications for psychiatric disorders involving dysregulated dopamine signaling.

NanoBiT Complementation to Monitor Agonist-Induced Adenosine A1 Receptor Internalization.

Receptor internalization in response to prolonged agonist treatment is an important regulator of G protein–coupled receptor (GPCR) function. The adenosine A1 receptor (A1AR) is one of the adenosine receptor family of GPCRs, and evidence for its agonist-induced internalization is equivocal. The recently developed NanoBiT technology uses split NanoLuc Luciferase to monitor changes in protein interactions. We have modified the human A1AR on the N-terminus with the small high-affinity HiBiT tag. In the presence of the large NanoLuc subunit (LgBiT), complementation occurs, reconstituting a full-length functional NanoLuc Luciferase. Here, we have used complemented luminescence to monitor the internalization of the A1AR in living HEK293 cells. Agonist treatment resulted in a robust decrease in cell-surface luminescence, indicating an increase in A1AR internalization. These responses were inhibited by the A1AR-selective antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), with an antagonist affinity that closely matched that measured using ligand binding with a fluorescent A1 receptor antagonist (CA200645). The agonist potencies for inducing A1AR internalization were very similar to the affinities previously determined by ligand binding, suggesting little or no amplification of the internalization response. By complementing the HiBiT tag to exogenous purified LgBiT, it was also possible to perform NanoBRET ligand-binding experiments using HiBiT–A1AR. This study demonstrates the use of NanoBiT technology to monitor internalization of the A1AR and offers the potential to combine these experiments with NanoBRET ligand-binding assays.

Shank Proteins Couple the Endocytic Zone to the Postsynaptic Density to Control Trafficking and Signaling of Metabotropic Glutamate Receptor 5

SummaryActivation of postsynaptic metabotropic glutamate receptors (mGluRs) modulates neuronal excitability and synaptic plasticity, while deregulation of mGluR signaling has been implicated in neurodevelopmental disorders. Overstimulation of mGluRs is restricted by the rapid endocytosis of receptors after activation. However, how membrane trafficking of mGluRs at synapses is controlled remains poorly defined. We find that in hippocampal neurons, the agonist-induced receptor internalization of synaptic mGluR5 is significantly reduced in Shank knockdown neurons. This is rescued by the re-expression of wild-type Shanks, but not by mutants unable to bind Homer1b/c, Dynamin2, or Cortactin. These effects are paralleled by a reduction in synapses associated with an endocytic zone. Moreover, a mutation in SHANK2 found in autism spectrum disorders (ASDs) similarly disrupts these processes. On the basis of these findings, we propose that synaptic Shank scaffolds anchor the endocytic machinery to govern the efficient trafficking of mGluR5 and to balance the surface expression of mGluRs to efficiently modulate neuronal functioning.

Heterodimerization of Mu Opioid Receptor Protomer with Dopamine D2 Receptor Modulates Agonist-Induced Internalization of Mu Opioid Receptor.

The interplay between the dopamine (DA) and opioid systems in the brain is known to modulate the additive effects of substances of abuse. On one hand, opioids serve mankind by their analgesic properties, which are mediated via the mu opioid receptor (MOR), a Class A G protein-coupled receptor (GPCR), but on the other hand, they pose a potential threat by causing undesired side effects such as tolerance and dependence, for which the exact molecular mechanism is still unknown. Using human embryonic kidney 293T (HEK 293T) and HeLa cells transfected with MOR and the dopamine D2 receptor (D2R), we demonstrate that these receptors heterodimerize, using an array of biochemical and biophysical techniques such as coimmunoprecipitation (co-IP), bioluminescence resonance energy transfer (BRET1), Fӧrster resonance energy transfer (FRET), and functional complementation of a split luciferase. Furthermore, live cell imaging revealed that D2LR, when coexpressed with MOR, slowed down internalization of MOR, following activation with the MOR agonist [D-Ala2, N-MePhe4, Gly-ol]-enkephalin (DAMGO).

Effect of fish oil on agonist-induced receptor internalization of the PG F2α receptor and cell signaling in bovine luteal cells in vitro

Many receptors span the plasma membrane allowing for signal transduction, converting extracellular signals into intracellular signals. Following ligand-induced activation, membrane-bound receptors are taken into endocytic vesicles, where they are targeted for degradation or recycled back to the plasma membrane. The objectives of the present study were to determine the influence of fish oil on (1) PGF2α-induced receptor internalization and trafficking of the PGF2α (FP) receptor, (2) cytoskeletal structural integrity, and (3) PGF2α-induced mitogen-activated protein kinase (MAPK) signaling in bovine luteal cells. Bovine ovaries were obtained from a local abattoir and corpora lutea (CL; n = 4–6) were digested using collagenase. For all experiments, cells were incubated in either BSA or fish oil–supplemented media for 72 h to allow incorporation of omega-3 fatty acids into biological membranes. Confocal microscopy was used to determine the influence of fish oil on PGF2α-induced receptor internalization and trafficking of the FP receptor and cytoskeletal structural integrity. In addition, Western blotting was used to determine the effects of fish oil on PGF2α-induced MAPK signaling in bovine luteal cells. Results from the present study demonstrate that fish oil disrupts the colocalization of Gαq with both caveola microdomains and FP receptor as well as PGF2α-induced MAPK signaling. This disruption of the FP receptor with the G-protein alpha subunit may be one mechanism by which a MAPK signaling is diminished following the addition of PGF2α. Furthermore, fish oil disrupts FP receptor internalization and endosomal protein trafficking without detectable changes in the cytoskeleton.

Src‐dependent phosphorylation of μ‐opioid receptor at Tyr 336 modulates opiate withdrawal

Opiate withdrawal/negative reinforcement has been implicated as one of the mechanisms for the progression from impulsive to compulsive drug use. Increase in the intracellular cAMP level and protein kinase A (PKA) activities within the neurocircuitry of addiction has been a leading hypothesis for opiate addiction. This increase requires the phosphorylation of μ‐opioid receptor (MOR) at Tyr336 by Src after prolonged opiate treatment in vitro. Here, we report that the Src‐mediated MOR phosphorylation at Tyr336 is a prerequisite for opiate withdrawal in mice. We observed the recruitment of Src in the vicinity of MOR and an increase in phosphorylated Tyr336 (pY336) levels during naloxone‐precipitated withdrawal. The intracerebroventricular or stereotaxic injection of a Src inhibitor (AZD0530), or Src shRNA viruses attenuated pY336 levels, and several somatic withdrawal signs. This was also observed in Fyn−/− mice. The stereotaxic injection of wild‐type MOR, but not mutant (Y336F) MOR, lentiviruses into the locus coeruleus of MOR −/− mice restored somatic withdrawal jumping. Regulating pY336 levels during withdrawal might be a future target for drug development to prevent opiate addictive behaviors.

Biased small-molecule ligands for selective inhibition of HIV-1 cell entry via CCR5.

Since the discovery of HIV's use of CCR5 as the primary coreceptor in fusion, the focus on developing small‐molecule receptor antagonists for inhibition hereof has only resulted in one single drug, Maraviroc. We therefore investigated the possibility of using small‐molecule CCR5 agonists as HIV‐1 fusion inhibitors. A virus‐free cell‐based fusion reporter assay, based on mixing “effector cells” (expressing HIV Env and luciferase activator) with “target cells” (expressing CD4, CCR5 wild type or a selection of well‐described mutations, and luciferase reporter), was used as fusion readout. Receptor expression was evaluated by ELISA and fluorescence microscopy. On CCR5 WT, Maraviroc and Aplaviroc inhibited fusion with high potencies (EC 50 values of 91 and 501 nM, respectively), whereas removal of key residues for both antagonists (Glu283Ala) or Maraviroc alone (Tyr251Ala) prevented fusion inhibition, establishing this assay as suitable for screening of HIV entry inhibitors. Both ligands inhibited HIV fusion on signaling‐deficient CCR5 mutations (Tyr244Ala and Trp248Ala). Moreover, the steric hindrance CCR5 mutation (Gly286Phe) impaired fusion, presumably by a direct hindrance of gp120 interaction. Finally, the efficacy switch mutation (Leu203Phe) – converting small‐molecule antagonists/inverse agonists to full agonists biased toward G‐protein activation – uncovered that also small‐molecule agonists can function as direct HIV‐1 cell entry inhibitors. Importantly, no agonist‐induced receptor internalization was observed for this mutation. Our studies of the pharmacodynamic requirements for HIV‐1 fusion inhibitors highlight the possibility of future development of biased ligands with selective targeting of the HIV–CCR5 interaction without interfering with the normal functionality of CCR5.

Agonist-induced internalization and desensitization of the apelin receptor

Apelin acts via the G protein-coupled apelin receptor (APJ) to mediate effects on cardiovascular and fluid homeostasis. G protein-coupled receptor (GPCR) trafficking has an important role in the regulation of receptor signalling pathways and cellular functions, however in the case of APJ the mechanisms and proteins involved in apelin-induced trafficking are not well understood. We generated a stable HEK-293 cell line expressing N-terminus HA-tagged mouse (m) APJ, and used a semi-automated imaging protocol to quantitate APJ trafficking and ERK1/2 activation following stimulation with [Pyr1]apelin-13. The mechanisms of [Pyr1]apelin-13-induced internalization and desensitization were explored using dominant-negative mutant (DNM) cDNA constructs of G protein-coupled receptor kinase 2 (GRK2), β-arrestin1, EPS15 and dynamin. The di-phosphorylated ERK1/2 (ppERK1/2) response to [Pyr1]apelin-13 desensitized during sustained stimulation, due to upstream APJ-specific adaptive changes. Furthermore, [Pyr1]apelin-13 stimulation caused internalization of mAPJ via clathrin coated vesicles (CCVs) and also caused a rapid reduction in cell surface and whole cell HA-mAPJ. Our data suggest that upon continuous agonist exposure GRK2-mediated phosphorylation targets APJ to CCVs that are internalized from the cell surface in a β-arrestin1-independent, EPS15- and dynamin-dependent manner. Internalization does not appear to contribute to the desensitization of APJ-mediated ppERK1/2 activation in these cells.

Analysis of Chemokine Receptor Trafficking by Site-Specific Biotinylation.

Chemokine receptors undergo internalization and desensitization in response to ligand activation. Internalized receptors are either preferentially directed towards recycling pathways (e.g. CCR5) or sorted for proteasomal degradation (e.g. CXCR4). Here we describe a method for the analysis of receptor internalization and recycling based on specific Bir A-mediated biotinylation of an acceptor peptide coupled to the receptor, which allows a more detailed analysis of receptor trafficking compared to classical antibody-based detection methods. Studies on constitutive internalization of the chemokine receptors CXCR4 (12.1% ± 0.99% receptor internalization/h) and CCR5 (13.7% ± 0.68%/h) reveals modulation of these processes by inverse (TAK779; 10.9% ± 0.95%/h) or partial agonists (Met-CCL5; 15.6% ± 0.5%/h). These results suggest an actively driven internalization process. We also demonstrate the advantages of specific biotinylation compared to classical antibody detection during agonist-induced receptor internalization, which may be used for immunofluorescence analysis as well. Site-specific biotinylation may be applicable to studies on trafficking of transmembrane proteins, in general.

PKCβII inhibits the ubiquitination of β-arrestin2 in an autophosphorylation-dependent manner

GPCR kinase 2 (GRK2)/β-arrestins and protein kinase A (PKA)/protein kinase C (PKC) mediate homologous and heterologous regulations of GPCRs, respectively. Conventional protein kinase C enzymes (PKCs), as exemplified by PKCβII, selectively inhibit internalization of dopamine D2 receptor and β2 adrenoceptor in a β-arrestin- but not GRK2-dependent manner. PKCβII interacts with β-arrestin2 upon autophosphorylation at T250, and inhibits the receptor internalization by decreasing the ubiquitination of β-arrestin2. PKCβII interferes with the interaction between β-arrestin2 and MDM2 in the cytosol, resulting in the redistribution of MDM2 to the nucleus. Subsequently, deubiquitination of β-arrestin2 and inhibition of agonist-induced receptor internalization follow. Thus, our study suggests that the extent of β-arrestin ubiquitination and the autophosphorylation status of PKCs determine PKCβII-mediated inhibition of homologous regulatory processes of GPCRs.

G protein beta 5 is targeted to D2-dopamine receptor-containing biochemical compartments and blocks dopamine-dependent receptor internalization.

G beta 5 (Gbeta5, Gβ5) is a unique G protein β subunit that is thought to be expressed as an obligate heterodimer with R7 regulator of G protein signaling (RGS) proteins instead of with G gamma (Gγ) subunits. We found that D2-dopamine receptor (D2R) coexpression enhances the expression of Gβ5, but not that of the G beta 1 (Gβ1) subunit, in HEK293 cells, and that the enhancement of expression occurs through a stabilization of Gβ5 protein. We had previously demonstrated that the vast majority of D2R either expressed endogenously in the brain or exogenously in cell lines segregates into detergent-resistant biochemical fractions. We report that when expressed alone in HEK293 cells, Gβ5 is highly soluble, but is retargeted to the detergent-resistant fraction after D2R coexpression. Furthermore, an in-cell biotin transfer proximity assay indicated that D2R and Gβ5 segregating into the detergent-resistant fraction specifically interacted in intact living cell membranes. Dopamine-induced D2R internalization was blocked by coexpression of Gβ5, but not Gβ1. However, the same Gβ5 coexpression levels had no effect on agonist-induced internalization of the mu opioid receptor (MOR), cell surface D2R levels, dopamine-mediated recruitment of β-arrestin to D2R, the amplitude of D2R-G protein coupling, or the deactivation kinetics of D2R-activated G protein signals. The latter data suggest that the interactions between D2R and Gβ5 are not mediated by endogenously expressed R7 RGS proteins.

Real‐time characterization of cannabinoid receptor 1 (CB1) allosteric modulators reveals novel mechanism of action

The cannabinoid receptor type 1 (CB1 ) has an allosteric binding site. The drugs ORG27569 {5-chloro-3-ethyl-N-[2-[4-(1-piperidinyl)phenyl]ethyl]-1H-indole-2-carboxamide} and PSNCBAM-1 {1-(4-chlorophenyl)-3-[3-(6-pyrrolidin-1-ylpyridin-2-yl)phenyl]urea} have been extensively characterized with regard to their effects on signalling of the orthosteric ligand CP55,940 {(-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol}, and studies have suggested that these allosteric modulators increase binding affinity but act as non-competitive antagonists in functional assays. To gain a deeper understanding of allosteric modulation of CB1 , we examined real-time signalling and trafficking responses of the receptor in the presence of allosteric modulators. Studies of CB1 signalling were carried out in HEK 293 and AtT20 cells expressing haemagglutinin-tagged human and rat CB1 . We measured real-time accumulation of cAMP, activation and desensitization of potassium channel-mediated cellular hyperpolarization and CB1 internalization. ORG27569 and PSNCBAM-1 produce a complex, concentration and time-dependent modulation of agonist-mediated regulation of cAMP levels, as well as an increased rate of desensitization of CB1 -mediated cellular hyperpolarization and a decrease in agonist-induced receptor internalization. Contrary to previous studies characterizing allosteric modulators at CB1, this study suggests that the mechanism of action is not non-competitive antagonism of signalling, but rather that enhanced binding results in an increased rate of receptor desensitization and reduced internalization, which results in time-dependent modulation of cAMP signalling. The observed effect of the allosteric modulators is therefore dependent on the time frame over which the signalling response occurs. This finding may have important consequences for the potential therapeutic application of these compounds.

Differential β-arrestin2 requirements for constitutive and agonist-induced internalization of the CB1 cannabinoid receptor

CB1 cannabinoid receptor (CB1R) undergoes both constitutive and agonist-induced internalization, but the underlying mechanisms of these processes and the role of β-arrestins in the regulation of CB1R function are not completely understood. In this study, we followed CB1R internalization using confocal microscopy and bioluminescence resonance energy transfer measurements in HeLa and Neuro-2a cells. We found that upon activation CB1R binds β-arrestin2 (β-arr2), but not β-arrestin1. Furthermore, both the expression of dominant-negative β-arr2 (β-arr2-V54D) and siRNA-mediated knock-down of β-arr2 impaired the agonist-induced internalization of CB1R. In contrast, neither β-arr2-V54D nor β-arr2-specific siRNA had a significant effect on the constitutive internalization of CB1R. However, both constitutive and agonist-induced internalization of CB1R were impaired by siRNA-mediated depletion of clathrin heavy chain. We conclude that although clathrin is required for both constitutive and agonist-stimulated internalization of CB1R, β-arr2 binding is only required for agonist-induced internalization of the receptor suggesting that the molecular mechanisms underlying constitutive and agonist-induced internalization of CB1R are different.

WITHDRAWN: Highly efficient agonist-induced internalization of somatostatin SST2 receptor in mouse dorsal root ganglion and spinal cord: Molecular evidence for mechanism of somatostatin inhibits neuropathic pain

WITHDRAWN: Highly efficient agonist-induced internalization of somatostatin SST2 receptor in mouse dorsal root ganglion and spinal cord: Molecular evidence for mechanism of somatostatin inhibits neuropathic pain

Agonist-Induced Receptor Internalization in Chinese Hamster Ovary Cells Stably Co-expressing β<sub>1</sub>- and β<sub>2</sub>-Adrenergic Receptors

β(1)- and β(2)-Adrenergic receptors (β(1)-AR and β(2)-AR) are co-expressed in numerous tissues, for example, heart and bladder. They play a very important role in the responses of a variety of organs to sympathetic nerve stimulation. Recent studies suggest that many G protein-coupled receptors, such as β(1)-AR, β(2)-AR, μ opioid receptor and δ opioid receptor, can form homo- and heterooligomers. Previous studies demonstrated that the β(1)-AR and β(2)-AR formed dimers in living HEK 293 cells. The aim of the present study is to investigate whether such heterooligomerization affect the agonist-induced receptor internalization in the CHO-K1 cells stably co-expressing β(1)-AR and β(2)-AR. Using co-immunoprecipitation, we confirmed that β(1)-AR and β(2)-AR formed heterooligomers in the CHO-K1 cells. In cells co-expressing β(1)-AR and β(2)-AR, 30% of β(1)-AR was internalized by isoproterenol, whereas only 20% of β(1)-AR was internalized in cells expressing the β(1)-AR alone. Heterooligomerization did not affect the ratio of internalized β(2)-AR. Salmeterol, a specific β(2)-AR agonist, broke β(1)-AR/β(2)-AR heterooligomers, and induced β(2)-AR-specific internalization in cells co-expressing β(1)-AR and β(2)-AR. The present study demonstrated that heterooligomerization between β(1)-AR and β(2)-AR accelerates the isoproterenol-promoted internalization of the β(1)-AR, and that salmeterol induces β(2)-AR-specific internalization in Chinese hamster ovary (CHO) cells stably co-expressing β(1)-AR and β(2)-AR.

Roles for NHERF1 and NHERF2 on the Regulation of C3a Receptor Signaling in Human Mast Cells

BackgroundThe anaphylatoxin C3a binds to the G protein coupled receptor (GPCR, C3aR) and activates divergent signaling pathways to induce degranulation and cytokine production in human mast cells. Adapter proteins such as the Na+/H+ exchange regulatory factor (NHERF1 and NHERF2) have been implicated in regulating functions of certain GPCRs by binding to the class I PDZ (PSD-95/Dlg/Zo1) motifs present on their cytoplasmic tails. Although C3aR possesses a class I PDZ motif, the possibility that it interacts with NHERF proteins to modulate signaling in human mast cells has not been determined.Methodology/Principal FindingsUsing reverse transcription PCR and Western blotting, we found that NHERF1 and NHERF2 are expressed in human mast cell lines (HMC-1, LAD2) and CD34+-derived primary human mast cells. Surprisingly, however, C3aR did not associate with these adapter proteins. To assess the roles of NHERFs on signaling downstream of C3aR, we used lentiviral shRNA to stably knockdown the expression of these proteins in human mast cells. Silencing the expression of NHERF1 and NHERF2 had no effect on C3aR desensitization, agonist-induced receptor internalization, ERK/Akt phosphorylation or chemotaxis. However, loss of NHERF1 and NHERF2 resulted in significant inhibition of C3a-induced mast cell degranulation, NF-κB activation and chemokine production.Conclusion/SignificanceThis study demonstrates that although C3aR possesses a class I PDZ motif, it does not associate with NHERF1 and NHERF2. Surprisingly, these proteins provide stimulatory signals for C3a-induced degranulation, NF-κB activation and chemokine generation in human mast cells. These findings reveal a new level of complexity for the functional regulation of C3aR by NHERFs in human mast cells.