Differential affinities of visual arrestin, beta arrestin1, and beta arrestin2 for G protein-coupled receptors delineate two major classes of receptors

Visual arrestin, betaarrestin1, and betaarrestin2 comprise a family of intracellular proteins that desensitize G protein-coupled receptors (GPCRs). In addition, betaarrestin1 and betaarrestin2 target desensitized receptors to clathrin-coated pits for endocytosis. Whether arrestins differ in their ability to interact with GPCRs in cells is not known. In this study, we visualize the interaction of arrestin family members with GPCRs in real time and in live cells using green fluorescent protein-tagged arrestins. In the absence of agonist, visual arrestin and betaarrestin1 were found in both the cytoplasm and nucleus of HEK-293 cells, whereas betaarrestin2 was found only in the cytoplasm. Analysis of agonist-mediated arrestin translocation to multiple GPCRs identified two major classes of receptors. Class A receptors (beta2 adrenergic receptor, mu opioid receptor, endothelin type A receptor, dopamine D1A receptor, and alpha1b adrenergic receptor) bound betaarrestin2 with higher affinity than betaarrestin1 and did not interact with visual arrestin. In contrast, class B receptors (angiotensin II type 1A receptor, neurotensin receptor 1, vasopressin V2 receptor, thyrotropin-releasing hormone receptor, and substance P receptor) bound both betaarrestin isoforms with similar high affinities and also interacted with visual arrestin. Switching the carboxyl-terminal tails of class A and class B receptors completely reversed the affinity of each receptor for the visual and non-visual arrestins. In addition, exchanging the betaarrestin1 and betaarrestin2 carboxyl termini reversed their extent of binding to class A receptors as well as their subcellular distribution. These results reveal for the first time marked differences in the ability of arrestin family members to bind GPCRs at the plasma membrane. Moreover, they show that visual arrestin can interact in cells with GPCRs other than rhodopsin. These findings suggest that GPCR signaling may be differentially regulated depending on the cellular complement of arrestin isoforms and the ability of arrestins to interact with other cellular proteins.     

HeLa、HEK293、SH-SY5Y细胞中的Tau蛋白

通过间接免疫荧光测定了HeLa、HEK-293、SH-SY5Y细胞内Tau蛋白的分布,观察到在细胞间期单克隆抗体Tau-1的荧光信号分布于细胞质和胞核中．特别是HeLa细胞,其胞核内具有相对较高的Tau蛋白免疫荧光信号．通过分离SH-SY5Y的细胞核,更为清楚地显示了Tau蛋白在细胞核中的分布,并且免疫荧光信号与DNA的Hoechst33258染色信号相重合．Western blotting的测定结果进一步证明了SH-SY5Y细胞的胞质和胞核中均含有Tau蛋白的不同异构体．以上结果提示,Tau蛋白不仅存在于神经、肌肉等细胞内,也存在于肿瘤细胞系,并且分布于间期的胞核中．     

The adenosine A2A receptor interacts with the actin-binding protein alpha-actinin

Recently, evidence has emerged that heptaspanning membrane or G protein-coupled receptors may be linked to intracellular proteins identified as regulators of receptor anchoring and signaling. Using a yeast two-hybrid screen, we identified alpha-actinin, a major F-actin-cross-linking protein, as a binding partner for the C-terminal domain of the adenosine A2A receptor (A2AR). Colocalization, co-immunoprecipitation, and pull-down experiments showed a close and specific interaction between A2AR and alpha-actinin in transfected HEK-293 cells and also in rat striatal tissue. A2AR activation by agonist induced the internalization of the receptor by a process that involved rapid beta-arrestin translocation from the cytoplasm to the cell surface. In the subsequent receptor traffic from the cell surface, the role of actin organization was shown to be crucial in transiently transfected HEK-293 cells, as actin depolymerization by cytochalasin D prevented its agonist-induced internalization. A2ADeltaCTR, a mutant version of A2AR that lacks the C-terminal domain and does not interact with alpha-actinin, was not able to internalize when activated by agonist. Interestingly, A2ADeltaCTR did not show aggregation or clustering after agonist stimulation, a process readily occurring with the wild-type receptor. These findings suggest an alpha-actinin-dependent association between the actin cytoskeleton and A2AR trafficking.     

Gαq-mediated plasma membrane translocation of sphingosine kinase-1 and cross-activation of S1P receptors

Sphingosine-1-phosphate (S1P), formed by sphingosine kinases (SphKs), regulates cellular proliferation and migration by acting as an agonist at specific receptors or intracellularly. Since S1P's effects are probably dependent on subcellular localization of its formation and degradation, we have studied the influence of G protein-coupled receptors on the localization of SphK1. Activation of G_q-coupled receptors induced a profound, rapid (half-life 3–5 s) and long-lasting (> 2 h) translocation of SphK1 to the plasma membrane. This was mimicked by expression of constitutively active G protein α-subunits specifically of the G_q family. Classical G_q signalling pathways, or phosphorylation at Ser²²⁵, phospholipase D and Ca²⁺/calmodulin were not involved in M₃ receptor-induced SphK1 translocation in HEK-293 cells. Translocation was associated with S1P receptor internalization, which was dependent on catalytic activity of SphK1 and S1P receptor binding and thus resulted from S1P receptor cross-activation. It is concluded that SphK1 is an important effector of G_q-coupled receptors, linking them via cross-activation of S1P receptors to G_i and G_12/13 signalling pathways.     

The Human Gonadotropin Releasing Hormone Type I Receptor Is a Functional Intracellular GPCR Expressed on the Nuclear Membrane

The mammalian type I gonadotropin releasing hormone receptor (GnRH-R) is a structurally unique G protein-coupled receptor (GPCR) that lacks cytoplasmic tail sequences and displays inefficient plasma membrane expression (PME). Compared to its murine counterparts, the primate type I receptor is inefficiently folded and retained in the endoplasmic reticulum (ER) leading to a further reduction in PME. The decrease in PME and concomitant increase in intracellular localization of the mammalian GnRH-RI led us to characterize the spatial distribution of the human and mouse GnRH receptors in two human cell lines, HEK 293 and HTR-8/SVneo. In both human cell lines we found the receptors were expressed in the cytoplasm and were associated with the ER and nuclear membrane. A molecular analysis of the receptor protein sequence led us to identify a putative monopartite nuclear localization sequence (NLS) in the first intracellular loop of GnRH-RI. Surprisingly, however, neither the deletion of the NLS nor the addition of the Xenopus GnRH-R cytoplasmic tail sequences to the human receptor altered its spatial distribution. Finally, we demonstrate that GnRH treatment of nuclei isolated from HEK 293 cells expressing exogenous GnRH-RI triggers a significant increase in the acetylation and phosphorylation of histone H3, thereby revealing that the nuclear-localized receptor is functional. Based on our findings, we conclude that the mammalian GnRH-RI is an intracellular GPCR that is expressed on the nuclear membrane. This major and novel discovery causes us to reassess the signaling potential of this physiologically and clinically important receptor.     

Differential regulation of the dopamine D2 and D3 receptors by G protein-coupled receptor kinases and beta-arrestins

The D(2) and D(3) receptors (D(2)R and D(3)R), which are potential targets for antipsychotic drugs, have a similar structural architecture and signaling pathway. Furthermore, in some brain regions they are expressed in the same cells, suggesting that differences between the two receptors might lie in other properties such as their regulation. In this study we investigated, using COS-7 and HEK-293 cells, the mechanism underlying the intracellular trafficking of the D(2)R and D(3)R. Activation of D(2)R caused G protein-coupled receptor kinase-dependent receptor phosphorylation, a robust translocation of beta-arrestin to the cell membrane, and profound receptor internalization. The internalization of the D(2)R was dynamin-dependent, suggesting that a clathrin-coated endocytic pathway is involved. In addition, the D(2)R, upon agonist-mediated internalization, localized to intracellular compartments distinct from those utilized by the beta(2)-adrenergic receptor. However, in the case of the D(3)R, only subtle agonist-mediated receptor phosphorylation, beta-arrestin translocation to the plasma membrane, and receptor internalization were observed. Interchange of the second and third intracellular loops of the D(2)R and D(3)R reversed their phenotypes, implicating these regions in the regulatory properties of the two receptors. Our studies thus indicate that functional distinctions between the D(2)R and D(3)R may be found in their desensitization and cellular trafficking properties. The differences in their regulatory properties suggest that they have distinct physiological roles in the brain.     

Dopamine D2 receptor-mediated Akt/PKB signalling: initiation by the D2S receptor and role in quinpirole-induced behavioural activation

The short and long isoforms of the dopamine D2 receptor (D2S and D2L respectively) are highly expressed in the striatum. Functional D2 receptors activate an intracellular signalling pathway that includes a cAMP-independent route involving Akt/GSK3 (glycogen synthase kinase 3). To investigate the Akt/GSK3 response to the seldom-studied D2S receptor, we established a rat D2S receptor-expressing cell line [HEK (human embryonic kidney)-293/rD2S]. We found that in HEK-293/rD2S cells, the D2/D3 agonists bromocriptine and quinpirole significantly induced Akt and GSK3 phosphorylation, as well as ERK1/2 (extracellular-signal-regulated kinase 1/2) activation. The D2S receptor-induced Akt signals were profoundly inhibited by the internalization blockers monodansyl cadaverine and concanavalin A. Activation of the D2S receptor in HEK-293/rD2S cells appeared to trigger Akt/phospho-Akt translocation to the cell membrane. In addition to our cell culture experiments, we studied D2 receptor-dependent Akt in vivo by systemic administration of the D2/D3 agonist quinpirole. The results show that quinpirole evoked Akt-Ser⁴⁷³ phosphorylation in the ventral striatum. Furthermore, intra-accumbens administration of wortmannin, a PI3K (phosphoinositide 3-kinase) inhibitor, significantly suppressed the quinpirole-evoked behavioural activation. Overall, we demonstrate that activation of the dopamine D2S receptor stimulates Akt/GSK3 signalling. In addition, in vivo Akt activity in the ventral striatum appears to play an important role in systemic D2/D3 agonist-induced behavioural activation.     

Subcellular localization of the aryl hydrocarbon receptor is modulated by the immunophilin homolog hepatitis B virus X-associated protein 2

The hepatitis B virus X-associated protein 2 (XAP2) is an immunophilin homolog and core component of the aryl hydrocarbon receptor (AhR). Immunophilins are components of many steroid receptor complexes, serving a largely unknown function. Transiently expressed AhR.YFP (yellow fluorescent protein) localized to the nuclei of COS-1 and NIH-3T3 cells. Co-expression of AhR.YFP with XAP2 restored cytoplasmic localization, which was reversed by 2,3,7, 8-tetrachlorodibenzo-p-dioxin treatment (TCDD). The effect of XAP2 on AhR localization was specific involving a nuclear localization signal-mediated pathway. Examination of the ratio of AhR to XAP2 in the AhR complex revealed that approximately 25% of transiently expressed AhR was associated with XAP2, in contrast with approximately 100% when the AhR and XAP2 were co-expressed. Strikingly, TCDD did not influence these ratios, suggesting that ligand binding initiates nuclear translocation prior to complex dissociation. Analysis of endogenous AhR in Hepa-1 cells revealed that approximately 40% of the AhR complex was associated with XAP2, predicting observed AhR localization to cytoplasm and nuclei. This study reveals a novel functional role for the immunophilin-like component of a soluble receptor complex and provides new insight into the mechanism of AhR-mediated signal transduction, demonstrating the existence of two structurally distinct and possibly functionally unique forms of the AhR.     

Studies on rabbit natural and recombinant tissue factors: intracellular retention and regulation of surface expression in cultured cells

Tissue factor (TF) is the most important trigger of blood coagulation in vascular pathology. Rabbit TF, with or without (delta C) its COOH-terminal intracellular tail, has been conjugated to green fluorescent protein (GFP) to study subcellular localization and other functions of TF. TF-GFP and TF delta C-GFP are associated with Na2CO3-resistant buoyant fractions in HEK-293 cells (lipid rafts); there is no morphological difference in the surface distribution of these or other GFP-labeled membrane proteins present in or excluded from rafts (confocal microscopy, HEK-293 cells). Endogenous TF expressed by rabbit aortic smooth muscle cells (SMCs) is also raft associated. Membranes from HEK-293 cells expressing recombinant TF-GFP or wild-type TF were equipotent to clot human plasma; however, TF delta C-GFP was approximately 20-fold more active (per membrane weight). Immunoblot confirmed that the deletion mutant is more abundantly expressed, and confocal microscopy showed that it has preferential membrane localization, whereas TF-GFP is mainly intracellular (nuclear lining and multiple granules). With a similar half-life (<4 h), the two constructions differ by their intracellular retention, lower for TF delta C-GFP. In serum-starved SMCs, the expression of endogenous TF was upregulated by interleukin-1 beta and/or FBS treatment (immunoblot, immunofluorescence, clotting assay). However, TF secretion or surface expression was not regulated by stimuli of physiological intensity (such as stimulation of the coexpressed kinin B1 receptors), although a calcium ionophore was highly active in this respect. TF is a raft-associated molecule whose surface expression (secretion) is apparently retarded or impaired by structural determinant(s) located in its COOH-terminal tail.     

A switching system regulating subcellular localization of nuclear proteins using a viral protease

We explored a novel approach to the functional regulation of nuclear proteins; altering their subcellular localization. To anchor a nuclear protein, beta-galactosidase with the nuclear localization signal of SV40 (nbeta-gal), within the cytoplasm, nbeta-gal was fused to the transmembrane domain of granulocyte colony-stimulating factor receptor (G-CSFR), a membrane protein. To liberate the nbeta-gal portion from the fusion protein, we used a protease derived from a plant virus, whose recognition sequence was inserted between the G-CSFR and nbeta-gal. Western analysis showed that the chimeric protein was cleaved in the presence of the protease in 293 cells and that the fusion protein without the recognition sequence remained intact. This chimeric protein was localized exclusively in the cytoplasm as visualized by X-gal staining and immunofluorescence microscopy. In contrast, when expressed together with the protease, beta-gal was predominantly detected in the nuclei. Moreover, we isolated 293-cell clones constitutively expressing the protease, indicating that this protease is not cytotoxic. These results suggest that the viral protease-mediated alteration of subcellular localization can potentially regulate the function of nuclear proteins.     

Evidence for NL1-independent nuclear translocation of the mineralocorticoid receptor

In the absence of hormone, corticosteroid receptors are primarily located in the cytoplasm, and they rapidly accumulate in the nucleus (t0.5 = 5 min) upon ligand binding. It is generally believed that the dissociation of hsp90 from the receptor is an absolute requirement for allowing its nuclear translocation. However, recent evidence suggests that hsp90 may remain associated with the glucocorticoid receptor during this process, and thus, the receptor nuclear localization signal (NLS) is not obscured by its presence. To determine the requirements for mineralocorticoid receptor (MR) nuclear transport, it was first shown that in rat kidney collecting duct cells, nuclear localization of MR in the presence of aldosterone was complete in 10 min. Although the hsp90 inhibitor radicicol delayed nuclear translocation, it did not prevent complete nuclear accumulation of MR at longer incubation times (t0.5 = 30-40 min). MR carbamylation generates a non-steroid-transformed receptor that, in contrast to native MR, is very stable in cell-free systems. In contrast to the full nuclear translocation of aldosterone-transformed MR, only a fraction of the carbamylated MR became nuclear in digitonin-permeabilized cells even though its NLS is exposed. Furthermore, while preincubation of permeabilized cells with NL1 peptide or anti-NL1 antibody fully inhibited the nuclear translocation of NL1-tagged albumin, neither treatment fully inhibited MR nuclear translocation. We postulate that there are at least two possible mechanisms for MR nuclear translocation. One of them is hsp90- and NL1-dependent, and the other functions in a manner that is independent of the classical pathway.     

Inhibition of nuclear import of LIMK2 in endothelial cells by protein kinase C-dependent phosphorylation at Ser-283

LIM kinases (LIMKs) are mainly in the cytoplasm and regulate actin dynamics through cofilin phosphorylation. Recently, it has been reported that nuclear localization of LIMKs can mediate suppression of cyclin D1 expression. Using immunofluorescence monitoring of enhanced green fluorescent protein-tagged LIMK2 in combination with photobleaching techniques and leptomycin B treatment, we demonstrate that LIMK2 shuttles between the cytoplasm and the nucleus in endothelial cells. Sequence analysis predicted two PKC phosphorylation sites in LIMK2 but not in LIMK1. One site at Ser-283 is present between the PDZ and the kinase domain, and the other site at Thr-494 is within the kinase domain. Activation of PKC by phorbol ester treatment of endothelial cells stimulated LIMK2 phosphorylation at Ser-283 and inhibited nuclear import of LIMK2 and the PDZ kinase construct of LIMK2 (amino acids 142-638) but not of LIMK1. The PKC-delta isoform phosphorylated LIMK2 at Ser-283 in vitro. Mutational analysis indicated that LIMK2 phosphorylation at Ser-283 but not Thr-494 was functional. Serum stimulation of endothelial cells also inhibited nuclear import of PDZK-LIMK2 by protein kinase C-dependent phosphorylation of Ser-283. Our study shows that phorbol ester and serum stimulation of endothelial cells inhibit nuclear import of LIMK2 but not LIMK1. This effect was dependent on PKC-delta-mediated phosphorylation of Ser-283. Since phorbol ester enhanced cyclin D1 expression and subsequent G1-to-S-phase transition of endothelial cells, we suggest that the PKC-mediated exclusion of LIMK2 from the nucleus might be a mechanism to relieve suppression of cyclin D1 expression by LIMK2.     

A functional enhanced green fluorescent protein (EGFP)-tagged angiotensin II at(1a) receptor recruits the endogenous Galphaq/11 protein to the membrane and induces its specific internalization independently of receptor-g protein coupling in HEK-293 cells

The angiotensin II (Ang II) AT(1A) receptor was tagged at its C terminus with the enhanced green fluorescent protein (EGFP), and the corresponding chimeric cDNA was expressed in HEK-293 cells. This tagged receptor presents wild-type pharmacological and signaling properties and can be immunodetected by Western blotting and immunoprecipitation using EGFP antibodies. Therefore, this EGFP-tagged AT(1A) receptor is the perfect tool for analyzing in parallel the subcellular distributions of the receptor and its interacting G protein and their trafficking using confocal microscopy. Morphological observation of both the fluorescent receptor and its cognate Galphaq/11 protein, identified by indirect immunofluorescence, and the development of a specific software for digital image analysis together allow examination and quantification of the cellular distribution of these proteins before and after the binding of different agonist or antagonist ligands. These observations result in several conclusions: 1) Expression of increasing amounts of the AT(1A) receptor at the cell surface is associated with a progressive recruitment of the cytosolic Galphaq/11 protein at the membrane; 2) Internalization of the EGFP-tagged AT(1A) induced by peptide ligands but not nonpeptide ligands is accompanied by a Galphaq/11 protein intracellular translocation, which presents a similar kinetic pattern but occurs predominantly in a different compartment; and 3) This Galphaq/11 protein cellular translocation is dependent on receptor internalization process, but not G protein coupling and signal transduction mechanisms, as assessed by pharmacological data using agonists and antagonists and the characterization of AT(1A) receptor mutants (D(74)N and Delta329) for which the coupling and internalization functions are modified.     

Heterooligomerization of human dopamine receptor 2 and somatostatin receptor 2 Co-immunoprecipitation and fluorescence resonance energy transfer analysis

Somatostatin and dopamine receptors are well expressed and co-localized in several brain regions, suggesting the possibility of functional interactions. In the present study we used a combination of pharmacological, biochemical and photobleaching fluorescence resonance energy transfer (pbFRET) to determine the functional interactions between human somatostatin receptor 2 (hSSTR2) and human dopamine receptor 2 (hD2R) in both co-transfected CHO-K1 or HEK-293 cells as well as in cultured neuronal cells which express both the receptors endogenously. In monotransfected CHO-K1 or HEK-293 cells, D2R exists as a preformed dimer which is insensitive to agonist or antagonist treatment. In control CHO-K1 cells stably co-transfected with hD2R and hSSTR2, relatively low FRET efficiency and weak expression in co-immunoprecipitate from HEK-293 cells suggest the absence of preformed heterooligomers. However, upon treatment with selective ligands, hD2R and hSSTR2 exhibit heterodimerization. Agonist-induced heterodimerization was accompanied by increased affinity for dopamine and augmented hD2R signalling as well as prolonged hSSTR2 internalization. In contrast, cultured striatal neurons display constitutive heterodimerization between D2R and SSTR2, which were agonist-independent. However, heterodimerization in neurons was completely abolished in the presence of the D2R antagonist eticlopride. These findings suggest that hD2R and hSSTR2 operate as functional heterodimers modulated by ligands in situ, which may prove to be a useful model in designing new therapeutic drugs.     

Immunologic analysis of human breast cancer progesterone receptors. 2. Structure, phosphorylation, and processing

We have used a monoclonal antibody (MAb) directed against chick oviduct progesterone receptors (PR), that cross-reacts with human PR, to analyze PR structure and phosphorylation. This MAb, designated PR-6, interacts only with B receptors (Mr 120,000) of T47D human breast cancer cells; it has no affinity for A receptors (Mr 94,000) or for proteolytic fragments from either protein. The antibody immunoprecipitates native B receptors and was used to study the structure of native untransformed 8S and transformed 4S receptors, using sucrose density gradient analysis, photoaffinity labeling, and gel electrophoresis. On molybdate-containing low-salt gradients, PR-6 complexes with 8S B receptors, causing their shift to the bottom of the gradient while A receptors remain at 8 S. Therefore, A and B receptors form separate 8S complexes, and we conclude that A and B do not dimerize in the holoreceptor. Similar gradient studies using salt-containing, molybdate-free buffers show that there are two forms of salt-transformed 4S receptors, comprising either A proteins or B proteins, suggesting that A and B are also not linked to one another in transformed PR. The independence of A- and B-receptor complexes was confirmed by the finding that purified, transformed B receptors bind well to DNA-cellulose. Since PR-6 cross-reacts with nuclear PR, it was used to analyze nuclear PR processing--a down-regulation step associated with receptor loss as measured by hormone binding. Insoluble nuclear receptors and soluble cytosol receptors were measured by immunoblotting following treatment of T47D cells for 5 min to 48 h with either R5020 or progesterone. From 8 to 48 h after R5020 treatment, immunoassayable receptors decreased in nuclei and were not recovered in cytosols. Nuclear receptors also decreased after progesterone treatment but replenished in cytosols between 8 and 24 h after the start of treatment. Thus, processing involves a true loss of nuclear receptor protein, and not just loss of hormone binding activity, and occurs after progesterone or R5020 treatment. This loss is chronic, however, only in R5020-treated cells. Additional studies focused on the covalent modifications of receptors. We previously described shifts in apparent molecular weight of nuclear PR following R5020 treatment using in situ photoaffinity labeling. To show whether these shifts can be explained by receptor phosphorylation, untreated cells and hormone-treated cells were metabolically labeled with [32P]orthophosphate, and the B receptors were isolated by immunoprecipitation with PR-6 and analyzed by sodium dodecyl sulfate (SDS) gel electrophoresis.(ABSTRACT TRUNCATED AT 400 WORDS)