Role of clathrin-mediated endocytosis in CXCR2 sequestration, resensitization, and signal transduction

CXCR2 is a seven-transmembrane receptor that transduces intracellular signals in response to the chemokines interleukin-8, melanoma growth-stimulatory activity/growth-regulatory protein, and other ELR motif-containing CXC chemokines by coupling to heterotrimeric GTP-binding proteins. In this study, we explored the mechanism responsible for ligand-induced CXCR2 endocytosis. Here, we demonstrate that dynamin, a component of clathrin-mediated endocytosis, is essential for CXCR2 endocytosis and resensitization. In HEK293 cells, dynamin I K44A, a dominant-negative mutant of dynamin that inhibits the clathrin-mediated endocytosis, blocks the ligand-stimulated CXCR2 sequestration. Furthermore, co-expression of dynamin I K44A significantly delays dephosphorylation of CXCR2 after ligand stimulation, suggesting that clathrin-mediated endocytosis plays an important role in receptor dephosphorylation and resensitization. In addition, ligand-mediated receptor down-regulation is attenuated when receptor internalization is inhibited by dynamin I K44A. Interestingly, inhibition of receptor endocytosis by dynamin I K44A does not affect the CXCR2-mediated stimulation of mitogen-activated protein kinase. Most significantly, our data indicate that the ligand-stimulated receptor endocytosis is required for CXCR2-mediated chemotaxis in HEK293 cells. Taken together, our findings suggest that clathrin-mediated CXCR2 internalization is crucial for receptor endocytosis, resensitization, and chemotaxis.     

beta-arrestins regulate interleukin-8-induced CXCR1 internalization.

The functional role of neutrophils during acute inflammatory responses is regulated by two high affinity interleukin-8 receptors (CXCR1 and CXCR2) that are rapidly desensitized and internalized upon binding their cognate chemokine ligands. The efficient re-expression of CXCR1 on the surface of neutrophils following agonist-induced internalization suggests that CXCR1 surface receptor turnover may involve regulatory pathways and intracellular factors similar to those regulating beta2-adrenergic receptor internalization and re-expression. To examine the internalization pathway utilized by ligand-activated CXCR1, a CXCR1-GFP construct was transiently expressed in two different cell lines, HEK 293 and RBL-2H3 cells. While interleukin-8 stimulation promoted CXCR1 sequestration in RBL-2H3 cells, receptor internalization in HEK 293 cells required co-expression of G protein-coupled receptor kinase 2 and beta-arrestin proteins. The importance of beta-arrestins in CXCR1 internalization was confirmed by the ability of a dominant negative beta-arrestin 1-V53D mutant to block internalization of CXCR1 in RBL-2H3 cells. A role for dynamin was also demonstrated by the lack of CXCR1 internalization in dynamin I-K44A dominant negative mutant-transfected RBL-2H3 cells. Agonist-promoted co-localization of transferrin and CXCR1-GFP in endosomes of RBL-2H3 cells confirmed that receptor internalization occurs via clathrin-coated vesicles. Our data provides a direct link between agonist-induced internalization of CXCR1 and a requirement for G protein-coupled receptor kinase 2, beta-arrestins, and dynamin during this process.     

Differential methylation and altered conformation of cytoplasmic and nuclear forms of protein phosphatase 2A during cell cycle progression

Protein phosphatase 2A (PP2A) appears to be involved in the regulation of many cellular processes. Control mechanisms that lead to the activation (and deactivation) of the various holoenzymes to initiate appropriate dephosphorylation events remain obscure. The core components of all PP2A holoenzymes are the catalytic (PP2Ac) and 63-65- kD regulatory (PR65) subunits. Monospecific and affinity-purified antibodies against both PP2Ac and PR65 show that these proteins are ubiquitously localized in the cytoplasm and the nucleus in nontransformed fibroblasts. As determined by quantitative immunofluorescence the core subunits of PP2A are twofold more concentrated in the nucleus than in the cytoplasm. Detailed analysis of synchronized cells reveals striking changes in the nuclear to cytoplasmic ratio of PP2Ac-specific immunoreactivity albeit the total amounts of neither PP2Ac nor PR65 in each compartment alters significantly during the cell cycle. Our results imply that differential methylation of PP2Ac occurs at the G0/G1 and G1/S boundaries. Specifically a demethylated form of PP2Ac is found in the cytoplasm of G1 cells, and in the nucleus of S and G2 cells. In addition nuclear PP2A holoenzymes appear to undergo conformational changes at the G0/G1 and G1/S boundaries. During mitosis PP2A is lost from the nuclear compartment, and unlike protein phosphatase 1 shows no specific association with the condensed chromatin.     

Identification of a motif in the carboxyl terminus of CXCR2 that is involved in adaptin 2 binding and receptor internalization

Agonist treatment of cells expressing the chemokine receptor, CXCR2, induces receptor phosphorylation and internalization through a dynamin-dependent mechanism. In the present study, we demonstrate that a carboxyl terminus-truncated mutant of CXCR2 (331T), which no longer undergoes agonist-induced phosphorylation, continues to undergo ligand-induced internalization in HEK293 cells. This mutant receptor exhibits reduced association with beta-arrestin 1 but continues to exhibit association with adaptin 2 alpha and beta subunits. Replacing Leu320-321 and/or Ile323-Leu324 with Ala (LL320,321AA, IL323,324AA, and LLIL320,321,323,324AAAA) in wild-type CXCR2 or 331T causes little change in ligand binding and signaling through Ca(2+) mobilization but greatly impairs the agonist-induced receptor sequestration and ligand-mediated chemotaxis. The LL320,321AA, IL323,324AA, and LLIL320,321,323,324AAAA mutants of CXCR2 exhibit normal binding to beta-arrestin 1 but exhibit decreased binding to adaptin 2alpha and beta. These data demonstrate a role for the LLKIL motif in the carboxyl terminus of CXCR2 in receptor internalization and cell chemotaxis and imply a role for adaptin 2 in the endocytosis of CXCR2.     

Baculovirus expression, purification, and characterization of human protein phosphatase 2A catalytic subunits alpha and beta

Protein phosphatase 2A (PP2A) contains a 36-kDa catalytic subunit (PP2Ac), a 65-kDa structural subunit (PR65/A), and a regulatory B subunit. The core enzyme consists of the structural and catalytic subunits. The catalytic subunit exists as two closely related isoforms, alpha and beta. Several natural toxins, including okadaic acid (OA) and microcystins, specifically inhibit PP2A. To obtain biologically active recombinant PP2A and to compare the properties of the PP2A catalytic subunit alpha and beta isoforms, we expressed human PP2Acalpha and cbeta in High Five insect cells. The recombinant PP2Acalpha and cbeta possess similar phosphatase activities using p-NPP and phosphopeptide as substrates and are strongly inhibited by OA and microcystin-LR to similar degrees. In addition, PP2Acalpha or cbeta was co-expressed with PR65/A and co-purified as a core dimer, PP2AD (Aalpha/calpha and Aalpha/cbeta) with PR65alpha/Aalpha. The recombinant PP2AD bound to the B subunit in vitro. These results show that the recombinant PP2Acalpha and cbeta are identical in their ability to associate with the A and B subunits, in their phosphatase activities, and in carboxyl-methylation. Furthermore, our results show that High Five insect cells can produce biologically active recombinant PP2A, which should be a valuable tool for detecting natural toxins and investigating the mechanism of PP2A catalysis and other protein interactions.     

Differential modes of regulation of cxc chemokine-induced internalization and recycling of human CXCR1 and CXCR2

Studies of human neutrophil IL-8 receptors, CXCR1 and CXCR2, have shown that the two receptors are differentially regulated by ELR⁺-CXC chemokines, that they differ functionally and may have diverse roles in mediating the inflammatory process. To elucidate the role of CXCR1 and CXCR2 in inflammation and to delineate the basis for the divergent regulation of these receptors by IL-8 and NAP-2, we characterized the IL-8- and NAP-2-induced mechanisms regulating the expression of each receptor, focusing on receptor internalization and recycling. Using HEK 293 cell transfectants, IL-8 was shown to induce significantly higher levels of CXCR2 internalization than NAP-2. Moreover, although CXCR2 bound IL-8 and NAP-2 with similarly high affinity, IL-8 functionally competed with and displaced NAP-2, and prompted high levels of internalization, similar to those induced by IL-8 alone. In a system providing an identical cellular milieu for reliable comparisons between CXCR1 and CXCR2, we have shown that the mechanisms controlling the internalization of CXCR1 diverge from those regulating CXCR2 internalization. Whereas IL-8-induced internalization of CXCR1 was profoundly dependent on a region of the carboxyl terminus expressing six phosphorylation sites, internalization of CXCR2 was primarily regulated by a membrane proximal domain of the carboxyl terminus that does not express phosphorylation sites. Analysis of receptor re-expression on the plasma membrane indicated that at early time points following removal of free ligand and incubation of the cells at 37°C, receptor recycling accounted for recovery of CXCR1 and CXCR2 expression, whereas at later time points other processes may be involved in receptor re-expression. Phosphorylation-independent mechanisms were shown to direct both receptors to the recycling pathway. The differential control of CXCR1 vs CXCR2 internalization by IL-8 and NAP-2, as well as by phosphorylation-mediated mechanisms, suggests that a chemokine- and receptor-specific mode of regulation of internalization may contribute to the divergent activities of these receptors.     

Intracellular cross-talk between the GPCR CXCR1 and CXCR2: role of carboxyl terminus phosphorylation sites

In the present study, we used the human chemokine receptors CXCR1 and CXCR2 as a model system for the study of intracellular cross-talk between two closely related G protein-coupled receptors (GPCR). In cells expressing either CXCR1 or CXCR2, exposure to the CXCL8 ligand resulted in prominent reduction in cell surface expression of the receptors. We have shown previously that the reduction in cell surface expression of CXCR1 and CXCR2, to be termed herein "down-regulation", is significantly lower in cells expressing both receptors together. Now we show that reduced receptor down-regulation was specific to the CXCR1+CXCR2 pair. Also, CXCR2 carboxyl terminus phosphorylation sites were required for inducing inhibition of CXCR1 down-regulation, and vice versa. Accordingly, phosphorylation of CXCR2 carboxyl terminus domain was intact when expressed together with CXCR1. Moreover, specific carboxyl terminus phosphorylation sites on each of the wild type receptors protected them from more severe inhibition of down-regulation, induced by joint expression with the other receptor. When concomitantly expressed, CXCR1 and CXCR2 were impaired in recycling to the plasma membrane, despite their undergoing intact dephosphorylation. Overall, we show that cross-talk between two GPCR is manifested by impairment of their intracellular trafficking, primarily of ligand-induced down-regulation, via carboxyl terminus phosphorylation sites.     

Eukaryotic translation termination factor 1 associates with protein phosphatase 2A and targets it to ribosomes

Purification of type 2A protein phosphatase (PP2A) from rabbit skeletal muscle resulted in the isolation of a trimeric phosphatase which is composed of a catalytic (PP2Ac), a structural (PR65alpha/Aalpha), and a regulatory (PR55alpha/Balpha) subunit, together with translation termination factor 1 (eRF1) and another protein of 55 kD (EMBO J., 15, 101-112). Yeast two-hybrid system analysis demonstrated that the eRF1 interacted with PP2Acalpha but not with PR65alpha/Aalpha or PR55alpha/Balpha. The N-terminal region of PP2Acalpha, comprising 50 amino acid residues, and the C-terminal part of eRF1, corresponding to an internal region between amino acids 338-381, were found to be necessary for eRF1--PP2Acalpha interaction in yeast. Immunoprecipitations using 12CA5 antibodies and extracts from COS1 cells transiently transfected with eRF1 tagged with 9-amino acid epitope from influenza hemagglutinin (HA) demonstrated the presence of eRF1--PP2Acalpha--PR65alpha/Aalpha complex in these cells. In addition, polysomes obtained from COS1 cells overexpressing HA--eRF1 displayed several-fold higher PP2A activity than control polysomes. No effect of either PP2Ac or dimeric and trimeric PP2A holoenzymes on the rate of translation termination was detected using an in vitro reconstituted translation termination assay. In summary, eRF1 appears to represent a novel PP2A-targeting subunit that brings this phosphatase in contact with putative ribosomal substrate(s). It remains to be established whether termination of translation requires dephosphorylation of participating protein factor(s).     

Vimentin Dephosphorylation by Protein Phosphatase 2A Is Modulated by the Targeting Subunit B55

The intermediate filament protein vimentin is a major phosphoprotein in mammalian fibroblasts, and reversible phosphorylation plays a key role in its dynamic rearrangement. Selective inhibition of type 2A but not type 1 protein phosphatases led to hyperphosphorylation and concomitant disassembly of vimentin, characterized by a collapse into bundles around the nucleus. We have analyzed the potential role of one of the major protein phosphatase 2A (PP2A) regulatory subunits, B55, in vimentin dephosphorylation. In mammalian fibroblasts, B55 protein was distributed ubiquitously throughout the cytoplasm with a fraction associated to vimentin. Specific depletion of B55 in living cells by antisense B55 RNA was accompanied by disassembly and increased phosphorylation of vimentin, as when type 2A phosphatases were inhibited using okadaic acid. The presence of B55 was a prerequisite for PP2A to efficiently dephosphorylate vimentin in vitro or to induce filament reassembly in situ. Both biochemical fractionation and immunofluorescence analysis of detergent-extracted cells revealed that fractions of PP2Ac, PR65, and B55 were tightly associated with vimentin. Furthermore, vimentin-associated PP2A catalytic subunit was displaced in B55-depleted cells. Taken together these data show that, in mammalian fibroblasts, the intermediate filament protein vimentin is dephosphorylated by PP2A, an event targeted by B55.     

The structure of Tap42/alpha4 reveals a tetratricopeptide repeat-like fold and provides insights into PP2A regulation

Physiological functions of protein phosphatase 2A (PP2A) are determined via the association of its catalytic subunit (PP2Ac) with diverse regulatory subunits. The predominant form of PP2Ac assembles into a heterotrimer comprising the scaffolding PR65/A subunit together with a variable regulatory B subunit. A distinct population of PP2Ac associates with the Tap42/alpha4 subunit, an interaction mutually exclusive with that of PR65/A. Tap42/alpha4 is also an interacting subunit of the PP2Ac-related phosphatases, PP4 and PP6. Tap42/alpha4, an essential protein in yeast and suppressor of apoptosis in mammals, contributes to critical cellular functions including the Tor signaling pathway. Here, we describe the crystal structure of the PP2Ac-interaction domain of Saccharomyces cerevisiae Tap42. The structure reveals an all alpha-helical protein with striking similarity to 14-3-3 and tetratricopeptide repeat (TPR) proteins. Mutational analyses of structurally conserved regions of Tap42/alpha4 identified a positively charged region critical for its interactions with PP2Ac. We propose a scaffolding function for Tap42/alpha4 whereby the interaction of PP2Ac at its N-terminus promotes the dephosphorylation of substrates recruited to the C-terminal region of the molecule.     

Biochemical characterization of recombinant subunits of type 2A protein phosphatase overexpressed in Pichia pastoris.

Methylotrophic yeast Pichia pastoris was used for a medium-scale expression of structural (PR65/A) and catalytic (PP2Ac) subunits of human type 2A protein phosphatase (PP2A). Constructs encoding these subunits, which were designed to introduce eight histidines at their N-termini, were introduced into the KM71 Pichia strain by homologous recombination. Recombinant proteins overproduced after methanol induction were purified from cell-free extracts by anion-exchange chromatography on DEAE-Sepharose, and Ni2+/nitrilotriacetate/agarose. In addition, chromatography on omega-aminohexyl-Sepharose was applied to purify recombinant (r)PR65/A. This purification scheme yielded approximately 5 mg and 100 microg of rPR65/A and rPP2Ac, respectively, from 1 L of the yeast culture. The specific activity of rPP2Ac measured with [32P]phosphorylase a [1.7 micromol.min-1.(mg protein)-1] and its inhibition by okadaic acid (IC50 = 0.66 nM) were similar to PP2A isolated from rabbit skeletal muscle. As demonstrated by immunodetection with methylation state-specific antibodies, recombinant PP2Ac was carboxymethylated at the last C-terminal leucine residue. Recombinant PP2A subunits were able to form a complex as demonstrated both by activity assays in the presence of protamine and by chromatography on protamine-agarose. In summary, P. pastoris provides a convenient heterologous system for the production of recombinant subunits of PP2A.     

Protein phosphatase 2A and neutral sphingomyelinase 2 regulate IRAK-1 protein ubiquitination and degradation in response to interleukin-1beta

The IL-1β signaling cascade is initiated by the phosphorylation of IL-1β receptor-associated kinase-1 (IRAK-1), followed by its ubiquitination and degradation. This paper investigates the regulation of IRAK-1 degradation in primary hepatocytes and in HEK cells overexpressing the IL-1β receptor. We provide evidence that protein phosphatase 2A (PP2A) is a negative regulator of the phosphorylation, Lys(48)-linked ubiquitination, and degradation of IRAK-1. PP2A catalytic activity increased within 30 min of stimulation with IL-1β. siRNA against PP2A catalytic subunit (PP2Ac) or treatment with pharmacological inhibitor, okadaic acid, enhanced IRAK-1 Lys(48)-linked ubiquitination and degradation. Direct interaction between PP2Ac and IRAK-1 was observed, suggesting that IRAK-1 might be a PP2A substrate. The mechanisms of PP2A activation by IL-1β involved neutral sphingomyelinase-2 (NSMase-2) and an accumulation of ceramide. Overexpression of NSMase-2 delayed IRAK-1 degradation in a PP2A-dependent manner, whereas NSMase-2 silencing had the opposite effect. The addition of sphingomyelinase, ceramide, or a proteasome inhibitor all led to retention of IRAK-1 at the cell membrane and to increased JNK phosphorylation. This study suggests that NSMase-2- and PP2A-dependent regulation of IRAK-1 degradation is a novel mechanism to fine tune the magnitude of IL-1β response.     

Interaction of nucleoredoxin with protein phosphatase 2A

A trimeric protein phosphatase 2A (PP2A(T55)) composed of the catalytic (PP2Ac), structural (PR65/A), and regulatory (PR55/B) subunits was isolated from rabbit skeletal muscle by thiophosphorylase affinity chromatography, and contained two additional proteins of 54 and 55 kDa, respectively. The 54 kDa protein was identified as eukaryotic translation termination factor 1 (eRF1) and as a PP2A interacting protein. The 55 kDa protein is now identified as nucleoredoxin (NRX). The formation of a complex between GST-NRX, PP2A(C) and PP2A(D) was demonstrated by pull-down experiments with purified forms of PP2A, and by immunoprecipitation of HA-tagged NRX expressed in HEK293 cells complexed endogenous PP2A subunits. Analysis of PP2A activity in the presence of GST-NRX showed that NRX competed with polycations for both stimulatory and inhibitory effects on different forms of PP2A.     

The carboxyl-terminal PDZ ligand motif of chemokine receptor CXCR2 modulates post-endocytic sorting and cellular chemotaxis

Adaptor protein interaction with specific peptide motifs found within the intracellular, carboxyl terminus of chemokine receptor CXCR2 has been shown to modulate intracellular trafficking and receptor function. Efficient ligand-induced internalization of this receptor is dependent on the binding of adaptor protein 2 to the specific LLKIL motif found within the carboxyl terminus (1). In this study we show that the carboxyl-terminal type 1 PDZ ligand motif (-STTL) of CXCR2 plays an essential role in both proper intracellular receptor trafficking and efficient cellular chemotaxis. First, we show that CXCR2 is sorted to and degraded in the lysosome upon long-term ligand stimulation. We also show that receptor degradation is not dependent upon receptor ubiquitination, but is instead modulated by the carboxyl-terminal type I PDZ ligand of CXCR2. Deletion of this ligand results in increased degradation, earlier co-localization with the lysosome, and enhanced sorting to the Rab7-positive late endosome. We also show that deletion of this ligand effects neither receptor internalization nor receptor recycling. Furthermore, we demonstrate that deletion of the PDZ ligand motif results in impaired chemotactic response. The data presented here demonstrate that the type I PDZ ligand of CXCR2 acts to both delay lysosomal sorting and facilitate proper chemotactic response.     

Essential role of dynamin in internalization of M2 muscarinic acetylcholine and angiotensin AT1A receptors

Most G protein-coupled receptors (GPCRs), including the M(1) muscarinic acetylcholine receptor (mAChR), internalize in clathrin-coated vesicles, a process that requires dynamin GTPase. The observation that some GPCRs like the M(2) mAChR and the angiotensin AT(1A) receptor (AT(1A)R) internalize irrespective of expression of dominant-negative K44A dynamin has led to the proposal that internalization of these GPCRs is dynamin-independent. Here, we report that, contrary to what is postulated, internalization of M(2) mAChR and AT(1A)R in HEK-293 cells is dynamin-dependent. Expression of N272 dynamin, which lacks the GTP-binding domain, or K535M dynamin, which is not stimulatable by phosphatidylinositol 4, 5-bisphosphate, strongly inhibits internalization of M(1) and M(2) mAChRs and AT(1A)Rs. Expression of kinase-defective K298M c-Src or Y231F,Y597F dynamin (which cannot be phosphorylated by c-Src) reduces M(1) mAChR internalization. Similarly, c-Src inhibitor PP1 as well as the generic tyrosine kinase inhibitor genistein strongly inhibit M(1) mAChR internalization. In contrast, M(2) mAChR internalization is not (or is only slightly) reduced by expression of these constructs or treatment with PP1 or genistein. Thus, dynamin GTPases are not only essential for M(1) mAChR but also for M(2) mAChR and AT(1A)R internalization in HEK-293 cells. Our findings also indicate that dynamin GTPases are differentially regulated by c-Src-mediated tyrosine phosphorylation.     

Rapid agonist-induced desensitization and internalization of the A(2B) adenosine receptor is mediated by a serine residue close to the COOH terminus

The G(s)-coupled rat A(2B) adenosine receptor (A(2B)-AR) was epitope-tagged at the NH(2) terminus with hemagglutinin (HA) and subjected to progressive deletions or point mutations of the COOH terminus in order to determine regions of the receptor that contribute to agonist-induced desensitization and internalization. When expressed stably in Chinese hamster ovary cells, a mutant receptor in which the final 2 amino acids were deleted, the Leu(330)-stop mutant, underwent rapid agonist-induced desensitization and internalization as did the wild type (WT) receptor. However, the Phe(328) and the Gln(325)-stop mutants were resistant to rapid agonist-induced desensitization and internalization. Co-expression of arrestin-2-green fluorescent protein (arrestin-2-GFP) with WT receptor or Leu(330)-stop mutant resulted in rapid translocation of arrestin-2-GFP from cytosol to membrane upon agonist addition. On the other hand, agonist activation of the Phe(328)-stop or Gln(325)-stop mutant did not result in translocation of arrestin-2-GFP from cytosol. A COOH terminus point mutant, S329G, was also unable to undergo rapid agonist-induced desensitization and internalization, indicating that Ser(329) is a critical residue for these processes. A further deletion mutant (Ser(326)-stop) unexpectedly underwent rapid agonist-induced desensitization and internalization. However, activation of this mutant did not promote translocation of arrestin-2-GFP from cytosol to membrane. In addition, whereas WT receptor internalization was markedly inhibited by co-expression of dominant negative mutants of arrestin-2 (arrestin-2-(319-418)), dynamin (dynamin K44A), or Eps-15 (EDelta95-295), Ser(326)-stop receptor internalization was only inhibited by dominant negative mutant dynamin. Taken together these results indicate that Ser(329), close to the COOH terminus of the rat A(2B)-AR, is critical for the rapid agonist-induced desensitization and internalization of the receptor. However, deletion of the COOH terminus also uncovers a motif that is able to redirect internalization of the receptor to an arrestin- and clathrin-independent pathway.