Formyl peptide receptor-mediated ERK1/2 activation occurs through G(i) and is not dependent on beta-arrestin1/2

Formyl peptide receptor (FPR) and C5a receptor (C5aR) are chemoattractant G protein-coupled receptors (GPCRs) involved in the innate immune response against bacterial infections and tissue injury. Like other GPCRs, they recruit beta-arrestin1/2 to the plasma membrane and activate the extracellular signal-regulated kinases 1 and 2 (ERK1/2). Previous studies with several GPCRs have suggested that beta-arrestins play an important role as signal transducers by scaffolding signaling molecules such as ERK1/2. This function of the beta-arrestins was not discovered until several years after their role in desensitization and endocytosis had been reported. In this study, we investigated the role of the beta-arrestins in the activation of ERK1/2 and receptor endocytosis. We took advantage of previously described mutants of FPR that have defects in G(i) coupling or beta-arrestin recruitment. The results obtained with the mutant FPRs, as well as experiments using an inhibitor of G(i) and cells overexpressing beta-arrestin2, showed that activation of ERK1/2 takes place through G(i) and is not affected by beta-arrestins. However, overexpression of beta-arrestin2 does enhance FPR sequestration from the cell surface, suggesting a role in desensitization, as shown for many other GPCRs. Experiments with CHO C5aR cells showed similar sensitivity to the G(i) inhibitor as CHO FPR cells, suggesting that the predominant activation of ERK1/2 through G protein may be a common characteristic among chemoattractant receptors.     

Multiple talents of the chemokine receptor-CXCR4

CXCR4 is a clinically relevant chemokine receptor that has first gained attention as one of the cofactors for HIV entry into target cells. Moreover, the receptor is involved in cancer cell migration to distant metastatic sites and immune effector recruitment in inflammatory diseases such as asthma and rheumatoid arthritis. Unfortunately, pharmacologic intervention is complicated by the vital function of CXCR4 in the organism. The most prominent of these functions is its role in stem cell homing. The CXCR4 chemokine ligand, produced by bone marrow stromal cells, leads both to migration of hematopoietic stem cells towards this niche and their retention in this compartment. As models of G-protein coupled receptor (GPCR) activation evolve, it becomes clear that multiple factors modulate the functional outcome of ligand binding to a receptor. Modulation of GPCR activity, for example by allosteric ligands, may permit more subtle therapeutic approaches adapted to long term treatment. In addition, GPCR signalling can be altered by hetero-oligomerization of GPCRs. In this perspective, it might be possible to achieve modulation of GPCR signalling by also targeting the oligomerization partner of a given receptor. This approach is described using the example of strategies that aim at the optimization of stem cell homing in the context of cord blood-derived hematopoietic stem cell transplantation.     

Plerixafor induces the rapid and transient release of stromal cell-derived factor-1 alpha from human mesenchymal stromal cells and influences the migration behavior of human hematopoietic progenitor cells

The interaction between the stromal cell-derived factor-1 alpha (SDF-1α, CXCL12) and its chemokine receptor CXCR4 has been reported to regulate stem cell migration, mobilization and homing. The CXCR4 antagonist plerixafor is highly efficient in mobilizing hematopoietic progenitor cells (HPCs). However, the precise regulatory mechanisms governing the CXCR4/SDF-1α axis between the bone marrow niche and HPCs remain unclear. In this study, we quantify the impact of plerixafor on the interaction between human bone marrow derived mesenchymal stromal cells (MSCs) and human CD34+ HPCs. An assessment of SDF-1α levels in the supernatant of MSC cultures revealed that exposure to plerixafor led to a transient increase but had no long-term effect. In Transwell experiments, we observed that the addition of SDF-1α significantly stimulated HPC migration; this stimulation was almost completely antagonized by the addition of plerixafor, confirming the direct impact of the CXCR4/SDF-1α interaction on the migration capacity of HPCs. We also developed a new microstructural niche model to determine the chemotactic sensitivity of HPCs. Time-lapse microscopy demonstrated that HPCs migrated actively along an SDF-1α gradient within the microchannels and the quantitative assessment of the required minimum gradient initiating this chemotaxis revealed a surprisingly high sensitivity of HPCs. These data demonstrate the fine-tuned balance of the CXCR4/SDF-1α axis and the synergistic effects of plerixafor on HPCs and MSCs, which most likely represent the key mechanisms for the consecutive mobilization of HPCs from the bone marrow niche into the circulating blood.     

Third Extracellular Loop (EC3)-N Terminus Interaction Is Important for Seven-transmembrane Domain Receptor Function: IMPLICATIONS FOR AN ACTIVATION MICROSWITCH REGION*

The canonical heptahelical bundle architecture of seven-transmembrane domain (7TM) receptors is intertwined by three intra- and three extracellular loops, whose local conformations are important in receptor signaling. Many 7TM receptors contain a cysteine residue in the third extracellular loop (EC3) and a complementary cysteine residue on the N terminus. The functional role of such EC3-N terminus conserved cysteine pairs remains unclear. This study explores the role of the EC3-N terminus cysteine pairs on receptor conformation and G protein activation by disrupting them in the chemokine receptor CXCR4, while engineering a novel EC3-N terminus cysteine pair into the complement factor 5a receptor (C5aR), a chemo attractant receptor that lacks it. Mutated CXCR4 and C5aRs were expressed in engineered yeast. Mutation of the cysteine pair with the serine pair (C28S/C274S) in constitutively active mutant CXCR4 abrogated the receptor activation, whereas mutation with the aromatic pair (C28F–C274F) or the salt bridge pair (C28R/C274E), respectively, rescued or retained the receptor activation in response to CXCL12. In this context, the cysteine pair (Cys³⁰ and Cys²⁷²) engineered into the EC3-N terminus (Ser³⁰ and Ser²⁷²) of a novel constitutively active mutant of C5aR restrained the constitutive signaling without affecting the C5a-induced activation. Further mutational studies demonstrated a previously unappreciated role for Ser²⁷² on EC3 of C5aR and its interaction with the N terminus, thus defining a new microswitch region within the C5aR. Similar results were obtained with mutated CXCR4 and C5aRs expressed in COS-7 cells. These studies demonstrate a novel role of the EC3-N terminus cysteine pairs in G protein-coupled receptor activation and signaling.     

Regulation of C3a receptor signaling in human mast cells by G protein coupled receptor kinases

Background

The complement component C3a activates human mast cells via its cell surface G protein coupled receptor (GPCR) C3aR. For most GPCRs, agonist-induced receptor phosphorylation leads to receptor desensitization, internalization as well as activation of downstream signaling pathways such as ERK1/2 phosphorylation. Previous studies in transfected COS cells overexpressing G protein coupled receptor kinases (GRKs) demonstrated that GRK2, GRK3, GRK5 and GRK6 participate in agonist-induced C3aR phosphorylation. However, the roles of these GRKs on the regulation of C3aR signaling and mediator release in human mast cells remain unknown.

Methodology/Principal Findings

We utilized lentivirus short hairpin (sh)RNA to stably knockdown the expression of GRK2, GRK3, GRK5 and GRK6 in human mast cell lines, HMC-1 and LAD2, that endogenously express C3aR. Silencing GRK2 or GRK3 expression caused a more sustained Ca²⁺ mobilization, attenuated C3aR desensitization, and enhanced degranulation as well as ERK1/2 phosphorylation when compared to shRNA control cells. By contrast, GRK5 or GRK6 knockdown had no effect on C3aR desensitization, but caused a significant decrease in C3a-induced mast cell degranulation. Interestingly, GRK5 or GRK6 knockdown rendered mast cells more responsive to C3a for ERK1/2 phosphorylation.

Conclusion/Significance

This study demonstrates that GRK2 and GRK3 are involved in C3aR desensitization. Furthermore, it reveals the novel finding that GRK5 and GRK6 promote C3a-induced mast cell degranulation but inhibit ERK1/2 phosphorylation via C3aR desensitization-independent mechanisms. These findings thus reveal a new level of complexity for C3aR regulation by GRKs in human mast cells.     

Regulation of the human chemokine receptor CCR1. Cross-regulation by CXCR1 and CXCR2

To investigate the regulation of the CCR1 chemokine receptor, a rat basophilic leukemia (RBL-2H3) cell line was modified to stably express epitope-tagged receptor. These cells responded to RANTES (regulated upon activation normal T expressed and secreted), macrophage inflammatory protein-1alpha, and monocyte chemotactic protein-2 to mediate phospholipase C activation, intracellular Ca(2+) mobilization and exocytosis. Upon activation, CCR1 underwent phosphorylation and desensitization as measured by diminished GTPase stimulation and Ca(2+) mobilization. Alanine substitution of specific serine and threonine residues (S2 and S3) or truncation of the cytoplasmic tail (DeltaCCR1) of CCR1 abolished receptor phosphorylation and desensitization of G protein activation but did not abolish desensitization of Ca(2+) mobilization. S2, S3, and DeltaCCR1 were also resistant to internalization, mediated greater phosphatidylinositol hydrolysis and sustained Ca(2+) mobilization, and were only partially desensitized by RANTES, relative to S1 and CCR1. To study CCR1 cross-regulation, RBL cells co-expressing CCR1 and receptors for interleukin-8 (CXCR1, CXCR2, or a phosphorylation-deficient mutant of CXCR2, 331T) were produced. Interleukin-8 stimulation of CXCR1 or CXCR2 cross-phosphorylated CCR1 and cross-desensitized its ability to stimulate GTPase activity and Ca(2+) mobilization. Interestingly, CCR1 cross-phosphorylated and cross-desensitized CXCR2, but not CXCR1. Ca(2+) mobilization by S3 and DeltaCCR1 were also cross-desensitized by CXCR1 and CXCR2 despite lack of receptor phosphorylation. In contrast to wild type CCR1, S3 and DeltaCCR1, which produced sustained signals, cross-phosphorylated and cross-desensitized responses to CXCR1 as well as CXCR2. Taken together, these results indicate that CCR1-mediated responses are regulated at several steps in the signaling pathway, by receptor phosphorylation at the level of receptor/G protein coupling and by an unknown mechanism at the level of phospholipase C activation. Moreover selective cross-regulation among chemokine receptors is, in part, a consequence of the strength of signaling (i.e. greater phosphatidylinositol hydrolysis and sustained Ca(2+) mobilization) which is inversely correlated with the receptor's susceptibility to phosphorylation. Since many chemokines activate multiple chemokine receptors, selective cross-regulation among such receptors may play a role in their immunomodulation.     

Distinct and shared roles of β-arrestin-1 and β-arrestin-2 on the regulation of C3a receptor signaling in human mast cells

Background

The complement component C3a induces degranulation in human mast cells via the activation of cell surface G protein coupled receptors (GPCR; C3aR). For most GPCRs, agonist-induced receptor phosphorylation leads to the recruitment of β-arrestin-1/β-arrestin-2; resulting in receptor desensitization and internalization. Activation of GPCRs also leads to ERK1/2 phosphorylation via two temporally distinct pathways; an early response that reflects G protein activation and a delayed response that is G protein independent but requires β-arrestins. The role of β-arrestins on C3aR activation/regulation in human mast cells, however, remains unknown.

Methodology/Principal Findings

We utilized lentivirus short hairpin (sh)RNA to stably knockdown the expression of β-arrestin-1 and β-arrrestin-2 in human mast cell lines, HMC-1 and LAD2 that endogenously expresses C3aR. Silencing β-arrestin-2 attenuated C3aR desensitization, blocked agonist-induced receptor internalization and rendered the cells responsive to C3a for enhanced NF-κB activity as well as chemokine generation. By contrast, silencing β-arrestin-1 had no effect on these responses but resulted in a significant decrease in C3a-induced mast cell degranulation. In shRNA control cells, C3a caused a transient ERK1/2 phosphorylation, which peaked at 5 min but disappeared by 10 min. Knockdown of β-arrestin-1, β-arrestin-2 or both enhanced the early response to C3a and rendered the cells responsive for ERK1/2 phosphorylation at later time points (10–30 min). Treatment of cells with pertussis toxin almost completely blocked both early and delayed C3a-induced ERK1/2 phosphorylation in β-arrestin1/2 knockdown cells.

Conclusion/Significance

This study demonstrates distinct roles for β-arrestins-1 and β-arrestins-2 on C3aR desensitization, internalization, degranulation, NF-κB activation and chemokine generation in human mast cells. It also shows that both β-arrestin-1 and β-arrestin-2 play a novel and shared role in inhibiting G protein-dependent ERK1/2 phosphorylation. These findings reveal a new level of complexity for C3aR regulation by β-arrestins in human mast cells.     

Complement component 5a receptor oligomerization and homologous receptor down-regulation

Most G-protein-coupled receptors (GPCRs) form di(oligo)-meric structures that constitute signaling and trafficking units and might be essential for receptor functions. Cell responses to complement C5a receptor (C5aR) are tightly controlled by receptor desensitization and internalization. To examine the implication of dimerization in C5aR regulation, we generated an NH(2)-terminally modified C5aR mutant, unable to bind C5a, and a phosphorylation-deficient mutant. Neither an intact NH(2) terminus nor the presence of COOH-terminal phosphorylation sites appeared to be required for the formation of C5aR dimers. Upon C5a stimulation, mutant receptors did not internalize when individually expressed. C5a stimulation of cells that co-expressed wild type C5aR together with either unliganded or phosphorylation-deficient mutant resulted in co-internalization of mutant receptors with C5aR. Unliganded GPCRs can be cross-phosphorylated within a heterologous receptor dimer or by second messenger-activated kinases. C5a stimulation of (32)P-labeled cells that co-expressed the unliganded mutant with either C5aR or the phosphorylation-deficient mutant did not induce phosphorylation of the unliganded mutant. We can thus postulate that, in the case of C5aR, the stimulation and phosphorylation of one monomer is enough to lead to dimer internalization. The existence and functional implication of di(oligo)mer formation may be important for an accurate C5aR down-regulation in pathological conditions.     

Arrestins block G protein-coupled receptor-mediated apoptosis

G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptors (GPCRs) activate numerous cellular signals through the combined actions of G proteins, GPCR kinases, and arrestins. Although arrestins have traditionally been thought of as mediating GPCR desensitization, they have now been shown to play important roles in the internalization, trafficking, and signaling of many GPCRs. We demonstrate that in cells devoid of arrestins, the stimulation of numerous GPCRs including the N-formyl peptide receptor (FPR) initiates rapid cell rounding, annexin V positivity, and caspase activation followed by cell death. The apoptotic response is initiated by G protein signaling and involves activation of phosphoinositide 3-kinase, mitogen-activated protein kinases, and c-Src resulting in cytochrome c release from mitochondria and ultimately caspase 9 and caspase 3 activation. Reconstitution with either arrestin-2 or arrestin-3 is completely sufficient to prevent FPR-mediated apoptosis. Surprisingly, a non-desensitizing and non-internalizing mutant of the FPR is unable to initiate apoptosis, indicating that receptor phosphorylation and internalization, but not solely chronic activation due to a lack of desensitization, are critical determinants for the induction of apoptosis by the FPR. We further demonstrate that this response is not unique to the FPR with numerous additional GPCRs, including the V2 vasopressin, angiotensin II (type 1A), and CXCR2 receptors, capable of initiating apoptosis upon stimulation, whereas GPCRs such as the beta(2)-adrenergic receptor and CXCR4 are not capable of initiating apoptotic signaling. These data demonstrate for the first time that arrestins play a critical and completely unexpected role in the suppression GPCR-mediated apoptosis, which we show is a common consequence of GPCR-mediated cellular activation in the absence of arrestins.     

Human complement 5a (C5a) anaphylatoxin receptor (CD88) phosphorylation sites and their specific role in receptor phosphorylation and attenuation of G protein-mediated responses. Desensitization of C5a receptor controls superoxide production but not receptor sequestration in HL-60 cells

Upon agonist binding, the anaphylatoxin human complement 5a receptor (C5aR) has previously been found to be phosphorylated on the six serine residues of its carboxyl-terminal tail (Giannini, E., Brouchon, L., and Boulay, F. (1995) J. Biol. Chem. 270, 19166-19172). To evaluate the precise roles that specific phosphorylation sites may play in receptor signaling, a series of mutants were expressed transiently in COS-7 cells and stably in the physiologically relevant myeloid HL-60 cells. Ser(334) was found to be a key residue that controls receptor phosphorylation. Phosphorylation of either of two serine pairs, namely Ser(332) and Ser(334) or Ser(334) and Ser(338), was critical for the phosphorylation of C5aR and its subsequent desensitization. Full phosphorylation and desensitization of C5aR were obtained when these serines were replaced by aspartic acid residues. The mutation S338A had no marked effect on the agonist-mediated phosphorylation of C5aR, but it allowed a sustained C5a-evoked calcium mobilization in HL-60 cells. These findings and the ability of the S314A/S317A/S327A/S332A mutant receptor to undergo desensitization indicate that the phosphorylation of Ser(334) and Ser(338) is critical and sufficient for C5aR desensitization. The lack of phosphorylation was found to result not only in a sustained calcium mobilization and extracellular signal-regulated kinase 2 activity but also in the enhancement of the C5a-mediated respiratory burst in neutrophil-like HL-60 cells. For instance, the nonphosphorylatable S332A/S334A mutant receptor triggered a 1.8-2-fold higher production of superoxide as compared with the wild-type receptor. Interestingly, although the desensitization of this mutant was defective, it was sequestered with the same time course and the same efficiency as the wild-type receptor. Thus, in myeloid HL-60 cells, desensitization and sequestration of C5aR appear to occur through divergent molecular mechanisms.     

Adaptor Protein-2 Interaction with Arrestin Regulates GPCR Recycling and Apoptosis

G protein-coupled receptors (GPCRs) are integral to cellular function in nearly all physiologic and many pathologic processes. GPCR signaling represents an intricate balance between receptor activation, inactivation (desensitization, internalization and degradation) and resensitization (recycling and de novo synthesis). Complex formation between phosphorylated GPCRs, arrestins and an ever-increasing number of effector molecules is known to regulate cellular function. Previous studies have demonstrated that, although N -formyl peptide receptor (FPR) internalization occurs in the absence of arrestins, FPR recycling is arrestin-dependent. Furthermore, FPR stimulation in the absence of arrestins leads to receptor accumulation in perinuclear endosomes and apoptosis. In this study, we show that the interaction of GPCR-bound arrestin with adaptor protein-2 (AP-2) is a critical anti-apoptotic event. In addition, AP-2 associates with the receptor-arrestin complex in perinuclear endosomes and is required for proper post-endocytic GPCR trafficking. Finally, we observed that depletion of endogenous AP-2 results in the initiation of apoptosis upon stimulation of multiple GPCRs, including P2Y purinergic receptors and CXCR2, but not CXCR4. We propose a model in which the abnormal accumulation of internalized GPCR-arrestin complexes in recycling endosomes, resulting from defective arrestin-AP-2 interactions, leads to the specific initiation of aberrant signaling pathways and apoptosis.     

Circulating CXCR4-positive stem/progenitor cells compete for SDF-1-positive niches in bone marrow,muscle and neural tissues: an alternative hypothesis to stem cell plasticity

The trans-differentiation hypothesis of adult tissue-specific stem cells has been recently questioned because of insufficient proof that the so-called plasticity experiments were performed on pure populations of tissue-specific stem cells. It was shown recently, for example, that the formation of haematopoietic colonies by muscle cells depended on the presence of haematopoietic stem/progenitor cells residing within the muscle tissue and hence was not related to the plasticity of the muscle stem cells. The explanation for the presence in, or homing into, muscles of haematopoietic stem cells is, however, not clear. In our study, we hypothesised that muscle tissues secrete stromal-derived factor (SDF)- 1, an alpha-chemokine for haematopoietic stem cells (HSC), which could attract HSC circulating in peripheral blood into muscle tissue. We found, using RT-PCR and immunocytochemistry, that SDF-1 was expressed in human heart and skeletal muscles. Moreover, muscle satellite cells, which are pivotal for regeneration of muscle, highly expressed on their surface CXCR4, a G-protein-coupled receptor that binds SDF-1. To determine whether the CXCR4 receptor is functional on muscle satellite/progenitor cells, we stimulated murine satellite cells (the C2C12 cell line) with SDF-1 and demonstrated the phosphorylation of p42/44 MAPK and AKT serine-threonine kinase in these cells. Moreover, we showed that SDF-1 gradient chemoattracts these cells. We postulate that the CXCR4-positive muscle satellite and CXCR4-positive HSC circulating in the peripheral blood compete for occupancy of SDF-1-positive stem cell niches that are present in bone marrow and muscle tissues. Thus, we suggest that competition for common niches by various circulating CXCR4-positive stem cells and their ability to home to the SDF-1-positive niches in various organs, is a better explanation than stem cell plasticity of why (i) haematopoietic colonies can be cultured from muscles and (ii) early muscle progenitors could be cultured from bone marrow.     

Turning receptors on and off with intracellular pepducins: new insights into G-protein-coupled receptor drug development

G-protein-coupled receptors (GPCRs) are a large family of remarkably versatile membrane proteins that are attractive therapeutic targets because of their involvement in a vast range of normal physiological processes and pathological diseases. Upon activation, intracellular domains of GPCRs mediate signaling to G-proteins, but these domains have yet to be effectively exploited as drug targets. Cell-penetrating lipidated peptides called pepducins target specific intracellular loops of GPCRs and have recently emerged as effective allosteric modulators of GPCR activity. The lipid moiety facilitates translocation across the plasma membrane, where pepducins then specifically modulate signaling of their cognate receptor. To date, pepducins and related lipopeptides have been shown to specifically modulate the activity of diverse GPCRs and other membrane proteins, including protease-activated receptors (PAR1, PAR2, and PAR4), chemokine receptors (CXCR1, CXCR2, and CXCR4), sphingosine 1-phosphate receptor-3 (S1P3), the melanocortin-4 receptor, the Smoothened receptor, formyl peptide receptor-2 (FPR2), the relaxin receptor (LGR7), G-proteins (Gα(q/11/o/13)), muscarinic acetylcholine receptor and vanilloid (TRPV1) channels, and the GPIIb integrin. This minireview describes recent advances made using pepducin technology in targeting diverse GPCRs and the use of pepducins in identifying potential novel drug targets.     

SDF-1 and CXCR4 in normal and malignant hematopoiesis

Over recent years it has become apparent that the chemokine SDF-1 and its receptor CXCR4 play pivotal roles in normal hematopoiesis. They are essential for the normal ontogeny of hematopoiesis during embryogenesis and continue to play a key role in retaining hematopoietic progenitors within the bone marrow microenvironment in the adult. As a result of this role disruption of SDF-1/CXCR4 interactions results in mobilization of hematopoietic progenitors and standard mobilization protocols disrupt this axis. Similarly SDF-1/CXCR4 interactions are required for homing and engraftment of hematopoietic stem cells during transplantation. SDF-1 regulates the localisation of leukemic cells and like their normal counterparts most leukemic cells respond to SDF-1 with increased adhesion, survival and proliferation. However in some instances leukemic cell responses to SDF-1 can be disregulated, the impact of which on the progression of disease in not known. In this review we discuss the pleiotropic roles of SDF-1/CXCR4 interactions in human hematopoietic stem cell ontogeny, bone marrow homing and engraftment, mobilization and how these interactions impact on malignant hematopoiesis.     

Na+/H+ Exchanger Regulatory Factor-1 Is Involved in Chemokine Receptor Homodimer CCR5 Internalization and Signal Transduction but Does Not Affect CXCR4 Homodimer or CXCR4-CCR5 Heterodimer

Chemokine receptors are members of the G protein-coupled receptor (GPCR) family. CCR5 is also the principal co-receptor for macrophage-tropic strains of human immunodeficiency virus, type 1 (HIV-1), and efforts have been made to develop ligands to inhibit HIV-1 infection by promoting CCR5 receptor endocytosis. Given the nature of GPCRs and their propensity to form oligomers, one can consider ligand-based therapies as unselective in terms of the oligomeric composition of complexes. For example, a ligand targeting a CCR5 homomer could likely induce signal transduction on a heteromeric CCR5-CXCR4. Other avenues could therefore be explored. We identified a receptor adaptor interacting specifically with one receptor complex but not others. NHERF1, an adaptor known for its role in desensitization, internalization, and regulation of the ERK signaling cascade for several GPCRs, interacts via its PDZ2 domain with the CCR5 homodimer but not with the CXCR4-CCR5 heterodimer or CXCR4 homodimer. To further characterize this interaction, we also show that NHERF1 increases the CCR5 recruitment of arrestin2 following stimulation. NHERF1 is also involved in CCR5 internalization, as we demonstrate that co-expression of constructs bearing the PDZ2 domain can block CCR5 internalization. We also show that NHERF1 potentiates RANTES (regulated on activation normal T cell expressed and secreted)-induced ERK1/2 phosphorylation via CCR5 activation and that this activation requires NHERF1 but not arrestin2. Taken together, our results suggest that oligomeric receptor complexes can associate specifically with partners and that in this case NHERF1 could represent an interesting new target for the regulation of CCR5 internalization and potentially HIV infection.     

Functional potentials of human hematopoietic progenitor cells are maintained by mesenchymal stromal cells and not impaired by plerixafor

Background aimsMesenchymal stromal cells (MSCs) resemble an essential component of the bone marrow niche for maintenance of stemness of hematopoietic progenitor cells (HPCs). Perturbation of the C-X-C chemokine receptor type 4 (CXCR4)/stromal cell-derived factor-1α (SDF-1α) axis by plerixafor (AMD3100) mobilizes HPCs from their niche; however, little is known about how plerixafor affects interaction of HPCs and MSCs in vitro.MethodsWe monitored cell division kinetics, surface expression of CD34 and CXCR4, migration behavior and colony-forming frequency of HPCs on co-culture with MSCs either with or without exposure to plerixafor.ResultsCo-culture with MSCs significantly accelerated cell division kinetics of HPCs. Despite this, the proportion of CD34⁺ cells was significantly increased on co-culture, whereas the expression of CXCR4 was reduced. In addition, co-culture with MSCs led to significantly higher colony-forming capacity and enhanced migration rate of HPCs compared with mono-culture conditions. The composition of MSC sub-populations—and conversely their hematopoiesis supportive functions—may be influenced by culture conditions. We compared the stromal function of MSCs isolated with three different culture media. Overall, the supporting potentials of these MSC preparations were quite similar. Perturbation by the CXCR4-antagonist plerixafor reduced the cell division kinetics of HPCs on co-culture with MSCs. However, the progenitor cell potential of the HPCs as reflected by colony-forming capacity was not affected by plerixafor.ConclusionsThese results support the notion that the CXCR4/SDF-1α axis is critical for HPC-MSC interaction with regard to migration, adhesion and regulation of proliferation but not for maintenance of primitive progenitor cells.     

Cutting edge: Differential regulation of chemoattractant receptor-induced degranulation and chemokine production by receptor phosphorylation

Phosphorylation of G protein-coupled receptors and the subsequent recruitment of beta-arrestin play an important role in desensitization of receptor-mediated responses, including degranulation in leukocytes. In this study, we report that receptor phosphorylation also provides a stimulatory signal for CCR ligand 2 (CCL2) production. C3a stimulated degranulation in a basophilic leukemia RBL-2H3 cell expressing wild-type C3aR or a phosphorylation-deficient mutant (DeltaST-C3aR). In contrast, C3a caused CCL2 production only in C3aR but not DeltaST-C3aR cells. Furthermore, overexpression of G protein-coupled receptor kinase 2 resulted in enhancement of both ligand-induced receptor phosphorylation and CCL2 production but inhibition of degranulation. Agonist activation of C3aR, but not DeltaST-C3aR, led to the translocation of green fluorescent protein tagged beta-arrestin 2 from the cytoplasm to the plasma membrane. These data demonstrate that receptor phosphorylation, which provides a turn off signal for degranulation, is essential for CCL2 production.     

Hematopoietic capacity of preterm cord blood hematopoietic stem/progenitor cells

Full-term cord blood (TCB) hematopoietic stem/progenitor cells (HSC/HPCs) are used for stem cell transplantation and are well characterized. However, the properties of preterm cord blood (PCB) HSC/HPCs remain unclear. In the present study, we compared HSC/HPCs from TCB and PCB with respect to their expression of surface markers, homing capacity and ability to repopulate HSCs in the NOD/Shi-scid mice bone marrow. The proportion of CD34+CD38− cells was significantly higher in PCB. On the other hand, the engraftment rate of TCB CD34+ cells into NOD/Shi-scid mice was significantly higher than PCB CD34+ cells. The expression of VLA4 was stronger among TCB CD34+ cells than PCB CD34+ cells. Moreover, there was a positive correlation between the proportion of CD34+CXCR4+ cells and gestational age. These data suggest that the homing ability of HSCs increases during gestation, so that TCB may be a better source of HSCs for transplantation than PCB.     

Phosphorylation of C3a Receptor at Multiple Sites Mediates Desensitization, β-Arrestin-2 Recruitment and Inhibition of NF-κB Activity in Mast Cells

Background

Phosphorylation of G protein coupled receptors (GPCRs) by G protein coupled receptor kinases (GRKs) and the subsequent recruitment of β-arrestins are important for their desensitization. Using shRNA-mediated gene silencing strategy, we have recently shown that GRK2, GRK3 and β-arrestin-2 promote C3a receptor (C3aR) desensitization in human mast cells. We also demonstrated that β-arrestin-2 provides an inhibitory signal for NF-κB activation. C3aR possesses ten potential phosphorylation sites within its carboxyl terminus but their role on desensitization, β-arrestin recruitment and NF-κB activation has not been determined.

Methodology/Principal Findings

We utilized a site directed mutagenesis approach in transfected HEK293 cells to determine the role of receptor phosphorylation on β-arrestin-2 recruitment and RBL-2H3 cells for functional studies. We found that although Ala substitution of Ser475/479, Thr480/481 residues resulted in 58±3.8% decrease in agonist-induced C3aR phosphorylation there was no change in β-arrestin-2 binding or receptor desensitization. By contrast, Ala substitution of Thr463, Ser465, Thr466 and Ser470 led to 40±1.3% decrease in agonist-induced receptor phosphorylation but this was associated with 74±2.4% decreases in β-arrestin-2 binding, significantly reduced desensitization and enhanced NF-κB activation. Combined mutation of these Ser/Thr residues along with Ser459 (mutant MT7), resulted in complete loss of receptor phosphorylation and β-arrestin-2 binding. RBL-2H3 cells expressing MT7 responded to C3a for greater Ca²⁺ mobilization, degranulation and NF-κB activation when compared to the wild-type receptor. Interestingly, co-expression of MT7 with a constitutively active mutant of β-arrestin (R169E) inhibited C3a-induced degranulation by 28±2.4% and blocked NF-κB activation by 80±2.4%.

Conclusion/Significance

This study demonstrates that although C3a causes phosphorylation of its receptor at multiple sites, Ser459, Thr463, Ser465, Thr466 and Ser470 participate in C3aR desensitization, β-arrestin-2 recruitment and inhibition of NF-κB activity. Furthermore, β-arrestin-2 inhibits C3a-induced NF-κB activation via receptor desensitization-dependent and independent pathways.