scholarly journals CXCL12/CXCR4-Axis Dysfunctions: Markers of the Rare Immunodeficiency Disorder WHIM Syndrome

2010 ◽  
Vol 29 (3-4) ◽  
pp. 189-198 ◽  
Author(s):  
Françoise Bachelerie
Keyword(s):  
Bacterial Infections ◽  
Receptor Internalization ◽  
Gene Encoding ◽  
Whim Syndrome ◽  
Papillomavirus Infection ◽  
Immunodeficiency Disorder ◽  
T Cell Lymphopenia ◽  
Chemokine Cxcl12 ◽  
G Protein Coupled ◽  
Cxcr4 Axis

The WHIM syndrome features susceptibility to human Papillomavirus infection-induced warts and carcinomas, hypogammaglobulinemia, recurrent bacterial infections, B and T-cell lymphopenia, and neutropenia associated with retention of senescent neutrophils in the bone marrow (i.e. myelokathexis). This rare disorder is mostly linked to inherited heterozygous autosomal dominant mutations in the gene encodingCXCR4, a G protein coupled receptor with a unique ligand, the chemokine CXCL12/SDF-1. Some individuals who have full clinical forms of the syndrome carry a wild typeCXCR4gene. In spite of this genetic heterogeneity, leukocytes from WHIM patients share in common dysfunctions of the CXCR4-mediated signaling pathway upon exposure to CXCL12. Dysfunctions are characterized by impaired desensitization and receptor internalization, which are associated with enhanced responses to the chemokine. Our increasing understanding of the mechanisms that account for the aberrant CXCL12/CXCR4-mediated responses is beginning to provide insight into the pathogenesis of the disorder. As a result we can expect to identify markers of the WHIM syndrome, as well as other disorders with WHIM-like features that are associated with dysfunctions of the CXCL12/CXCR4 axis.

scholarly journals Adaptive Immunodeficiency in WHIM Syndrome

2018 ◽  
Vol 20 (1) ◽  
pp. 3 ◽  
Author(s):  
Shamik Majumdar ◽  
Philip Murphy
Keyword(s):  
Chemokine Receptor ◽  
Autosomal Dominant ◽  
Whim Syndrome ◽  
C Terminus ◽  
Immunodeficiency Disorder ◽  
Chemokine Ligand ◽  
Chemokine Receptor 4 ◽  
The Impact ◽  
G Protein Coupled ◽  
Increased Susceptibility

Cysteine-X-cysteine chemokine receptor 4 (CXCR4) is a broadly expressed and multifunctional G protein-coupled chemokine receptor critical for organogenesis, hematopoiesis, and antimicrobial host defense. In the hematopoietic system, the binding of CXCR4 to its cognate chemokine ligand, CXCL12, mediates leukocyte trafficking, distribution, survival, activation, and proliferation. Warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome is a rare, autosomal dominant, combined immunodeficiency disorder caused by mutations in the C-terminus of CXCR4 that prevent receptor downregulation and therefore result in pathologically increased signaling. The “M” in the acronym WHIM refers to myelokathexis, the retention of neutrophils in the bone marrow resulting in neutropenia, which explains in part the increased susceptibility to bacterial infection. However, WHIM patients also present with B and T lymphopenia, which may explain the susceptibility to human papillomavirus (HPV), the cause of warts. The impact of WHIM mutations on lymphocytes and adaptive immunity has received less attention than myelokathexis and is the focus of this review.

scholarly journals GRKs as Modulators of Neurotransmitter Receptors

Cells ◽  
2020 ◽  
Vol 10 (1) ◽  
pp. 52
Author(s):  
Eugenia V. Gurevich ◽  
Vsevolod V. Gurevich
Keyword(s):  
G Protein ◽  
General Model ◽  
G Protein Coupled Receptors ◽  
Receptor Internalization ◽  
Neurotransmitter Receptors ◽  
Receptor Subtype ◽  
Receptor Subtypes ◽  
Receptor Kinase ◽  
G Protein Coupled ◽  
Homologous Desensitization

Many receptors for neurotransmitters, such as dopamine, norepinephrine, acetylcholine, and neuropeptides, belong to the superfamily of G protein-coupled receptors (GPCRs). A general model posits that GPCRs undergo two-step homologous desensitization: the active receptor is phosphorylated by kinases of the G protein-coupled receptor kinase (GRK) family, whereupon arrestin proteins specifically bind active phosphorylated receptors, shutting down G protein-mediated signaling, facilitating receptor internalization, and initiating distinct signaling pathways via arrestin-based scaffolding. Here, we review the mechanisms of GRK-dependent regulation of neurotransmitter receptors, focusing on the diverse modes of GRK-mediated phosphorylation of receptor subtypes. The immediate signaling consequences of GRK-mediated receptor phosphorylation, such as arrestin recruitment, desensitization, and internalization/resensitization, are equally diverse, depending not only on the receptor subtype but also on phosphorylation by GRKs of select receptor residues. We discuss the signaling outcome as well as the biological and behavioral consequences of the GRK-dependent phosphorylation of neurotransmitter receptors where known.

CCK receptor phosphorylation exposes regulatory domains affecting phosphorylation and receptor trafficking

2000 ◽  
Vol 279 (6) ◽  
pp. C1986-C1992 ◽  
Author(s):  
Rammohan V. Rao ◽  
Eileen L. Holicky ◽  
Susan M. Kuntz ◽  
Laurence J. Miller
Keyword(s):  
G Protein ◽  
Chinese Hamster ◽  
G Protein Coupled Receptors ◽  
Receptor Internalization ◽  
Ovary Cell ◽  
Guanine Nucleotide Binding Protein ◽  
Receptor Phosphorylation ◽  
Regulatory Domains ◽  
Ovary Cell Line ◽  
G Protein Coupled

Agonist-stimulated phosphorylation of guanine nucleotide-binding protein (G protein)-coupled receptors has been recognized as an important mechanism for desensitization by interfering with coupling of the activated receptor with its G protein. We recently described a mutant of the CCK receptor that modified two of five key sites of phosphorylation (S260,264A) and eliminated agonist-stimulated receptor phosphorylation, despite normal ligand binding and signaling (20). As expected, this nonphosphorylated mutant had impaired rapid desensitization but was ultimately able to be desensitized by normal receptor internalization. Here we demonstrate that this mutant receptor is also defective in resensitization, with abnormal recycling to the cell surface. To explore this, another receptor mutant was prepared, replacing the same serines with aspartates to mimic the charge of serine-phosphate (S260,264D). This mutant was expressed in a Chinese hamster ovary cell line and shown to bind CCK normally. It had accelerated kinetics of signaling and desensitization and was phosphorylated in response to agonist occupation, with all other normal sites of phosphorylation modified. It was internalized like wild-type receptors and was resensitized and trafficked normally. This provides evidence for an additional important function for phosphorylation of G protein-coupled receptors. Phosphorylation may induce a conformational change in the receptor to expose other potential sites of phosphorylation and to expose domains involved in the targeting and trafficking of endosomes. The hierarchical phosphorylation of these sites may play a key role in receptor regulation.

Identification of Rifampicin Resistance Mutations in Escherichia coli, Including an Unusual Deletion Mutation

2017 ◽  
Vol 27 (6) ◽  
pp. 356-362 ◽  
Author(s):  
Eugene Y. Wu ◽  
Angela K. Hilliker
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Bacterial Infections ◽  
Deletion Mutant ◽  
Deletion Mutation ◽  
Rifampicin Resistance ◽  
Clinical Settings ◽  
Resistance Mutations ◽  
Gene Encoding ◽  
Β Chain

Rifampicin is an effective antibiotic against mycobacterial and other bacterial infections, but resistance readily emerges in laboratory and clinical settings. We screened <i>Escherichia coli</i> for rifampicin resistance and identified numerous mutations to the gene encoding the β-chain of RNA polymerase (<i>rpoB</i>), including an unusual 9-nucleotide deletion mutation. Structural modeling of the deletion mutant indicates locations of potential steric clashes with rifampicin. Sequence conservation in the region near the deletion mutation suggests a similar mutation may also confer resistance during the treatment of tuberculosis.

scholarly journals The luminescent HiBiT peptide enables selective quantitation of G protein–coupled receptor ligand engagement and internalization in living cells

2020 ◽  
Vol 295 (15) ◽  
pp. 5124-5135 ◽  
Author(s):  
Michelle E. Boursier ◽  
Sergiy Levin ◽  
Kris Zimmerman ◽  
Thomas Machleidt ◽  
Robin Hurst ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Cell Surface ◽  
G Protein ◽  
Dynamic Range ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Receptor Internalization ◽  
Cell Surface Receptor ◽  
Cellular Interactions ◽  
G Protein Coupled

G protein–coupled receptors (GPCRs) are prominent targets to new therapeutics for a range of diseases. Comprehensive assessments of their cellular interactions with bioactive compounds, particularly in a kinetic format, are imperative to the development of drugs with improved efficacy. Hence, we developed complementary cellular assays that enable equilibrium and real-time analyses of GPCR ligand engagement and consequent activation, measured as receptor internalization. These assays utilize GPCRs genetically fused to an N-terminal HiBiT peptide (1.3 kDa), which produces bright luminescence upon high-affinity complementation with LgBiT, an 18-kDa subunit derived from NanoLuc. The cell impermeability of LgBiT limits signal detection to the cell surface and enables measurements of ligand-induced internalization through changes in cell-surface receptor density. In addition, bioluminescent resonance energy transfer is used to quantify dynamic interactions between ligands and their cognate HiBiT-tagged GPCRs through competitive binding with fluorescent tracers. The sensitivity and dynamic range of these assays benefit from the specificity of bioluminescent resonance energy transfer and the high signal intensity of HiBiT/LgBiT without background luminescence from receptors present in intracellular compartments. These features allow analyses of challenging interactions having low selectivity or affinity and enable studies using endogenously tagged receptors. Using the β-adrenergic receptor family as a model, we demonstrate the versatility of these assays by utilizing the same HiBiT construct in analyses of multiple aspects of GPCR pharmacology. We anticipate that this combination of target engagement and proximal functional readout will prove useful to the study of other GPCR families and the development of new therapeutics.

Mutations in the gene encoding neutrophil elastase in congenital and cyclic neutropenia

Blood ◽  
2000 ◽  
Vol 96 (7) ◽  
pp. 2317-2322 ◽  
Author(s):  
David C. Dale ◽  
Richard E. Person ◽  
Audrey Anna Bolyard ◽  
Andrew G. Aprikyan ◽  
Cindy Bos ◽  
...  
Keyword(s):  
Neutrophil Elastase ◽  
Rna Splicing ◽  
Bacterial Infections ◽  
Autosomal Dominant ◽  
Mononuclear Cells ◽  
Mutational Analysis ◽  
Congenital Neutropenia ◽  
Gene Encoding ◽  
Cyclic Neutropenia ◽  
Shwachman Diamond Syndrome

Abstract Congenital neutropenia and cyclic neutropenia are disorders of neutrophil production predisposing patients to recurrent bacterial infections. Recently the locus for autosomal dominant cyclic neutropenia was mapped to chromosome 19p13.3, and this disease is now attributable to mutations of the gene encoding neutrophil elastase (the ELA2 gene). The authors hypothesized that congenital neutropenia is also due to mutations of neutrophil elastase. Patients with congenital neutropenia, cyclic neutropenia, or Shwachman-Diamond syndrome were referred to the Severe Chronic Neutropenia International Registry. Referring physicians provided hematologic and clinical data. Mutational analysis was performed by sequencing polymerase chain reaction (PCR)-amplified genomic DNA for each of the 5 exons of the neutrophil ELA2 gene and 20 bases of the flanking regions. RNA from bone marrow mononuclear cells was used to determine if the affected patients expressed both the normal and the abnormal transcript. Twenty-two of 25 patients with congenital neutropenia had 18 different heterozygous mutations. Four of 4 patients with cyclic neutropenia and 0 of 3 patients with Shwachman-Diamond syndrome had mutations. For 5 patients with congenital neutropenia having mutations predicted to alter RNA splicing or transcript structure, reverse transcriptase-PCR showed expression of both normal and abnormal transcripts. In cyclic neutropenia, the mutations appeared to cluster near the active site of the molecule, whereas the opposite face was predominantly affected by the mutations found in congenital neutropenia. This study indicates that mutations of the gene encoding neutrophil elastase are probably the most common cause for severe congenital neutropenia as well as the cause for sporadic and autosomal dominant cyclic neutropenia.

scholarly journals Mammalian Type I Gonadotropin-Releasing Hormone Receptors Undergo Slow, Constitutive, Agonist-Independent Internalization

Endocrinology ◽  
2007 ◽  
Vol 149 (3) ◽  
pp. 1415-1422 ◽  
Author(s):  
Adam J. Pawson ◽  
Elena Faccenda ◽  
Stuart Maudsley ◽  
Zhi-Liang Lu ◽  
Zvi Naor ◽  
...  
Keyword(s):  
G Protein ◽  
Regulatory Elements ◽  
Gnrh Receptor ◽  
G Protein Coupled Receptors ◽  
Receptor Internalization ◽  
Type I ◽  
Receptor Desensitization ◽  
Gnrh Receptors ◽  
Carboxyl Terminal ◽  
G Protein Coupled

Regulatory elements present in the cytoplasmic carboxyl-terminal tails of G protein-coupled receptors contribute to agonist-dependent receptor desensitization, internalization, and association with accessory proteins such as β-arrestin. The mammalian type I GnRH receptors are unique among the rhodopsin-like G protein-coupled receptors because they lack a cytoplasmic carboxyl-terminal tail. In addition, they do not recruit β-arrestin, nor do they undergo rapid desensitization. By measuring the internalization of labeled GnRH agonists, previous studies have reported that mammalian type I GnRH receptors undergo slow agonist-dependent internalization. In the present study, we have measured the internalization of epitope-tagged GnRH receptors, both in the absence and presence of GnRH stimulation. We demonstrate that mammalian type I GnRH receptors exhibit a low level of constitutive agonist-independent internalization. Stimulation with GnRH agonist did not significantly enhance the level of receptor internalization above the constitutive level. In contrast, the catfish GnRH and rat TRH receptors, which have cytoplasmic carboxyl-terminal tails, displayed similar levels of constitutive agonist-independent internalization but underwent robust agonist-dependent internalization, as did chimeras of the mammalian type I GnRH receptor with the cytoplasmic carboxyl-terminal tails of the catfish GnRH receptor or the rat TRH receptor. When the carboxyl-terminal Tyr325 and Leu328 residues of the mammalian type I GnRH receptor were replaced with alanines, these two mutant receptors underwent significantly impaired internalization, suggesting a function for the Tyr-X-X-Leu sequence in mediating the constitutive agonist-independent internalization of mammalian type I GnRH receptors. These findings provide further support for the underlying notion that the absence of the cytoplasmic carboxyl-terminal tail of the mammalian type I GnRH receptors has been selected for during evolution to prevent rapid receptor desensitization and internalization to allow protracted GnRH signaling in mammals.

scholarly journals Peptide-binding induced inhibition of chemokine CXCL12

RSC Advances ◽  
2017 ◽  
Vol 7 (34) ◽  
pp. 21298-21307
Author(s):  
Hongyang Duan ◽  
Ling Zhu ◽  
Jiaxi Peng ◽  
Mo Yang ◽  
Hanyi Xie ◽  
...  
Keyword(s):  
Binding Affinity ◽  
Peptide Binding ◽  
Inhibitory Effect ◽  
High Binding ◽  
Significant Inhibitory Effect ◽  
Chemokine Cxcl12 ◽  
Cxcr4 Axis ◽  
High Binding Affinity

A designed peptide (W4) has a significant inhibitory effect on the CXCL12/CXCR4 axis by targeting CXCL12 with high binding affinity.

scholarly journals A pH-Sensitive Fluor, CypHerTM 5, Used to Monitor Agonist-Induced G Protein-Coupled Receptor Internalization in Live Cells

BioTechniques ◽  
2002 ◽  
Vol 33 (5) ◽  
pp. 1152-1157 ◽  
Author(s):  
E.J. Adie ◽  
S. Kalinka ◽  
L. Smith ◽  
M.J. Francis ◽  
A. Marenghi ◽  
...  
Keyword(s):  
G Protein ◽  
Ph Sensitive ◽  
Receptor Internalization ◽  
Live Cells ◽  
G Protein Coupled Receptor ◽  
G Protein Coupled

scholarly journals Clathrin and GRK2/3 inhibitors block δ-opioid receptor internalization in myenteric neurons and inhibit neuromuscular transmission in the mouse colon

2019 ◽  
Vol 317 (2) ◽  
pp. G79-G89 ◽  
Author(s):  
Jesse J. DiCello ◽  
Pradeep Rajasekhar ◽  
Emily M. Eriksson ◽  
Ayame Saito ◽  
Arisbel B. Gondin ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
G Protein ◽  
Opioid Receptor ◽  
Neuromuscular Transmission ◽  
G Protein Coupled Receptors ◽  
Receptor Internalization ◽  
Enteric Neurons ◽  
Myenteric Neurons ◽  
Gut Function ◽  
G Protein Coupled

Endocytosis is a major mechanism through which cellular signaling by G protein-coupled receptors (GPCRs) is terminated. However, recent studies demonstrate that GPCRs are internalized in an active state and continue to signal from within endosomes, resulting in effects on cellular function that are distinct to those arising at the cell surface. Endocytosis inhibitors are commonly used to define the importance of GPCR internalization for physiological and pathophysiological processes. Here, we provide the first detailed examination of the effects of these inhibitors on neurogenic contractions of gastrointestinal smooth muscle, a key preliminary step to evaluate the importance of GPCR endocytosis for gut function. Inhibitors of clathrin-mediated endocytosis (Pitstop2, PS2) or G protein-coupled receptor kinase-2/3-dependent phosphorylation (Takeda compound 101, Cmpd101), significantly reduced GPCR internalization. However, they also attenuated cholinergic contractions through different mechanisms. PS2 abolished contractile responses by colonic muscle to SNC80 and morphine, which strongly and weakly internalize δ-opioid and μ-opioid receptors, respectively. PS2 did not affect the increased myogenic contractile activity following removal of an inhibitory neural influence (tetrodotoxin) but suppressed electrically evoked neurogenic contractions. Ca2+ signaling by myenteric neurons in response to exogenous ATP was unaffected by PS2, suggesting inhibitory actions on neurotransmitter release rather than neurotransmission. In contrast, Cmpd101 attenuated contractions to the cholinergic agonist carbachol, indicating direct effects on smooth muscle. We conclude that, although PS2 and Cmpd101 are effective blockers of GPCR endocytosis in enteric neurons, these inhibitors are unsuitable for the study of neurally mediated gut function due to their inhibitory effects on neuromuscular transmission and smooth muscle contractility. NEW & NOTEWORTHY Internalization of activated G protein-coupled receptors is a major determinant of the type and duration of subsequent downstream signaling events. Inhibitors of endocytosis effectively block opioid receptor internalization in enteric neurons. The clathrin-dependent endocytosis inhibitor Pitstop2 blocks effects of opioids on neurogenic contractions of the colon in an internalization-independent manner. These inhibitors also significantly impact cholinergic neuromuscular transmission. We conclude that these tools are unsuitable for examination of the contribution of neuronal G protein-coupled receptor endocytosis to gastrointestinal motility.

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