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Biomedical subjects

R M Richardson

Publications and source records attributed to R M Richardson.

At least 19 recordsLinked to original sources
PubMed2006

Isolation of neuronal progenitor cells from the adult human neocortex.

BACKGROUND: The reliability of harvesting neuronal progenitor cells (NPCs) from the adult human neocortex has not been established, with respect to preparing autologous cell cultures for transplantation in stroke and traumatic brain injured patients. METHOD: Enriched NPC cultures have been generated from nonneurogenic regions of the adult rodent brain by buoyancy-dependent fractionation, but the feasibility of using such a method to isolate NPCs from the adult human cortex has not been reported previously. To determine if a starter population of human adult cortical NPCs could be isolated for in vitro expansion using this method, tissue samples from five patients undergoing cortical resection for either epilepsy or trauma were assayed. FINDINGS: Cultured cells generated from all patients predominately expressed both the NPC marker nestin and neuron-specific beta-tubulin III. The presence of NPCs was verified by in vitro BrdU/beta-tubulin III co-labeling and increasing beta-tubulin expression in differentiating conditions. Despite the formation of aggregates in monolayer culture, cell proliferation as measured by BrdU incorporation was not as prevalent as that reported from rodent cultures generated by this protocol. CONCLUSIONS: NPCs isolated from the adult human neocortex using this method expressed beta-tubulin III in larger percentages than has been previously reported for NPCs isolated using other methods. As such, these data suggest the possibility of culturing dividing neuroblasts from the adult neocortex for further manipulation as transplantable cells.

Adolescent↗
PubMed2005

Novel self-assembly of amphiphilic copolymers into nanotubes: characterization by small-angle neutron scattering.

The self-assembly into nanotubes in solution of an amphiphilic copolymer is characterized by small-angle neutron scattering (SANS). This study confirmed the shape and the size of the tubular association and the 3-D association of the tubes predicted by molecular orbital theory. Moreover, the characterization of the stability of the association has revealed that the addition of a very small amount of salt to the solution increases the size of the association. When more salt is added, the size of the association decreases, and the structure is altered. The association was found to be independent of temperature and therefore is very stable.

Chemistry↗
PubMed2005

Calculation of the birefringences of nematic liquid crystals at optical and infrared wavelengths.

In this paper we critically examine computational methods for predicting the birefringences of nematic liquid crystals, with a view to screening molecules for potential use in infrared applications. Using the liquid-crystal 5CB as a test molecule, we calculate molecular electronic polarizabilities using ab initio quantum-mechanical techniques and a wide range of basis sets. We show that the polarizabilities tend to a limiting value as the quality of the basis set is improved. However, the biggest hurdle remains the determination of the refractive index from the polarizability data. We examine several methods for performing this conversion and conclude that the simplest equation, due to Vuks, is adequate for predicting the birefringence, given the uncertainties involved in other parameters. The agreement between calculation and experiment is best described as "semiquantitative." We perform similar calculations for a wide range of nematic liquid crystals at both 589 and 1550 nm, taking into account the likely impact of molecular vibrations at the longer wavelength. We demonstrate that there is a simple scale factor, for conventional nematics, between the birefringence at visible wavelengths and in the infrared. Thus knowledge of the birefringence at optical wavelengths, as widely available in the literature, is a good guide to the usefulness of conventional nematic liquid crystals as active elements for optical switching in the telecommunications industry.

Journal Article↗
PubMed2001

A liquid-crystalline silsesquioxane dendrimer exhibiting chiral nematic and columnar mesophases.

A hexadecamer, first-generation, octasilsesquioxane liquid-crystalline dendrimer was synthesized by a platinum-catalyzed hydrosilylation reaction of the parent first-generation vinyl octasilsesquioxane dendrimer with a modified, laterally substituted mesogen. The structure and purity of the octasilsesquioxane substrate was confirmed by 1H, 13C, and 29 Si NMR spectroscopy, microanalysis, and size exclusion chromatography (SEC). The mesogenic substrate was found to exhibit only a chiral nematic phase, whereas the resulting hexadecamer dendrimer displays enantiotropic chiral nematic, disordered hexagonal columnar, and disordered rectangular columnar phases, with a glass transition below room temperature. The lateral or side-on attachment of the mesogen to the dendritic core was found to be a key design feature in the formation of the mesophases.

Journal Article↗
PubMed2001

RGS4 inhibits platelet-activating factor receptor phosphorylation and cellular responses.

To define the role of regulators of G-protein signaling (RGS) in chemoattractant-mediated responses, RGS4 and the receptors for platelet-activating factor (PAFR), formylated peptides (FR), or interleukin-8 (CXCR1) were stably coexpressed in a rat basophilic leukemia (RBL-2H3) cell line. The data demonstrate that RGS4 inhibited responses by PAFR (i.e., phosphoinositide (PI) hydrolysis, Ca2+ mobilization) but not by FR or CXCR1. An N-terminal 33 amino acid deletion mutant of RGS4 (DeltaRGS4), deficient in GAP (GTPase activating protein) activity and plasma membrane localization, had no effect on either PAFR, FR, or CXCR1. RGS4, but not DeltaRGS4, also blocked phosphorylation of PAFR by platelet-activating factor (PAF) and, unexpectedly, by phorbol 12-myristate 13-acetate (PMA); it also blocked cross-phosphorylation by formylmethionylleucylphenylalanine (fMLP). A point mutant of RGS4 (N88S), deficient in GAP activity but not membrane localization, partially blocked PAFR phosphorylation but had no effect on PAFR-mediated PI hydrolysis and Ca2+ mobilization. Truncation of the cytoplasmic tail of PAFR (mPAFR) resulted in a loss of its susceptibility to inhibition by RGS4. Taken together, the data indicate that of the receptors studied, RGS4 selectively inhibited responses to PAFR, which preferentially couples to Gq. At the level of expression studied, RGS4 did not inhibit FR or CXCR1 which activates Gi to transduce cellular signals. Since the tail-deleted mutant of PAFR was not affected by RGS4, and RGS4 blocked homologous as well as heterologous phosphorylation of this receptor, it is possible that RGS4 interferes sterically with the cytoplasmic tail of PAFR. Thus, in addition to stimulating the GTPase activity of Galpha, RGS4 prevents G protein activation by PAFR and the homologous and heterologous phosphorylation of this receptor.

Animals↗
PubMed2001

Nonpeptide CXCR2 antagonist prevents neutrophil accumulation in hyperoxia-exposed newborn rats.

Neutrophil influx in lung injury is controlled in part by chemokines acting through the receptor, CXCR2. To avoid adverse effects of steroids typically used to modify inflammation, we evaluated the effects of competitive blockade of CXCR2 in rats on neutrophil function in vitro and on neutrophil influx in vivo in hyperoxia-induced newborn lung injury, a model of bronchopulmonary dysplasia. In vitro, SB-265610 antagonizes rat cytokine-induced neutrophil chemoattractant-1 (CINC-1)-induced calcium mobilization, IC50 = 3.7 nM, and rat neutrophil chemotaxis in a concentration-dependent manner, IC50 = 70 nM. In vivo, newborn rats exposed to 95% O2 for 8 days had increased lung neutrophil content. Injection with 1 to 3 mg/kg SB-265610 on days 3 to 5 reduced hyperoxia-induced neutrophil accumulation in bronchoalveolar lavage and whole lung myeloperoxidase accumulation at the highest doses. To determine whether these effects might be due in part to increased neutrophil apoptosis, peripheral neutrophils were cultured with and without SB-265610. Apoptosis was assessed by morphology, viability, and terminal transferase deoxyuridine triphosphatidyl nucleotide nick-end labeling. Treatment of neutrophils with CINC-1 reduced apoptosis compared with untreated neutrophils. SB-265610 reduced the antiapoptotic effect of CINC-1 to the levels of those untreated with CINC-1. A selective CXCR2 antagonist may be useful in diseases where neutrophil-mediated exacerbation is present.

Animals↗
PubMed2000

Combined X-Ray and fully leaky guided mode studies of the smectic layer and optic tensor configuration in a ferroelectric liquid-crystal cell.

X-ray scattering together with optical characterization using fully leaky guided modes have been used for the first time to study the same ferroelectric liquid-crystal cell. This enables direct calculation of an accurate cone and chevron description of the liquid-crystal director profile since the layer structure and optic tensor configuration are both well known.

Journal Article↗
PubMed2000

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.

Amino Acid Sequence↗
PubMed2000

Comparative mortality of hemodialysis and peritoneal dialysis in Canada.

BACKGROUND: Comparisons of mortality rates in patients on hemodialysis versus those on peritoneal dialysis have been inconsistent. We hypothesized that comorbidity has an important effect on differential survival in these two groups of patients. METHODS: Eight hundred twenty-two consecutive patients at 11 Canadian institutions with irreversible renal failure had an extensive assessment of comorbid illness collected prospectively, immediately prior to starting dialysis therapy. The cohort was assembled between March 1993 and November 1994; vital status was ascertained as of January 1, 1998. RESULTS: The mean follow-up was 24 months. Thirty-four percent of patients at baseline, 50% at three months, and 51% at six months used peritoneal dialysis. Values for a previously validated comorbidity score were higher for patients on hemodialysis at baseline (4.0 vs. 3.1, P < 0.001), three months (3.7 vs. 3.2, P = 0.001), and six months (3.6 vs. 3.2, P = 0.005). The overall mortality was 41%. The unadjusted peritoneal dialysis/hemodialysis mortality hazard ratios were 0.65 (95% CI, 0. 51 to 0.83, P = 0.0005), 0.84 (95% CI, 0.66 to 1.06, P = NS), and 0. 83 (95% CI, 0.64 to 1.08, P = NS) based on the modality of dialysis in use at baseline, three months, and six months, respectively. When adjusted for age, sex, diabetes, cardiac failure, myocardial infarction, peripheral vascular disease, malignancy, and acuity of renal failure, the corresponding hazard ratios were 0.79 (95% CI, 0. 62 to 1.01, P = NS), 1.00 (95% CI, 0.78 to 1.28, P = NS), and 0.95 (95% CI, 0.73 to 1.24, P = NS). Adjustment for a previously validated comorbidity score resulted in hazard ratios of 0.74 (95% CI, 0.58 to 0.94, P = 0.01), 0.94 (95% CI, 0.74 to 1.19, P = NS), and 0.88 (95% CI, 0.68 to 1.13, P = NS) at baseline, three months, and six months. There was no survival advantage for either modality in any of the major subgroups defined by age, sex, or diabetic status. CONCLUSIONS: The apparent survival advantage of peritoneal dialysis in Canada is due to lower comorbidity and a lower burden of acute onset end-stage renal disease at the inception of dialysis therapy. Hemodialysis and peritoneal dialysis, as practiced in Canada in the 1990s, are associated with similar overall survival rates.

Canada↗
PubMed2000

Comparative hospitalization of hemodialysis and peritoneal dialysis patients in Canada.

BACKGROUND: Most comparisons of hemodialysis (HD) and peritoneal dialysis (PD) have used mortality as an outcome. Relatively few studies have directly compared the hospitalization rates, an outcome of perhaps equal importance, of patients using these different dialysis modalities. METHODS: Eight hundred twenty-two consecutive patients at 11 Canadian institutions with irreversible renal failure had an extensive assessment of comorbid illness and initial mode of dialysis collected prospectively immediately prior to starting dialysis therapy. The cohort was assembled between March 1993 and November 1994. The mean follow-up was 24 months. Admission data were used to compare hospitalization rates in HD and PD. RESULTS: Thirty-four percent of patients at baseline and 50% at three months used PD. Twenty-five percent of HD and 32% of PD patients switched dialysis modality at least once after their first treatment (P = NS). Nine percent of HD patients and 30% of PD patients switched modality after three months (P < 0. 001). Total comorbidity was higher in HD patients at baseline (P < 0. 001) and at three months (P = 0.001). The overall hospitalization rate was 40.2 days per 1000 patient days after baseline and 38.0 days per 1000 patient days after three months. When an adjustment was made for baseline comorbid conditions, patients on PD had a lower rate of hospitalization in intention-to-treat analysis according to the type of dialysis in use at baseline (RR 0.85, 95% CI, 0.82 to 0.87, P < 0.001), but a higher rate according to the type of dialysis in use three months after study entry (RR 1.31, 95% CI, 1.27 to 1.34, P < 0.001). In analyses based on the amount of time actually spent on each treatment modality, PD was associated with a higher rate of hospitalization when analyzed according to the type of dialysis in use at baseline (RR 1.10, 95% CI, 1.07 to 1.13, P < 0.001) and according to the type of dialysis in use three months after study entry (RR 1.26, 95% CI, 1.23 to 1.30, P < 0.001). CONCLUSIONS: Conclusions regarding comparative hospitalization rates are heavily dependent on the analytic starting point and on whether intention-to-treat or treatment-received analyses are used. When early treatment switches are accounted for, HD is associated with a lower rate of hospitalization than PD, but the effect is modest.

Canada↗
PubMed2000

Function and regulation of chemoattractant receptors.

Phagocyte migration and activation at sites of inflammation is mediated through chemoattractant receptors that are coupled to G-proteins. Early studies from our laboratory demonstrated G-protein-mediated phospholipase C activation by chemoattractants. Recently, this laboratory developed cellular and animal models to allow biochemical, cell biological and molecular genetic approaches to be used in determining the mechanisms of chemoattractant receptor function, regulation, and cross regulation. These studies provided evidence that chemoattractant receptors activate distinct pathways for chemotaxis and exocytosis and cross-regulate each other's function at multiple levels. A major site of regulation is through phosphorylation of receptors by G-protein-coupled receptor kinases and by protein kinase C. In addition, the activation of phospholipase C by chemoattractants is also regulated at additional sites distal to receptor phosphorylation. These may include modulation of G-protein activation by regulators of G-protein signaling (RGS) and modification of phospholipase C. Phosphorylation of phospholipase Cbeta3 by both protein kinase A and protein kinase C has been demonstrated. The function and regulation of chemoattractant receptors are also being examined in mouse models. In these studies, mice deficient in leukotriene B4 receptors have been generated by targeted gene disruption. These mice displayed reduced neutrophil accumulation in certain inflammation models and sex-related differences in platelet-activating-factor induced anaphylaxis.

Animals↗
PubMed1999

Chemoattractant receptors activate distinct pathways for chemotaxis and secretion. Role of G-protein usage.

Human leukocyte chemoattractant receptors activate chemotactic and cytotoxic pathways to varying degrees and also activate different G-proteins depending on the receptor and the cell-type. To determine the relationship between G-protein usage and the biological and biochemical responses activated, receptors for the chemoattractants formyl peptides (FR), platelet-activating factor (PAFR), and leukotriene B(4) (BLTR) were transfected into RBL-2H3 cells. Pertussis toxin (Ptx) served as a Galpha(i) inhibitor. These receptors were chosen to represent the spectrum of G(i) usage as Ptx had differential effects on their ability to induce calcium mobilization, phosphoinositide hydrolysis, and exocytosis with complete inhibition of all responses by FR, intermediate effects on BLTR, and little effect on PAFR. Ptx did not affect ligand-induced phosphorylation of PAFR and BLTR but inhibited phosphorylation of FR. In contrast, chemotaxis to formylmethionylleucylphenylalanine, leukotriene B(4), and platelet-activating factor was completely blocked by Ptx. Wortmannin, a phosphotidylinositol 3-kinase inhibitor, also completely blocked ligand-induced chemotaxis by all receptors but did not affect calcium mobilization or phosphoinositide hydrolysis; however, it partially blocked the exocytosis response to formylmethionylleucylphenylalanine and the platelet-activating factor. Membrane ruffling and pseudopod extension via the BLTR was also completely inhibited by both Ptx and wortmannin. These data suggest that of the chemoattractant receptors studied, G-protein usage varies with FR being totally dependent on G(i), whereas BLTR and PAFR utilize both G(i) and a Ptx-insensitive G-protein. Both Ptx-sensitive and -insensitive G-protein usage can mediate the activation of phospholipase C, mobilization of intracellular calcium, and exocytosis by chemoattractant receptors. Chemotaxis, however, had an absolute requirement for a G(i)-mediated pathway.

Animals↗
PubMed1998

Differential cross-regulation of the human chemokine receptors CXCR1 and CXCR2. Evidence for time-dependent signal generation.

Neutrophils and transfected RBL-2H3 cells were used to investigate the mechanism of cross-regulation of the human interleukin-8 (IL-8) receptors CXCR1 and CXCR2 by chemoattractants. In neutrophils, Ca2+ mobilization by the CXCR2-specific chemokine, growth-related oncogene alpha (Groalpha), was desensitized by prior exposure to the chemoattractants N-formylated peptides (fMLP) or a complement cleavage product (C5a). In contrast, growth-related oncogene alpha did not desensitize the latter receptors. To investigate this phenomenon, CXCR2 was stably expressed in RBL-2H3 cells and mediated phosphoinositide hydrolysis, Ca2+ mobilization, chemotaxis, and secretion. In cells co-expressing CXCR2 and receptors for either C5a (C5aR) or fMLP (FR), CXCR2 was cross-phosphorylated and cross-desensitized by C5a and fMLP. However, neither C5aR nor FR was cross-phosphorylated or cross-desensitized by CXCR2 activation, although CXCR1 did mediate this process. Receptor internalization induced by IL-8 was more rapid and occurred at lower doses with CXCR2 than CXCR1, although both receptors mediated equipotent chemotaxis and exocytosis in RBL. Truncation of the cytoplasmic tail of CXCR2 (331T) prolonged its signaling relative to CXCR2, increased its resistance to internalization, and induced phospholipase D activation. 331T was resistant to homologous phosphorylation and cross-phosphorylation but not cross-desensitization of its Ca2+ mobilization by fMLP or C5a, indicating an inhibitory site distal to receptor/G protein coupling. In contrast to CXCR2, stimulation of 331T cross-desensitized Ca2+ mobilization by both FR and C5aR. CXCR2 and the mutant 331T induced phospholipase C beta3 phosphorylation to an extent equivalent to that of CXCR1. Taken together, these results suggest that CXCR1 and CXCR2 bind IL-8 to produce a group of equipotent responses, but their ability to generate other signals, including receptor internalization, cross-desensitization, and phospholipase D activation, are very different. The latter phenomena apparently require prolonged receptor activation, which in the case of CXCR2 is precluded by rapid receptor phosphorylation and internalization. Thus, receptors coupling to identical G proteins may trigger different cellular responses dependent on the length of their signaling time, which can be regulated by receptor phosphorylation.

Animals↗
PubMed1998

Differential regulation of formyl peptide and platelet-activating factor receptors. Role of phospholipase Cbeta3 phosphorylation by protein kinase A.

Formylated peptides (e.g. n-formyl-Met-Leu-Phe (fMLP)) and platelet-activating factor (PAF) mediate chemotactic and cytotoxic responses in leukocytes through receptors coupled to G proteins that activate phospholipase C (PLC). In RBL-2H3 cells, fMLP utilizes a pertussis toxin (ptx)-sensitive G protein to activate PLC, whereas PAF utilizes a ptx-insensitive G protein. Here we demonstrate that fMLP, but not PAF, enhanced intracellular cAMP levels via a ptx-sensitive mechanism. Protein kinase A (PKA) inhibition by H-89 enhanced inositol phosphate formation stimulated by fMLP but not PAF. Furthermore, a membrane-permeable cAMP analog 8-(4-chlorophenylthio)-cAMP (cpt-cAMP) inhibited phosphoinositide hydrolysis and secretion stimulated by fMLP but not PAF. Both cpt-cAMP and fMLP stimulated PLCbeta3 phosphorylation in intact RBL cells. The purified catalytic subunit of PKA phosphorylated PLCbeta3 immunoprecipitated from RBL cell lysate. Pretreatment of intact cells with cpt-cAMP and fMLP, but not PAF, resulted in an inhibition of subsequent PLCbeta3 phosphorylation by PKA in vitro. These data demonstrate that fMLP receptor, which couples to a ptx-sensitive G protein, activates both PLC and cAMP production. The resulting PKA activation phosphorylates PLCbeta3 and appears to block the ability of Gbetagamma to activate PLC. Thus, both fMLP and PAF generate stimulatory signals for PLCbeta3, but only fMLP produces a PKA-dependent inhibitory signal. This suggests a novel mechanism for the bidirectional regulation of receptors which activate PLC by ptx-sensitive G proteins.

Cell Line↗
PubMed1998

Multiple signaling pathways of human interleukin-8 receptor A. Independent regulation by phosphorylation.

Interleukin-8 (IL-8) receptor A (CXCR1) couples to a pertussis toxin-sensitive G protein to mediate phospholipase Cbeta (PLCbeta) activation and cellular responses. Responses to CXCR1 are attenuated by prior exposure of neutrophils to either IL-8, a cleavage product of the fifth component of complement (C5a) or n-formylated peptides (formylmethionylleucylphenylalanine, fMLP). To characterize the role of receptor phosphorylation in the regulation of the CXCR1, a phosphorylation-deficient mutant, M2CXCR1, was constructed. This receptor, stably expressed in RBL-2H3 cells, coupled more efficiently to G protein and stimulated enhanced phosphoinositide hydrolysis, cAMP production, exocytosis, and phospholipase D activation, and was resistant to IL-8-induced receptor internalization. The rate and total amount of ligand stimulated actin polymerization remained unchanged, but interestingly, chemotaxis was decreased by approximately 30% compared with the wild type receptor. To study the role of receptor phosphorylation in cross-desensitization of chemoattractant receptors, M2CXCR1 was coexpressed with cDNAs encoding receptors for either fMLP (FR), C5a (C5aR), or platelet-activating factor (PAFR). Both C5aR and PAFR were cross-phosphorylated upon M2CXCR1 activation, resulting in attenuated guanosine 5'-3'-O-(thio)triphosphate (GTPgammaS) binding in membranes. In contrast, FR and M2CXCR1 were resistant to cross-phosphorylation and cross-inhibition of GTPgammaS binding by other receptors. Despite the resistance of M2CXCR1 to cross-phosphorylation and receptor/G protein uncoupling, its susceptibility to cross-desensitization of its Ca2+ response by fMLP and C5a, was equivalent to CXCR1. Regardless of the enhancement in certain receptor functions in M2CXCR1 compared with the wild type CXCR1, the mutated receptors mediated equivalent PLCbeta3 phosphorylation and cross-desensitization of Ca2+ mobilization by FR, C5aR, and PAFR. The results herein indicate that phosphorylation of CXCR1 regulates some, but not all of the receptors functions. While receptor phosphorylation inhibits G protein turnover, PLC activation, Ca2+ mobilization and secretion, it is required for normal chemotaxis and receptor internalization. Since phosphorylation of CXCR1 had no effect on its ability to induce phosphorylation of PLCbeta3 or to mediate class-desensitization, these activities may be mediated by independently regulated pathways.

Antigens, CD↗
PubMed1997

Regulation of human chemokine receptors CXCR4. Role of phosphorylation in desensitization and internalization.

Members of the chemokine receptor family CCR5 and CXCR4 have recently been shown to be involved in the entry of human immunodeficiency virus (HIV) into target cells. Here, we investigated the regulation of CXCR4 in rat basophilic leukemia cells (RBL-2H3) stably transfected with wild type (Wt CXCR4) or a cytoplasmic tail deletion mutant (DeltaCyto CXCR4) of CXCR4. The ligand, stromal cell derived factor-1 (SDF-1) stimulated higher G-protein activation, inositol phosphate generation, and a more sustained calcium elevation in cells expressing DeltaCyto CXCR4 relative to Wt CXCR4. SDF-1 and phorbol 12-myristate 13-acetate (PMA), but not a membrane permeable cAMP analog induced rapid phosphorylation as well as desensitization of Wt CXCR4. Phosphorylation of DeltaCyto CXCR4 was not detected under any of these conditions. Despite lack of receptor phosphorylation, calcium mobilization by SDF-1 in DeltaCyto CXCR4 cells was partially desensitized by prior treatment with SDF-1. Of interest, the rapid release of calcium was inhibited without affecting the sustained calcium elevation, indicating independent regulatory pathways for these processes. PMA completely inhibited phosphoinositide hydrolysis and calcium mobilization in Wt CXCR4 but only partially inhibited these responses in DeltaCyto CXCR4. cAMP also partially inhibited these responses in both Wt CXCR4 and DeltaCyto CXCR4. SDF-1, PMA, and cAMP caused phosphorylation of phospholipase Cbeta3 in Wt and DeltaCyto CXCR4 cells. Both SDF-1 as well as PMA induced rapid internalization of Wt CXCR4. SDF-1 but not PMA induced internalization of DeltaCyto CXCR4 albeit at reduced levels relative to Wt CXCR4. These results indicate that signaling and internalization of CXCR4 are regulated by receptor phosphorylation dependent and independent mechanisms. Desensitization of CXCR4 signaling, independent of receptor phosphorylation, appears to be a consequence of the phosphorylation of phospholipase Cbeta3.

Amino Acid Sequence↗
PubMed1997

Chemoattractant receptor-induced phosphorylation of L-selectin.

The selectin adhesion molecules and chemoattractant receptors synergistically regulate leukocyte migration into lymphoid tissues and sites of inflammation, but little is known about how these families of receptors modulate each other's function. In this study, L-selectin was found to be phosphorylated in lymphoblastoid cell lines, and phosphorylation was enhanced by phorbol ester (phorbol 12-myristate 13-acetate (PMA)) treatment. Interactions between L-selectin and chemoattractant receptors were therefore examined using transfected rat basophilic leukemia cell lines (RBL-2H3) that expressed human L-selectin along with human leukocyte chemoattractant receptors. L-selectin was rapidly phosphorylated in cells treated with chemoattractants, thrombin, IgE receptor agonists, or PMA. Pertussis toxin or the protein kinase C inhibitor, staurosporine, completely blocked chemoattractant receptor-induced phosphorylation of L-selectin. PMA-induced phosphorylation was on serine residues within the cytoplasmic tail of L-selectin that have been well conserved during recent evolution. Although L-selectin phosphorylation was not essential for basal levels of adhesion through L-selectin in transformed cell lines, the rapid increase in ligand binding activity of L-selectin that occurs following leukocyte activation was blocked by staurosporine. These results demonstrate that L-selectin can be phosphorylated following engagement of chemoattractant receptors and suggest that this may be a physiologically relevant mechanism for the synergistic regulation of these receptors during leukocyte migration.

Animals↗