Undifferentiated U937 cells transfected with chemoattractant receptors: a model system to investigate chemotactic mechanisms and receptor structure/function relationships

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The human N-formylpeptide receptor (FPR) represents one of the most thoroughly studied leukocyte chemoattractant receptors. Despite this, little is known about the molecular mechanisms involved in the activation and desensitization of this receptor. To assess the role of phosphorylation in receptor function, U937 promonocytic cells were stably transfected to express the recombinant human FPR. Three mutant forms of the FPR lacking specific serine and threonine residues in the receptor C terminus were studied with respect to activation and desensitization. Replacement of all 11 serine and threonine residues within the C terminus by alanine and glycine residues (⌬ST) resulted in a receptor capable of ligand binding and G protein activation similar to the wild-type receptor. However, whereas the wild-type FPR was phosphorylated on both serine and threonine residues upon exposure to agonist and displayed a significantly reduced ability to stimulate G protein-mediated GTP hydrolysis upon subsequent exposure to agonist, ⌬ST demonstrated a complete lack of phosphorylation and displayed little alteration in its ability to stimulate G protein-mediated GTP hydrolysis upon a subsequent exposure to agonist. In addition to desensitization of G protein-mediated GTP hydrolysis, calcium mobilization was assayed to test whether desensitization occurred at a site distal to G protein activation. However, as observed with G protein activation, ⌬ST underwent no desensitization of the calcium mobilization response upon a second exposure to agonist. To define more precisely the role of specific serine and threonine residues, two additional mutants were analyzed. Neutrophils possess a large number of cell-surface G proteincoupled receptors that respond to structurally diverse ligands such as N-formylpeptides, complement components C5a and C3a, platelet-activating factor, and chemokines such as IL-8 1 (1). Receptor activation results in the stimulation of phospholipases, the mobilization of intracellular calcium, and the activation of a multitude of protein kinases culminating in functions such as chemotaxis, phagocytosis, superoxide production, and degranulation. Following an initial exposure to ligand, resulting in transient cell activation, neutrophils rapidly become unresponsive to continued or subsequent stimulation. This process of cellular acquiescence in the presence of agonist is termed desensitization and has been characterized for many hormonal (2) and neurotransmitter (3) receptors. Although the complex mechanisms involved in this process are poorly characterized, one of the early events has been suggested to involve receptor phosphorylation (4).G protein-coupled receptor kinases are a family of protein kinases that rapidly phosphorylate seven transmembrane receptors in a ligand-dependent manner (5-7). Following phosphorylation and the possible association with accessory proteins, such as arrestin, receptors are no longer capable of effectively activating G proteins, a process termed homologous desensitization (8). G protein-...

Section: Resultsmentioning

confidence: 58%

mentioning

confidence: 91%

Desensitization of N-Formylpeptide Receptor-mediated Activation Is Dependent upon Receptor Phosphorylation

Prossnitz

1997

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“…To investigate the potential interaction between the FPR and arrestins, we utilized a model promonocytic cell line, U937, stably transfected with the wild type FPR (23). Analysis of whole cell lysates by Western blot revealed that this cell line predominantly expresses arrestin-2 (data not shown).…”

Section: Resultsmentioning

confidence: 99%

Arrestin Binding to the G Protein-coupled N-Formyl Peptide Receptor Is Regulated by the Conserved “DRY” Sequence

Bennett

Maestas

Prossnitz³

2000

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Following activation by ligand, the N-formyl peptide receptor (FPR) undergoes processing events initiated by phosphorylation that lead to receptor desensitization and internalization. Our previous results have shown that FPR internalization can occur in the absence of receptor desensitization, suggesting that FPR desensitization and internalization are controlled by distinct mechanisms. More recently, we have provided evidence that internalization of the FPR occurs via a mechanism that is independent of the actions of arrestin, dynamin, and clathrin. In the present report, we demonstrate that stimulation of the FPR with agonist leads to a significant translocation of arrestin-2 from the cytosol to the membrane. Fluorescence microscopy revealed that the translocated arrestin-2 is highly colocalized with the ligand-bound FPR. A D71A mutant FPR, which does not undergo activation or phosphorylation in response to ligand, did not colocalize with arrestin-2. Surprisingly, an R123G mutant FPR, which does not bind G protein but does become phosphorylated and subsequently internalized, also did not bind arrestin. These results indicate that arrestin binding is not required for FPR internalization and demonstrate for the first time that a common motif, the conserved "DRY" domain of G protein-coupled receptors, is essential for phosphorylation-dependent arrestin binding, as well as G protein activation.

“…Cells were grown in RPMI 1640 (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum, 100 units/ml penicillin, 100 g/ml streptomycin, 10 mM HEPES, pH 7.4, 100 g/ml ciprofloxacin, 2 mM L-glutamine, at 37°C in a humidified atmosphere of 5% CO 2 and 95% air. Site-directed mutants of formyl peptide receptor in the human monoblastoid line U937 constitutively expressing human VLA-4 integrin were prepared as described (19). High expressors were selected using the MoFlo Flow Cytometer (Cytomation, Inc., Fort Collins, CO).…”

Section: Materials-thementioning

confidence: 99%

Real-time Analysis of Very Late Antigen-4 Affinity Modulation by Shear

Zwartz¹,

Chigaev²,

Dwyer

et al. 2004

Shear promotes endothelial recruitment of leukocytes, cell activation, and transmigration. Mechanical stress on cells caused by shear can induce a rapid integrin conformational change and activation, followed by an increase in binding to the extracellular matrix. The molecular mechanism of increased avidity is unknown. We have shown previously that the affinity of the ␣ 4 ␤ 1 integrin, very late antigen-4 (VLA-4), measured with an LDV-containing small molecule, varies with cellular avidity, measured from cell disaggregation rates. In this study, we measured in real time affinity changes of VLA-4 in response to shear. The resulting affinity was comparable with the state mediated by receptor signaling and corresponded in time with intracellular Ca 2؉ responses. Ca Leukocytes are recruited to endothelial cells in a multistep process using selectin and integrin adhesion molecules (1, 2). These molecules allow a cell to tether, roll, adhere, and transmigrate along and across an endothelial layer. Selectin and some integrin molecules and their associated ligands mediate tethering and rolling interactions. Firm adhesion is mediated by vascular ligands of the immunoglobulin superfamily such as vascular cell adhesion molecule 1 (VCAM-1) 1 and their associated integrins (1, 2). The adhesive strength or avidity (3) of cells expressing integrins can be rapidly modulated by chemokines and chemoattractants, which also regulate leukocyte recruitment and migration across vascular endothelium. The rapid changes in avidity have been attributed to changes in the number of interacting molecules or valency due to molecular redistribution or clustering and to changes in the affinity of the individual receptor-ligand bonds (3-10).Physiological shear can also regulate leukocyte traffic by stimulating mechanosensors on neutrophils, monocytes, lymphocytes, erythrocytes, and platelets (see Ref. 11 and references therein). Shear arises from bifurcating blood vessels or rapid changes in blood vessel diameters. Shear acting on leukocytes, bound to endothelial cells, produces mechanical stress on the cells or their receptors, regulating cell growth and proliferation, protein synthesis, gene expression, and blood cell recruitment (12, 13). Integrins (such as ␣ v ␤ 3 , ␣ 5 ␤ 1 , ␣ 4 ␤ 1 , and ␣ 2 ␤ 1 ) on endothelial cells can act as mechanosensors to changes in blood flow (13,14) and trigger an intracellular signaling pathway involving focal adhesion kinase and mitogen-activated protein kinase cascades. How shear specifically induces blood cell adhesiveness or recruitment through mechanosensors is unknown. Indirect evidence shows that increased integrin binding to the extracellular matrix occurs when shear acts on cells or their mechanosensors to induce intracellular signaling. For example, intracellular signaling leads to conformational changes and activation of ␣ v ␤ 3 on endothelial cells and ␣ 4 ␤ 1 and ␣ 5 ␤ 1 integrins on monocytic cells (15, 16). Shear acting on endothelial cells affects the GTPase Rho signaling pathway and in monocyti...