scholarly journals Ligand binding directly stimulates ubiquitination of the inositol 1,4,5-trisphosphate receptor

2000 ◽  
Vol 348 (3) ◽  
pp. 551-556 ◽  
Author(s):  
Chang-Cheng ZHU ◽  
Richard J. H. WOJCIKIEWICZ
Keyword(s):  
Ligand Binding ◽  
Muscarinic Receptor ◽  
Receptor Activation ◽  
Type I ◽  
Wild Type ◽  
Down Regulation ◽  
Mutant Type ◽  
Human Neuroblastoma ◽  
Surface Receptors ◽  
Defective Mutant

Down-regulation of the Ins(1,4,5)P3 receptor is an adaptive response to the activation of certain phosphoinositidase C-linked cell-surface receptors. It is manifested as a profound decline in cellular Ins(1,4,5)P3 receptor content, occurs with a half-time of 0.5-2 h and is due to accelerated proteolysis. It has been shown that this process is mediated by the ubiquitin/proteasome pathway and is therefore initiated by Ins(1,4,5)P3 receptor ubiquitination. To investigate the role of ligand binding in Ins(1,4,5)P3 receptor ubiquitination, we expressed ‘exogenous’ wild-type and ligand-binding-defective mutant type I Ins(1,4,5)P3 receptors in human neuroblastoma SH-SY5Y cells, in which muscarinic receptor activation elicits Ins(1,4,5)P3 receptor down-regulation. We found (1) that exogenous wild-type Ins(1,4,5)P3 receptors are efficiently ubiquitinated in response to muscarinic receptor stimulation, (2) that exogenous ligand binding-defective mutant Ins(1,4,5)P3 receptors are resistant to ubiquitination, (3) that this resistance is not caused by the removal of potential ubiquitin-conjugating sites in the mutated region, and (4) that in heterotetramers of exogenous mutant receptors and ‘endogenous’ receptors, only the latter are targeted for ubiquitination. These results indicate that the binding of Ins(1,4,5)P3 directly stimulates ubiquitination of the Ins(1,4,5)P3 receptor and that the targeting of Ins(1,4,5)P3 receptors for ubiquitination is a highly specific process. We therefore propose that an Ins(1,4,5)P3-binding-induced conformational change in the receptor exposes a degradation signal that leads to ubiquitination.

scholarly journals Mechanisms of Transforming Growth Factor-β Receptor Endocytosis and Intracellular Sorting Differ between Fibroblasts and Epithelial Cells

2001 ◽  
Vol 12 (3) ◽  
pp. 675-684 ◽  
Author(s):  
Jules J.E. Doré ◽  
Diying Yao ◽  
Maryanne Edens ◽  
Nandor Garamszegi ◽  
Elizabeth L. Sholl ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epithelial Cells ◽  
Ligand Binding ◽  
Transforming Growth Factor ◽  
Point Mutations ◽  
Type I ◽  
Receptor Complex ◽  
Type Ii ◽  
Down Regulation ◽  
Β Receptor

Transforming growth factor-βs (TGF-β) are multifunctional proteins capable of either stimulating or inhibiting mitosis, depending on the cell type. These diverse cellular responses are caused by stimulating a single receptor complex composed of type I and type II receptors. Using a chimeric receptor model where the granulocyte/monocyte colony-stimulating factor receptor ligand binding domains are fused to the transmembrane and cytoplasmic signaling domains of the TGF-β type I and II receptors, we wished to describe the role(s) of specific amino acid residues in regulating ligand-mediated endocytosis and signaling in fibroblasts and epithelial cells. Specific point mutations were introduced at Y182, T200, and Y249 of the type I receptor and K277 and P525 of the type II receptor. Mutation of either Y182 or Y249, residues within two putative consensus tyrosine-based internalization motifs, had no effect on endocytosis or signaling. This is in contrast to mutation of T200 to valine, which resulted in ablation of signaling in both cell types, while only abolishing receptor down-regulation in fibroblasts. Moreover, in the absence of ligand, both fibroblasts and epithelial cells constitutively internalize and recycle the TGF-β receptor complex back to the plasma membrane. The data indicate fundamental differences between mesenchymal and epithelial cells in endocytic sorting and suggest that ligand binding diverts heteromeric receptors from the default recycling pool to a pathway mediating receptor down-regulation and signaling.

scholarly journals Proteasomes Regulate the Duration of Erythropoietin Receptor Activation by Controlling Down-regulation of Cell Surface Receptors

2000 ◽  
Vol 275 (24) ◽  
pp. 18375-18381 ◽  
Author(s):  
Frédérique Verdier ◽  
Pierre Walrafen ◽  
Nathalie Hubert ◽  
Stany Chrétien ◽  
Sylvie Gisselbrecht ◽  
...  
Keyword(s):  
Cell Surface ◽  
Receptor Activation ◽  
Erythropoietin Receptor ◽  
Cell Surface Receptors ◽  
Down Regulation ◽  
Surface Receptors

scholarly journals Plasma Membrane Localization of Gαz Requires Two Signals

1998 ◽  
Vol 9 (1) ◽  
pp. 1-14 ◽  
Author(s):  
Janine Morales ◽  
C. Simone Fishburn ◽  
Paul T. Wilson ◽  
Henry R. Bourne
Keyword(s):  
Plasma Membrane ◽  
G Protein ◽  
Protein Localization ◽  
Receptor Activation ◽  
Α Subunit ◽  
Surface Receptors ◽  
Defective Mutant ◽  
Specific Localization ◽  
Membrane Attachment ◽  
Stable Association

Three covalent attachments anchor heterotrimeric G proteins to cellular membranes: the α subunits are myristoylated and/or palmitoylated, whereas the γ chain is prenylated. Despite the essential role of these modifications in membrane attachment, it is not clear how they cooperate to specify G protein localization at the plasma membrane, where the G protein relays signals from cell surface receptors to intracellular effector molecules. To explore this question, we studied the effects of mutations that prevent myristoylation and/or palmitoylation of an epitope-labeled α subunit, αz. Wild-type αz (αz-WT) localizes specifically at the plasma membrane. A mutant that incorporates only myristate is mistargeted to intracellular membranes, in addition to the plasma membrane, but transduces hormonal signals as well as does αz-WT. Removal of the myristoylation site produced a mutant αz that is located in the cytosol, is not efficiently palmitoylated, and does not relay the hormonal signal. Coexpression of βγ with this myristoylation defective mutant transfers it to the plasma membrane, promotes its palmitoylation, and enables it to transmit hormonal signals. Pulse-chase experiments show that the palmitate attached to this myristoylation-defective mutant turns over much more rapidly than does palmitate on αz-WT, and that the rate of turnover is further accelerated by receptor activation. In contrast, receptor activation does not increase the slow rate of palmitate turnover on αz-WT. Together these results suggest that myristate and βγ promote stable association with membranes not only by providing hydrophobicity, but also by stabilizing attachment of palmitate. Moreover, palmitoylation confers on αz specific localization at the plasma membrane.

Down-regulation of phospholipase C-β1 following chronic muscarinic receptor activation

1998 ◽  
Vol 346 (1) ◽  
pp. R1-R2 ◽  
Author(s):  
Scott D Sorensen ◽  
Daniel A Linseman ◽  
Stephen K Fisher
Keyword(s):  
Phospholipase C ◽  
Muscarinic Receptor ◽  
Receptor Activation ◽  
Down Regulation

Proteasomes regulate the duration of erythropoietin receptor activation by controlling down-regulation of cell surface receptors

2000 ◽  
Vol 28 (5) ◽  
pp. A284-A284
Author(s):  
F. Verdier ◽  
P. Walrafen ◽  
N. Hubert ◽  
S. Chretien ◽  
S. Gisselbrecht ◽  
...  
Keyword(s):  
Cell Surface ◽  
Receptor Activation ◽  
Erythropoietin Receptor ◽  
Cell Surface Receptors ◽  
Down Regulation ◽  
Surface Receptors

scholarly journals Structure and function of the cytoskeleton of a Dictyostelium myosin-defective mutant.

1990 ◽  
Vol 110 (2) ◽  
pp. 367-378 ◽  
Author(s):  
Y Fukui ◽  
A De Lozanne ◽  
J A Spudich
Keyword(s):  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Insertional Mutagenesis ◽  
Chain Gene ◽  
Mitotic Spindles ◽  
Wild Type ◽  
Surface Receptors ◽  
Dictyostelium Myosin ◽  
Thick Filaments ◽  
Defective Mutant

To study the role of conventional myosin in nonmuscle cells, we determined the cytoskeletal organization and physiological responses of a Dictyostelium myosin-defective mutant. Dictyostelium hmm cells were created by insertional mutagenesis of the myosin heavy chain gene (De Lozanne, A., and J. A. Spudich. 1987. Science (Wash. DC). 236: 1086-1091). Western blot analysis using different mAbs confirms that hmm cells express a truncated myosin fragment of 140 kD (HMM-140 protein) instead of the normal 243-kD myosin heavy chain (MHC). Spontaneous revertants appear at a frequency less than 4 x 10(-5), which synthesize normal myosin and are capable of forming thick filaments. In hmm cells, the HMM-140 protein is diffusely distributed in the cytoplasm, indicating that it cannot assemble into thick filaments. The actin distribution in these mutant cells appears similar to that of wild-type cells. However, there is a significant abnormality in the organization of cytoplasmic microtubules, which penetrate into lamellipodial regions. The microtubule networks consist of approximately 13 microtubules on average and their pattern is abnormal. Although hmm cells can form mitotic spindles, mitosis is not coordinated with normal furrow formation. The hmm cells are clearly defective in the contractile events that lead to normal cytokinesis. The retraction of different regions of the cell can result in the occasional pinching off of part of the cell. This process is not coupled with formation of mitotic spindles. There is no specific accumulation of HMM-140 in such constrictions, whereas 73% of such cells show actin concentrated in these regions. The mutant hmm cells are also deficient in capping of Con-A-bound surface receptors, but instead internalize this complex into the cytoplasm. The hmm cells display active phagocytosis of bacteria. Whereas actin is concentrated in the phagocytic cups, HMM-140 protein is not localized in these regions. cAMP, a chemoattractant that induces drastic rounding up and formation of surface blebs in wild type cells, does not induce rounding up in the hmm cells. A Triton-permeabilized cell model of the wild-type amebae contracts on reactivation with Mg-ATP, whereas a model of the hmm cell shows no detectable contraction. Our data demonstrate that the conventional myosin participates in the significant cortical motile activities of Dictyostelium cells, which include rounding up, constriction of cleavage furrows, capping surface receptors, and establishing cell polarity.

M3-receptor knockout mice: muscarinic receptor function in atria, stomach fundus, urinary bladder, and trachea

2002 ◽  
Vol 282 (5) ◽  
pp. R1443-R1449 ◽  
Author(s):  
Peter W. Stengel ◽  
Masahisa Yamada ◽  
Jürgen Wess ◽  
Marlene L. Cohen
Keyword(s):  
Smooth Muscle ◽  
Urinary Bladder ◽  
Muscarinic Receptor ◽  
Knockout Mice ◽  
Smooth Muscle Contraction ◽  
Receptor Activation ◽  
Muscarinic Agonist ◽  
Wild Type ◽  
Atrial Rate ◽  
Stomach Fundus

Negative chronotropic and smooth muscle contractile responses to the nonselective muscarinic agonist carbamylcholine were compared in isolated tissues from M3-muscarinic receptor knockout and wild-type mice. Carbamylcholine (10−8–3.0 × 10−5 M) induced a concentration-dependent decrease in atrial rate that was similar in atria from M3-receptor knockout and wild-type mice, indicating that M3 receptors were not involved in muscarinic receptor-mediated atrial rate decreases. In contrast, the M3 receptor was a major muscarinic receptor involved in smooth muscle contraction of stomach fundus, urinary bladder, and trachea, although differences existed in the extent of M3-receptor involvement among the tissues. Contraction to carbamylcholine was virtually abolished in urinary bladder from M3-receptor knockout mice, suggesting that contraction was predominantly due to M3-receptor activation. However, ∼50–60% maximal contraction to carbamylcholine occurred in stomach fundus and trachea from M3-receptor knockout mice, indicating that contraction in these tissues was also due to M2-receptor activation. High concentrations of carbamylcholine relaxed the stomach fundus from M3-receptor knockout mice by M1-receptor activation. Thus M3-receptor knockout mice provided unambiguous evidence that M3 receptors 1) play no role in carbamylcholine-induced atrial rate reduction, 2) are the predominant receptor mediating carbamylcholine-induced urinary bladder contractility, and 3) share contractile responsibility with M2 receptors in mouse stomach fundus and trachea.

scholarly journals Adenosine activation of A2B receptor(s) is essential for stimulated epithelial ciliary motility and clearance

2011 ◽  
Vol 301 (2) ◽  
pp. L171-L180 ◽  
Author(s):  
Diane S. Allen-Gipson ◽  
Michael R. Blackburn ◽  
Daniel J. Schneider ◽  
Hui Zhang ◽  
DeAndre L. Bluitt ◽  
...  
Keyword(s):  
Knockout Mice ◽  
Adenosine Receptor ◽  
Beat Frequency ◽  
Receptor Activation ◽  
Breakdown Product ◽  
Protein Kinase Activity ◽  
Wild Type ◽  
Surface Receptors ◽  
Ciliary Motility ◽  
Adenosine Receptor Agonist

Mucociliary clearance, vital to lung clearance, is dependent on cilia beat frequency (CBF), coordination of cilia, and the maintenance of periciliary fluid. Adenosine, the metabolic breakdown product of ATP, is an important modulator of ciliary motility. However, the contributions of specific adenosine receptors to key airway ciliary motility processes are unclear. We hypothesized that adenosine modulates ciliary motility via activation of its cell surface receptors (A1, A2A, A2B, or A3). To test this hypothesis, mouse tracheal rings (MTRs) excised from wild-type and adenosine receptor knockout mice (A1, A2A, A2B, or A3, respectively), and bovine ciliated bronchial epithelial cells (BBECs) were stimulated with known cilia activators, isoproterenol (ISO; 10 μM) and/or procaterol (10 μM), in the presence or absence of 5′-(N-ethylcarboxamido) adenosine (NECA), a nonselective adenosine receptor agonist [100 nM (A1, A2A, A3); 10 μM (A2B)], and CBF was measured. Cells and MTRs were also stimulated with NECA (100 nM or 10 μM) in the presence and absence of adenosine deaminase inhibitor, erythro-9- (2-hydroxy-3-nonyl) adenine hydrochloride (10 μM). Both ISO and procaterol stimulated CBF in untreated cells and/or MTRs from both wild-type and adenosine knockout mice by ∼3 Hz. Likewise, CBF significantly increased ∼2–3 Hz in BBECs and wild-type MTRs stimulated with NECA. MTRs from A1, A2A, and A3 knockout mice stimulated with NECA also demonstrated an increase in CBF. However, NECA failed to stimulate CBF in MTRs from A2B knockout mice. To confirm the mechanism by which adenosine modulates CBF, protein kinase activity assays were conducted. The data revealed that NECA-stimulated CBF is mediated by the activation of cAMP-dependent PKA. Collectively, these data indicate that purinergic stimulation of CBF requires A2B adenosine receptor activation, likely via a PKA-dependent pathway.

scholarly journals Distinct Endocytic Responses of Heteromeric and Homomeric Transforming Growth Factor β Receptors

1997 ◽  
Vol 8 (11) ◽  
pp. 2133-2143 ◽  
Author(s):  
Robert A. Anders ◽  
Sandra L. Arline ◽  
Jules J.E. Doré ◽  
Edward B. Leof
Keyword(s):  
Growth Factor ◽  
Ligand Binding ◽  
Transforming Growth Factor ◽  
Transforming Growth Factor Β ◽  
Cell Surface Receptor ◽  
Type I ◽  
Type Ii ◽  
Down Regulation ◽  
Tgfβ Receptors ◽  
Tgfβ Receptor

Transforming growth factor β (TGFβ) family ligands initiate a cascade of events capable of modulating cellular growth and differentiation. The receptors responsible for transducing these cellular signals are referred to as the type I and type II TGFβ receptors. Ligand binding to the type II receptor results in the transphosphorylation and activation of the type I receptor. This heteromeric complex then propagates the signal(s) to downstream effectors. There is presently little data concerning the fate of TGFβ receptors after ligand binding, with conflicting reports indicating no change or decreasing cell surface receptor numbers. To address the fate of ligand-activated receptors, we have used our previously characterized chimeric receptors consisting of the ligand binding domain from the granulocyte/macrophage colony-stimulating factor α or β receptor fused to the transmembrane and cytoplasmic domain of the type I or type II TGFβ receptor. This system not only provides the necessary sensitivity and specificity to address these types of questions but also permits the differentiation of endocytic responses to either homomeric or heteromeric intracellular TGFβ receptor oligomerization. Data are presented that show, within minutes of ligand binding, chimeric TGFβ receptors are internalized. However, although all the chimeric receptor combinations show similar internalization rates, receptor down-regulation occurs only after activation of heteromeric TGFβ receptors. These results indicate that effective receptor down-regulation requires cross-talk between the type I and type II TGFβ receptors and that TGFβ receptor heteromers and homomers show distinct trafficking behavior.

Functional Analysis of Leukemia-Associated Mutations Involving the Heterodimerization Domain of NOTCH1.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 843-843
Author(s):  
Cheryll Sanchez-Irizarry ◽  
Michael Malecki ◽  
Woojoong Lee ◽  
Mina Xu ◽  
Stephen C. Blacklow ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Reporter Gene ◽  
Cleavage Site ◽  
Transmembrane Domain ◽  
Regulatory Region ◽  
Receptor Activation ◽  
Luciferase Reporter ◽  
Type I ◽  
Gamma Secretase ◽  
Gain Of Function

Abstract NOTCH1 is a type I transmembrane receptor that regulates T cell development via a signaling pathway that relies on regulated proteolysis. During its maturation, most NOTCH1 is cleaved at a position 70 amino acids external to the transmembrane domain by a furin-like protease, creating extracellular (NEC) and transmembrane (NTM) subunits that are held together non-covalently by a juxtamembranous heterodimerization (HD) domain. Ligand-binding to NEC promotes cleavage by i) metalloproteases at a site in the ectodomain of NTM, followed by ii) gamma-secretase within the transmembrane domain. This releases the NOTCH1 intracellular domain (ICN1), allowing it to translocate to the nucleus and activate target genes. Normally, proteolysis is constrained prior to ligand-binding by an extracellular negative regulatory region consisting of 3 iterated LNR repeats and the N- and C-terminal portions of the HD domain, which flank the furin cleavage site. Recent work has shown that human T-ALL is frequently associated with gain of function mutations that map to the HD domain of NOTCH1. These mutations are distributed in both parts of the HD domain and include point mutations, short insertions, and deletions, suggesting that there might be variation in their relative strength and the mechanisms by which they act. To investigate these issues, we introduced 16 of the HD domain mutations found in primary T-ALLs or T-ALL cell lines into a full-length NOTCH1 cDNA, and tested their ability to activate a NOTCH sensitive luciferase reporter gene. Except for the "mutation" R1609S, which was found in only one primary T-ALL sample, all of the mutations stimulated NOTCH1 signaling. These increases in signaling were abolished by a gamma-secretase inhibitor and were associated with increased rates of metalloprotease-mediated cleavage, indicating that activation proceeds through the normal series of proteolytic events. The mutations also caused gains in function when introduced into NOTCH1 polypeptides lacking the ligand-binding region of NEC, indicating that the HD domain mutations can cause ligand-independent receptor activation. Since NEC dissociation can lead to activation of NOTCH signaling (and is a proposed mechanism for normal ligand-mediated NOTCH activation), one simple way for HD domain mutations to act is through the destabilization of NOTCH1 heterodimers. To test this model, each mutation was introduced into soluble NOTCH1 mini-receptors bearing N-terminal FLAG and C-terminal HA tags. When expressed transiently, the normal NOTCH1 mini-receptor was secreted into conditioned media as a furin-processed heterodimer. Certain activating HD domain mutations, such as L1601P, resulted in complete dissociation of the furin-processed mini-receptor subunits under native conditions, and all other HD domain mutations save one were more sensitive to urea-induced dissociation than normal NOTCH1. The exception was an unusual insertional mutation (identified in the P12-Ichikawa cell line) that introduces a 14 amino acid direct repeat sequence at a position immediately N-terminal of the metalloprotease cleavage site. We hypothesize that this mutation, which was associated with the greatest increases in signaling in NOTCH1 reporter gene assays, displaces protective HD and LNR domain residues and thereby unveils the metalloprotease cleavage site. We conclude that most T-ALL-associated HD domain mutations confer ligand-independent gain-of-function on NOTCH1 receptors, but vary in strength and are likely to act through several distinct mechanisms.

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