scholarly journals Signal transduction by Fc gamma RIII (CD16) is mediated through the gamma chain.

1992 ◽  
Vol 175 (5) ◽  
pp. 1381-1390 ◽  
Author(s):  
U Wirthmueller ◽  
T Kurosaki ◽  
M S Murakami ◽  
J V Ravetch
Keyword(s):  
Signal Transduction ◽  
Cell Surface ◽  
Cell Activation ◽  
Receptor Activation ◽  
Alpha Subunit ◽  
Wild Type ◽  
Cytoplasmic Domains ◽  
Gamma Chain ◽  
Glycosyl Phosphatidylinositol ◽  
Mutant Forms

To determine the functional role of the two isoforms of Fc gamma RIII (CD16) (IIIA, IIIB), the signal transduction capabilities of wild-type and mutant forms of these receptors were analyzed in transfected lymphoid, myeloid, and fibroblastic cell lines. Functional reconstitution of receptor signalling was observed in hematopoietic T and mast cells, and was absent in nonhematopoietic (CHO) cells. Fc gamma RIIIA, a hetero-oligomeric receptor composed of a ligand-binding subunit alpha and dimeric gamma chains, generated both proximal and distal responses in Jurkat and P815 cells, typical of what is seen in natural killer cells and macrophages upon receptor activation. In contrast, Fc gamma RIIIB, which is normally attached to the cell surface via a glycosyl-phosphatidylinositol anchor, was incapable of transducing signals. After crosslinking, Fc gamma RIIIA signalling was dependent only upon the gamma chain. Fc gamma RIIIA chimeras in which the alpha subunit transmembrane and cytoplasmic domains were substituted with the corresponding gamma chain sequences functioned as well as wild-type hetero-oligomeric receptors. These data indicate that the ability of the Fc gamma RIIIA complex to activate the appropriate pathways for cell activation is cell-type restricted and independent of the transmembrane and cytoplasmic domains of the alpha subunit. The presence of the gamma chain is responsible for the assembly of, as well as the signal transduction by, the functional cell surface complex.

scholarly journals Heterologous transmembrane and cytoplasmic domains direct functional chimeric influenza virus hemagglutinins into the endocytic pathway.

1986 ◽  
Vol 102 (4) ◽  
pp. 1271-1283 ◽  
Author(s):  
M G Roth ◽  
C Doyle ◽  
J Sambrook ◽  
M J Gething
Keyword(s):  
Influenza Virus ◽  
Cell Surface ◽  
Dna Sequences ◽  
Acquired Resistance ◽  
Endocytic Pathway ◽  
Wild Type ◽  
Cytoplasmic Domains ◽  
Coated Pits ◽  
Herpes Simplex Virus 1 ◽  
Surface Receptors

Chimeric genes were created by fusing DNA sequences encoding the ectodomain of the influenza virus hemagglutinin (HA) to DNA coding for the transmembrane and cytoplasmic domains of either the G glycoprotein of vesicular stomatitis virus or the gC glycoprotein of Herpes simplex virus 1. CV-1 cells infected with SV40 vectors carrying the recombinant genes expressed large amounts of the chimeric proteins, HAG or HAgC on their surfaces. Although the ectodomains of HAG and HAgC differed in their immunological properties from that of HA, the chimeras displayed the biological functions characteristic of the wild-type protein. Both HAG and HAgC bound erythrocytes as efficiently as HA did and, after brief exposure to an acidic environment, induced the fusion of erythrocyte and CV-1 cell membranes. However, the behavior of HAG and HAgC at the cell surface differed from that of HA in several important respects. HAG and HAgC were observed to collect in coated pits whereas wild-type HA was excluded from those structures. In the presence of chloroquine, which inhibits the exit of receptors from endosomes, HAG and HAgC accumulated in intracellular vesicles. By contrast, chloroquine had no effect on the location of wild-type HA. HAG and HAgC labeled at the cell surface exhibited a temperature-dependent acquisition of resistance to extracellular protease at a rate similar to the rates of internalization observed for many cell surface receptors. HA acquired resistance to protease at a rate at least 20-fold slower. We conclude that HAG and HAgC are efficiently routed into the endocytic pathway and HA is not. However, like HA, HAG was degraded slowly, raising the possibility that HAG recycles to the plasma membrane.

Endogenous Lectins as Cell Surface Transducers

2000 ◽  
Vol 20 (4) ◽  
pp. 213-237 ◽  
Author(s):  
Eric Hébert
Keyword(s):  
Signal Transduction ◽  
Cell Surface ◽  
Growth Factor Receptor ◽  
Receptor Activation ◽  
Carbohydrate Binding ◽  
Animal Cells ◽  
Endogenous Lectins ◽  
Natural Killer Receptors ◽  
Carbohydrate Binding Proteins ◽  
Transfer Of Information

Interactions between cells or between cell and substratum involve specificreceptors and their ligands. Among the various cell surface receptorsidentified during the last decades, the carbohydrate-binding proteins, e.g., lectins are of peculiar interest because glycolipids, glycoproteinsand proteoglycans have been shown to interact with lectins on the surfaceof animal cells. Animal lectins are recognized as molecules playingimportant roles in a variety of biological processes through binding toglycoconjugates and lectin-like receptors such as selectins, sialoadhesins(CD22, CD33), natural killer receptors (NKR-P1, CD69 and CD94/NKG2), hyaluronate receptors (CD44, RHAMM, ICAM-1), B-cell associated antigen(CD23, CD72), γ2 leukocyte integrin (CD11b/CD18) or the well-knownreceptors for mannose, mannose-6-phosphate or asialoglycoprotein havebeen suggested to be able to mediate the transfer of information fromthe outside to the inside of the cell. This review focuses on the mostrecent advances in our understanding of the molecular basis of “outside-in” signaling mediated by lectins. Lectin-likereceptors are involved in signal transduction in a great variety of ways;at the molecular level, they mimic in most of the cases the function ofgrowth factor receptor either coupled to tyrosine kinase activity or toheterotrimeric G protein. They lead to a multiplicity of cellular eventsfollowing their activation depending on factors such as cellular type, species and/or tissue. Nevertheless the potential of surface lectins astransducers is emphasized by the observation that in a few cases lectin-likereceptors induce either novel signal transduction mechanism or newintracellular events with regards to what it has been observed as aconsequence of growth factor receptor activation. This observation bringsthe idea that lectins may offer, as cell surface transducers, an alternativeor additional signaling potential to cell.

scholarly journals A pathway for targeting soluble misfolded proteins to the yeast vacuole.

1996 ◽  
Vol 135 (3) ◽  
pp. 623-633 ◽  
Author(s):  
E Hong ◽  
A R Davidson ◽  
C A Kaiser
Keyword(s):  
Cell Surface ◽  
Secretory Pathway ◽  
Carboxypeptidase Y ◽  
Misfolded Protein ◽  
Secreted Protein ◽  
Wild Type ◽  
Salvage Pathway ◽  
Lambda Repressor ◽  
Hybrid Proteins ◽  
Mutant Forms

We have evaluated the fate of misfolded protein domains in the Saccharomyces cerevisiae secretory pathway by fusing mutant forms of the NH2-terminal domain of lambda repressor protein to the secreted protein invertase. The hybrid protein carrying the wild-type repressor domain is mostly secreted to the cell surface, whereas hybrid proteins with amino acid substitutions that cause the repressor domain to be thermodynamically unstable are retained intracellularly. Surprisingly, the retained hybrids are found in the vacuole, where the repressor moiety is degraded by vacuolar proteases. The following observations indicate that receptor-mediated recognition of the mutant repressor domain in the Golgi lumen targets these hybrid fusions to the vacuole. (a) The invertase-repressor fusions, like wild-type invertase, behave as soluble proteins in the ER lumen. (b) Targeting to the vacuole is saturable since overexpression of the hybrids carrying mutant repressor increases the fraction of fusion protein that appears at the cell surface. (c) Finally, deletion of the VPS10 gene, which encodes the transmembrane Golgi receptor responsible for targeting carboxypeptidase Y to the vacuole, causes the mutant hybrids to be diverted to the cell surface. Together these findings suggest that yeast have a salvage pathway for degradation of nonnative luminal proteins by receptor-mediated transport to the vacuole.

scholarly journals Constitutive Activation and Inactivation of Mutations Inducing Cell Surface Loss of Receptor and Impairing of Signal Transduction of Agonist-Stimulated Eel Follicle-Stimulating Hormone Receptor

2020 ◽  
Vol 21 (19) ◽  
pp. 7075
Author(s):  
Munkhzaya Byambaragchaa ◽  
Jeong-Soo Kim ◽  
Hong-Kyu Park ◽  
Dae-Jung Kim ◽  
Sun-Mee Hong ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Cell Surface ◽  
Hormone Receptor ◽  
Follicle Stimulating Hormone ◽  
Chinese Hamster ◽  
Cell Surface Receptor ◽  
Enzyme Linked Immunosorbent Assay ◽  
Wild Type ◽  
Surface Receptor ◽  
Stimulating Hormone

In the present study, we investigated the signal transduction of mutants of the eel follicle-stimulating hormone receptor (eelFSHR). Specifically, we examined the constitutively activating mutant D540G in the third intracellular loop, and four inactivating mutants (A193V, N195I, R546C, and A548V). To directly assess functional effects, we conducted site-directed mutagenesis to generate mutant receptors. We measured cyclic adenosine monophosphate (cAMP) accumulation via homogeneous time-resolved fluorescence assays in Chinese hamster ovary (CHO-K1) cells and investigated cell surface receptor loss using an enzyme-linked immunosorbent assay in human embryonic kidney (HEK) 293 cells. The cells expressing eelFSHR-D540G exhibited a 23-fold increase in the basal cAMP response without agonist treatment. The cells expressing A193V, N195I, and A548V mutants had completely impaired signal transduction, whereas those expressing the R546C mutant exhibited little increase in cAMP responsiveness and a small increase in signal transduction. Cell surface receptor loss in the cells expressing inactivating mutants A193V, R546C, and A548V was clearly slower than in the cell expressing the wild-type eelFSHR. However, cell surface receptor loss in the cells expressing inactivating mutant N195I decreased in a similar manner to that of the cells expressing the wild-type eelFSHR or the activating mutant D540G, despite the completely impaired cAMP response. These results provide important information regarding the structure–function relationships of G protein-coupled receptors during signal transduction.

Interaction of a non-peptide agonist with angiotensin II AT1 receptor mutants

2002 ◽  
Vol 80 (5) ◽  
pp. 413-417 ◽  
Author(s):  
Claudio M Costa-Neto ◽  
Ayumi A Miyakawa ◽  
João B Pesquero ◽  
Laerte Oliveira ◽  
Siv A Hjorth ◽  
...  
Keyword(s):  
Angiotensin Ii ◽  
Inositol Phosphate ◽  
Receptor Activation ◽  
Peptide Ligand ◽  
Wild Type ◽  
Angiotensin Ii Receptor ◽  
Extracellular Region ◽  
Preferential Binding ◽  
Peptide Agonist ◽  
Mutant Forms

To identify residues of the rat AT1A angiotensin II receptor involved with signal transduction and binding of the non-peptide agonist L-162,313 (5,7-dimethyl-2-ethyl-3-[[4-[2(n-butyloxycarbonylsulfonamido)-5-isobutyl-3-thienyl]phenyl]methyl]imidazol[4,5,6]-pyridine) we have performed ligand binding and inositol phosphate turnover assays in COS-7 cells transiently transfected with the wild-type and mutant forms of the receptor. Mutant receptors bore modifications in the extracellular region: T88H, Y92H, G196I, G196W, and D278E. Compound L-162,313 displaced [125I]-Sar1,Leu8-AngII from the mutants G196I and G196W with IC50 values similar to that of the wild-type. The affinity was, however, slightly affected by the D278E mutation and more significantly by the T88H and Y92H mutations. In inositol phosphate turnover assays, the ability of L-162,313 to trigger the activation cascade was compared with that of angiotensin II. These assays showed that the G196W mutant reached a relative maximum activation exceeding that of the wild-type receptor; the efficacy was slightly reduced in the G196I mutant and further reduced in the T88H, Y92H, and D278E mutants. Our data suggest that residues of the extracellular domain of the AT1 receptor are involved in the binding of the non-peptide ligand, or in a general receptor activation phenomenon that involves conformational modifications for a preferential binding of agonists or antagonists. Key words: angiotensin, receptor, GPCR, non-peptide agonist, transduction.

scholarly journals The effects of clathrin inactivation on localization of Kex2 protease are independent of the TGN localization signal in the cytosolic tail of Kex2p.

1996 ◽  
Vol 7 (11) ◽  
pp. 1667-1677 ◽  
Author(s):  
K Redding ◽  
M Seeger ◽  
G S Payne ◽  
R S Fuller
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Heavy Chain ◽  
Intracellular Transport ◽  
Chain Gene ◽  
Wild Type ◽  
Clathrin Heavy Chain ◽  
Localization Signal ◽  
Mutant Forms ◽  
Kex2 Protease

Localization of Kex2 protease (Kex2p) to the yeast trans-Golgi network (TGN) requires a TGN localization signal (TLS) in the Kex2p C-terminal cytosolic tail. Mutation of the TLS accelerates transport of Kex2p to the vacuole by an intracellular (SEC1-independent) pathway. In contrast, inactivation of the clathrin heavy-chain gene CHC1 results in transport of Kex2p and other Golgi membrane proteins to the cell surface. Here, the relationship of the two localization defects was assessed by examining the effects of a temperature-sensitive CHC1 allele on trafficking of wild-type (WT) and TLS mutant forms of Kex2p. Inactivation of clathrin by shifting chc1-ts cells to 37 degrees C caused WT and TLS mutant forms of Kex2p to behave identically. All forms of Kex2p appeared at the plasma membrane within 30-60 min of the temperature shift. TLS mutant forms of Kex2p were stabilized, their half-lives increasing to that of wild-type Kex2p. After inactivation of clathrin heavy chain, vacuolar protease-dependent degradation of all forms of Kex2p was blocked by a sec1 mutation, which is required for secretory vesicle fusion to the plasma membrane, indicating that transport to the cell surface was required for degradation by vacuolar proteolysis. Finally, after clathrin inactivation, all forms of Kex2p were degraded in part by a vacuolar protease-independent pathway. After inactivation of both chc1-ts and sec1-ts, Kex2 was degraded exclusively by this pathway. We conclude that the effects of clathrin inactivation on Kex2p localization are independent of the Kex2p C-terminal cytosolic tail. Although these results neither prove nor rule out a direct interaction between the Kex2 TLS and a clathrin-dependent structure, they do imply that clathrin is required for the intracellular transport of Kex2p TLS mutants to the vacuole.

SH2 domain–mediated targeting, but not localization, of Syk in the plasma membrane is critical for FcεRI signaling

Blood ◽  
2001 ◽  
Vol 97 (5) ◽  
pp. 1352-1359 ◽  
Author(s):  
Kiyonao Sada ◽  
Juan Zhang ◽  
Reuben P. Siraganian
Keyword(s):  
Signal Transduction ◽  
Tyrosine Phosphorylation ◽  
T Cell Activation ◽  
Protein Tyrosine Kinase ◽  
Cell Activation ◽  
Sh2 Domain ◽  
Wild Type ◽  
Downstream Signaling ◽  
Intracellular Signals ◽  
Signaling Events

Aggregation of the high-affinity IgE receptor induces the tyrosine phosphorylation of subunits of the receptor and the subsequent association with the receptor of the cytosolic protein tyrosine kinase Syk. The current experiments examined the functional importance of membrane association of Syk and the role of the SH2 domain in receptor-mediated signal transduction. Wild-type Syk and chimeric Syk molecules with the c-Src myristylation sequence at the amino-terminus were expressed in a Syk-negative mast cell line. Chimeric Syk with the myristylation sequence was membrane associated, and a small fraction was constitutively colocalized with FcεRI, Lyn, and LAT (linker for T-cell activation) in the glycolipid-enriched microdomains or rafts. However, even under these conditions, the tyrosine phosphorylation of Syk and the downstream propagation of signals required FcεRI aggregation. This chimeric Syk was less active than wild-type Syk in FcεRI-mediated signal transduction. In contrast, a truncated membrane-associated form of Syk that lacked the SH2 domains was not tyrosine phosphorylated by receptor aggregation and failed to transduce intracellular signals. These findings suggest that SH2 domain–mediated membrane translocation of Syk is essential for the FcεRI-mediated activation of Syk for downstream signaling events leading to histamine release. Furthermore, the localization of Syk in glycolipid-enriched microdomains by itself is not enough to generate or enhance signaling events.

scholarly journals BPAG1e Maintains Keratinocyte Polarity through β4 Integrin–mediated Modulation of Rac 1 and Cofilin Activities

2009 ◽  
Vol 20 (12) ◽  
pp. 2954-2962 ◽  
Author(s):  
Kevin J. Hamill ◽  
Susan B. Hopkinson ◽  
Philip DeBiase ◽  
Jonathan C.R. Jones
Keyword(s):  
Signal Transduction ◽  
Cell Surface ◽  
Bullous Pemphigoid ◽  
Wild Type ◽  
Expression Levels ◽  
Bullous Pemphigoid Antigen ◽  
Affect Expression ◽  
Α6β4 Integrin ◽  
And Migration ◽  
Β4 Integrin

α6β4 integrin, a component of hemidesmosomes, also plays a role in keratinocyte migration via signaling through Rac1 to the actin-severing protein cofilin. Here, we tested the hypothesis that the β4 integrin-associated plakin protein, bullous pemphigoid antigen 1e (BPAG1e) functions as a scaffold for Rac1/cofilin signal transduction. We generated keratinocyte lines exhibiting a stable knockdown in BPAG1e expression. Knockdown of BPAG1e does not affect expression levels of other hemidesmosomal proteins, nor the amount of β4 integrin expressed at the cell surface. However, the amount of Rac1 associating with β4 integrin and the activity of both Rac1 and cofilin are significantly lower in BPAG1e-deficient cells compared with wild-type keratinocytes. In addition, keratinocytes deficient in BPAG1e exhibit loss of front-to-rear polarity and display aberrant motility. These defects are rescued by inducing expression of constitutively active Rac1 or active cofilin. These data indicate that the BPAG1e is required for efficient regulation of keratinocyte polarity and migration by determining the activation of Rac1.

scholarly journals Increase in cell-surface localization of parathyroid hormone receptor by cytoskeletal protein 4.1G

2005 ◽  
Vol 392 (1) ◽  
pp. 75-81 ◽  
Author(s):  
Masaki Saito ◽  
Maki Sugai ◽  
Yuriko Katsushima ◽  
Teruyuki Yanagisawa ◽  
Jun Sukegawa ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Parathyroid Hormone ◽  
Cell Surface ◽  
Plasma Membranes ◽  
Receptor Activation ◽  
Dominant Negative ◽  
Cytoskeletal Protein ◽  
C Terminus ◽  
Surface Localization ◽  
Cell Surface Localization

The cell-surface localization of GPCRs (G-protein-coupled receptors) has emerged as one of critical factors of the GPCR-mediated signal transduction. It has been reported that the C-termini of GPCRs contain the sequences for sorting the receptors to cell surface. In the present study, we have searched for proteins that interact with the C-terminus of PTH (parathyroid hormone)/PTH-related protein receptor (PTHR) by using the yeast two-hybrid system, and identified a cytoskeletal protein 4.1G (generaltype 4.1 protein) as an interactant with the C-terminus. Immunohistochemical study revealed that both PTHR and 4.1G were co-localized on plasma membranes, when they were transiently expressed in COS-7 cells. When 4.1G or the C-terminal domain of 4.1G (4.1G-CTD), a dominant-negative form of 4.1G, was co-expressed with PTHR in COS-7 cells, 4.1G, but not 4.1G-CTD, facilitated the cell-surface localization of PTHR, determined by cell-surface biotinylation assay. PTH-(1–34) caused phosphorylation of ERK (extracellular-signal-regulated kinase) 1/2 in PTHR-expressed cells mainly mediated through EGF (epidermal growth factor) receptor. The phosphorylation was enhanced by the expression of 4.1G, but not 4.1G-CTD. PTH-(1–34) elevated [Ca2+]i (intracellular Ca2+ concentration) independent of EGF receptor activation, and the elevation was enhanced by the expression of 4.1G, but not 4.1G-CTD. These data indicate that 4.1G facilitates the cell-surface localization of PTHR through its interaction with the C-terminus of the receptor, resulting in the potentiation of PTHR-mediated signal transduction.

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