scholarly journals Cell surface delivery and structural re-organization by pharmacological chaperones of an oligomerization-defective α1b-adrenoceptor mutant demonstrates membrane targeting of GPCR oligomers

2008 ◽  
Vol 417 (1) ◽  
pp. 161-172 ◽  
Author(s):  
Meritxell Canals ◽  
Juan F. Lopez-Gimenez ◽  
Graeme Milligan
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Surface ◽  
Ligand Binding ◽  
Transmembrane Domain ◽  
Early Stage ◽  
Mutant Cell ◽  
Critical Role ◽  
Wild Type ◽  
Pharmacological Chaperones ◽  
Mutant Receptor

Many G-protein-coupled receptors, including the α1b-adrenoceptor, form homo-dimers or oligomers. Mutation of hydrophobic residues in transmembrane domains I and IV alters the organization of the α1b-adrenoceptor oligomer, with transmembrane domain IV playing a critical role. These mutations also result in endoplasmic reticulum trapping of the receptor. Following stable expression of this α1b-adrenoceptor mutant, cell surface delivery, receptor function and structural organization were recovered by treatment with a range of α1b-adrenoceptor antagonists that acted at the level of the endoplasmic reticulum. This was accompanied by maturation of the mutant receptor to a terminally N-glycosylated form, and only this mature form was trafficked to the cell surface. Co-expression of the mutant receptor with an otherwise wild-type form of the α1b-adrenoceptor that is unable to bind ligands resulted in this wild-type variant also being retained in the endoplasmic reticulum. Ligand-induced cell surface delivery of the mutant α1b-adrenoceptor now allowed co-recovery to the plasma membrane of the ligand-binding-deficient mutant. These results demonstrate that interactions between α1b-adrenoceptor monomers occur at an early stage in protein synthesis, that ligands of the α1b-adrenoceptor can act as pharmacological chaperones to allow a structurally compromised form of the receptor to pass cellular quality control, that the mutated receptor is not inherently deficient in function and that an oligomeric assembly of ligand-binding-competent and -incompetent forms of the α1b-adrenoceptor can be trafficked to the cell surface by binding of a ligand to only one component of the receptor oligomer.

Analysis of Structural Signals Conferring Localisation of Pig OST48 to the Endoplasmic Reticulum

2001 ◽  
Vol 382 (7) ◽  
pp. 1039-1047 ◽  
Author(s):  
Birgit Hardt ◽  
Raquel Aparicio ◽  
Wilhelm Breuer ◽  
Ernst Bause
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Surface ◽  
Transmembrane Domain ◽  
Membrane Topology ◽  
Type I ◽  
Catalytic Subunits ◽  
Hybrid Proteins ◽  
Lysine Residues ◽  
Typical Type ◽  
Hydrophobic Transmembrane Domain

Abstract Pig liver oligosaccharyltransferase (OST) is a heterooligomeric protein complex responsible for the cotranslational transfer of GlcNAc[2]Man[9]Glc[3] from Dol PP onto specific asparagine residues in the nascent polypeptide. OST48, one of the catalytic subunits in this complex, exerts a typical type I membrane topology, containing a large luminal domain, a hydrophobic transmembrane domain and a short cytosolic peptide tail. Because OST48 is found within the endoplasmic reticulum (ER) when overexpressed in COS-1 cells, we carried out experiments to identify structural signals potentially capable of directing ERtargeting, using OST48 mutants and hybrid proteins consisting of individual OST48 domains and Man[9] mannosidase. Immunofluorescence microscopy showed that OST48 mutants in which the Cterminal lysine-3 or lysine-5, but not lysine-7, had been replaced by leucine (OST48?K) could be detected on the cell surface. This indicates that these two lysine residues are sufficient for conferring ERresidency on OST48. The doublelysine motif operates only when exposed cytosolically, where it acts as a relocation signal rather than causing retention. OST48?K-3, when coexpressed in COS-1 cells together with myctagged ribophorin I, was quantitatively retained in the ER. By contrast, coexpression in the presence of ribophorin I resulted in no reduction of cell surface fluorescence for the OMO?K-5 chimera containing the cytosolic and transmembrane domain of OST48 attached to the Cterminus of the Man[9]mannosidase luminal domain. Thus ERlocalisation of OST48 is probably brought about by complex formation with ribophorin I and this most likely involves the luminal domains of both proteins. Consequently, the doublelysine motif in the cytosolic domain of OST48 is unlikely to have a primary function except being involved in recapture of molecules which have escaped from the ER.

scholarly journals Mutations of the Rous sarcoma virus env gene that affect the transport and subcellular location of the glycoprotein products.

1984 ◽  
Vol 99 (6) ◽  
pp. 2011-2023 ◽  
Author(s):  
J W Wills ◽  
R V Srinivas ◽  
E Hunter
Keyword(s):  
Endoplasmic Reticulum ◽  
Amino Acid ◽  
Golgi Apparatus ◽  
Cell Surface ◽  
Nuclear Membrane ◽  
Rous Sarcoma Virus ◽  
Cytoplasmic Domain ◽  
Wild Type ◽  
Rous Sarcoma ◽  
Sarcoma Virus

The envelope glycoproteins of Rous sarcoma virus (RSV), gp85 and gp37, are anchored in the membrane by a 27-amino acid, hydrophobic domain that lies adjacent to a 22-amino acid, cytoplasmic domain at the carboxy terminus of gp37. We have altered these cytoplasmic and transmembrane domains by introducing deletion mutations into the molecularly cloned sequences of a proviral env gene. The effects of the mutations on the transport and subcellular localization of the Rous sarcoma virus glycoproteins were examined in monkey (CV-1) cells using an SV40 expression vector. We found, on the one hand, that replacement of the nonconserved region of the cytoplasmic domain with a longer, unrelated sequence of amino acids (mutant C1) did not alter the rate of transport to the Golgi apparatus nor the appearance of the glycoprotein on the cell surface. Larger deletions, extending into the conserved region of the cytoplasmic domain (mutant C2), resulted in a slower rate of transport to the Golgi apparatus, but did not prevent transport to the cell surface. On the other hand, removal of the entire cytoplasmic and transmembrane domains (mutant C3) did block transport and therefore did not result in secretion of the truncated protein. Our results demonstrate that the C3 polypeptide was not transported to the Golgi apparatus, although it apparently remained in a soluble, nonanchored form in the lumen of the rough endoplasmic reticulum; therefore, it appears that this mutant protein lacks a functional sorting signal. Surprisingly, subcellular localization by internal immunofluorescence revealed that the C3 protein (unlike the wild type) did not accumulate on the nuclear membrane but rather in vesicles distributed throughout the cytoplasm. This observation suggests that the wild-type glycoproteins (and perhaps other membrane-bound or secreted proteins) are specifically transported to the nuclear membrane after their biosynthesis elsewhere in the rough endoplasmic reticulum.

A GPI-Anchored Co-Receptor for TFPI Controls Its Intracellular Trafficking and Endothelial Surface Expression.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1927-1927
Author(s):  
Anna C. Cunningham ◽  
Charles M. Mansbach ◽  
Alan E. Mast
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Surface ◽  
Tissue Factor ◽  
Cell Lines ◽  
Surface Expression ◽  
Resistant Cell ◽  
Wild Type ◽  
Endothelial Surface ◽  
Cellular Processing ◽  
Resistant Cells

Abstract Tissue factor pathway inhibitor (TFPI) is the major endogenous inhibitor of tissue factor initiated blood coagulation and a is key regulator of the development of intravascular thrombosis. TFPI indirectly binds to the endothelial surface through tight association with a GPI-anchored co-receptor. The location of recombinant TFPI expression in mammalian cells varies depending on the cell line used. In cell lines that do not produce endogenous TFPI, CHO and HEK293 cells, recombinant TFPI is secreted into the culture media. However, in a cell line that produces endogenous surface TFPI, EaHy926 cells, recombinant TFPI is expressed on the cell surface. These data suggest that TFPI is expressed on the surface of cells that produce the GPI-anchored co-receptor and is secreted by cells that do not. To further investigate the function of the co-receptor in TFPI cellular trafficking we developed aerolysin resistant ECV304 and EaHy926 cells lines. Both of these cell lines produce endogenous, surface associated TFPI. The cell lines were mutated with ethyl methanesulfonate and selected with aerolysin. Mutant cells lacking surface GPI-anchored proteins are resistant to the toxic effects of aerolysin and survive. The morphology and growth rate of the two aerolysin resistant cell lines are identical to that of the wild-type cells. They were first characterized to rule out the presence of a mutation that could directly alter cellular metabolism of TFPI. Sequencing of TFPI cDNA indicates that no mutations are present in the TFPI exons. Analysis of mRNA by real time PCR demonstrates that the aerolysin resistant cells make similar amounts of TFPI mRNA as their wild-type counterparts. Thus, transcription and translation of TFPI appear identical to the wild-type cells. In addition, the two independently derived cell lines have very similar phenotypes, as described below, indicating that the aerolysin resistant cell lines have a defect in GPI-anchor biosynthesis but not additional random mutations that could alter cellular processing of TFPI. Characterization of protein expression by flow cytometry indicates that the aerolysin resistant cell lines do not express GPI-anchored proteins (CD59, uPAR) or TFPI on the cell surface but do have wild-type surface expression of transmembrane proteins (CD9, tissue factor). Interestingly, instead of being secreted, western blot analysis of cellular lysates indicates that TFPI is degraded within the aerolysin resistant cells in a manner similar to that observed for GPI-anchored proteins. Intracellular degradation of TFPI is prevented by brefeldin A indicating that degradation takes place in a post endoplasmic reticulum compartment. Pepstatin A, but not MG-132, also prevents degradation, indicating that degradation is lysosomal rather than proteosomal. It appears that binding of TFPI to its co-receptor occurs early in cellular processing, likely within the endoplasmic reticulum. Cellular trafficking of TFPI is controlled by its co-receptor, which has not yet been identified. The co-receptor directs TFPI to the cell surface in wild-type endothelial cells or to be degraded in aerolysin resistant cells. In the absence if its co-receptor TFPI is secreted. Therefore, regulation of co-receptor expression provides a mechanism for the production of cell associated TFPI, as occurs in endothelial cells, versus soluble TFPI, as may occur in megakaryocytes/platelets.

scholarly journals Mutations in Multiple Domains Activate Paramyxovirus F Protein-Induced Fusion

2004 ◽  
Vol 78 (16) ◽  
pp. 8513-8523 ◽  
Author(s):  
Shaguna Seth ◽  
Andrew L. Goodman ◽  
Richard W. Compans
Keyword(s):  
Amino Acid ◽  
Transmembrane Domain ◽  
Critical Role ◽  
Syncytium Formation ◽  
Simian Virus ◽  
Wild Type ◽  
F Protein ◽  
Simian Virus 5 ◽  
Partial Suppression ◽  
Multiple Domains

ABSTRACT SER virus, a paramyxovirus that is closely related to simian virus 5 (SV5), is unusual in that it fails to induce syncytium formation. The SER virus F protein has an unusually long cytoplasmic tail (CT), and it was previously observed that truncations or specific mutations of this domain result in enhanced syncytium formation. In addition to the long CT, the SER F protein has nine amino acid differences from the F protein of SV5. We previously observed only a partial suppression of fusion in a chimeric SV5 F protein with a CT derived from SER virus, indicating that these other amino acid differences between the SER and SV5 F proteins also play a role in regulating the fusion phenotype. To examine the effects of individual amino acid differences, we mutated the nine SER residues individually to the respective residues of the SV5 F protein. We found that most of the mutants were expressed well and were transported to the cell surface at levels comparable to that of the wild-type SER F protein. Many of the mutants showed enhanced lipid mixing, calcein transfer, and syncytium formation even in the presence of the long SER F protein CT. Some mutants, such as the I310 M, T438S, M489I, T516V, and N529K mutants, also showed fusion at lower temperatures of 32, 25, and 18°C. The residue Asn529 plays a critical role in the suppression of fusion activity, as the mutation of this residue to lysine caused a marked enhancement of fusion. The effect of the N529K mutation on the enhancement of fusion by a previously described mutant, L539,548A, as well as by chimeric SV5/SER F proteins was also dramatic. These results indicate that activation to a fusogenic conformation is dependent on the interplay of residues in the ectodomain, the transmembrane domain, and the CT domain of paramyxovirus F proteins.

scholarly journals The chemical chaperone CFcor-325 repairs folding defects in the transmembrane domains of CFTR-processing mutants

2006 ◽  
Vol 395 (3) ◽  
pp. 537-542 ◽  
Author(s):  
Tip W. Loo ◽  
M. Claire Bartlett ◽  
Ying Wang ◽  
David M. Clarke
Keyword(s):  
Cystic Fibrosis ◽  
Endoplasmic Reticulum ◽  
Cell Surface ◽  
Hot Spot ◽  
Cross Linking ◽  
Cysteine Residues ◽  
Transmembrane Conductance Regulator ◽  
Wild Type ◽  
Transmembrane Conductance ◽  
Chemical Chaperone

Most patients with CF (cystic fibrosis) express a CFTR [CF TM (transmembrane) conductance regulator] processing mutant that is not trafficked to the cell surface because it is retained in the endoplasmic reticulum due to altered packing of the TM segments. CL4 (cytoplasmic loop 4) connecting TMs 10 and 11 is a ‘hot-spot’ for CFTR processing mutations. The chemical chaperone CFcor-325 (4-cyclohexyloxy-2-{1-[4-(4-methoxy-benezenesulphonyl)piperazin-1-yl]-ethyl}-quinazoline) rescued most CL4 mutants. To test if CFcor-325 promoted correct folding of the TMDs (TM domains), we selected two of the CL4 mutants (Q1071P and H1085R) for disulphide cross-linking analysis. Pairs of cysteine residues that were cross-linked in mature wild-type CFTR were introduced into mutants Q1071P and H1085R. The cross-linking patterns of the Q1071P or H1085R double cysteine mutants rescued with CFcor-325 were similar to those observed with mature wild-type double cysteine proteins. These results show that CFcor-325 rescued CFTR mutants by repairing the folding defects in the TMDs.

scholarly journals Microneme Rhomboid Protease TgROM1 Is Required for Efficient Intracellular Growth of Toxoplasma gondii

2008 ◽  
Vol 7 (4) ◽  
pp. 664-674 ◽  
Author(s):  
Fabien Brossier ◽  
G. Lucas Starnes ◽  
Wandy L. Beatty ◽  
L. David Sibley
Keyword(s):  
Toxoplasma Gondii ◽  
Cell Invasion ◽  
Secretory Pathway ◽  
Transmembrane Domain ◽  
Critical Role ◽  
Host Cells ◽  
Wild Type ◽  
Intracellular Growth ◽  
Adhesive Proteins

ABSTRACT Rhomboids are serine proteases that cleave their substrates within the transmembrane domain. Toxoplasma gondii contains six rhomboids that are expressed in different life cycle stages and localized to different cellular compartments. Toxoplasma rhomboid protein 1 (TgROM1) has previously been shown to be active in vitro, and the orthologue in Plasmodium falciparum processes the essential microneme protein AMA1 in a heterologous system. We investigated the role of TgROM1 to determine its role during in vitro growth of T. gondii. TgROM1 was localized in the secretory pathway of the parasite, including the Golgi apparatus and micronemes, which contain adhesive proteins involved in invasion of host cells. However, unlike other micronemal proteins, TgROM1 was not released onto the parasite surface during cell invasion, suggesting it does not play a critical role in cell invasion. Suppression of TgROM1 using the tetracycline-regulatable system revealed that ROM1-deficient parasites were outcompeted by wild-type T. gondii. ROM1-deficient parasites showed only modest decrease in invasion but replicated more slowly than wild-type cells. Collectively, these results indicate that ROM1 is required for efficient intracellular growth by T. gondii.

scholarly journals Endoplasmic Reticulum (ER) Reorganization and Intracellular Retention of CD58 Are Functionally Independent Properties of the Human Cytomegalovirus ER-Resident Glycoprotein UL148

2019 ◽  
Vol 94 (5) ◽  
Author(s):  
Christopher C. Nguyen ◽  
Anthony J. Domma ◽  
Hongbo Zhang ◽  
Jeremy P. Kamil
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Surface ◽  
Human Cytomegalovirus ◽  
Stress Responses ◽  
Immune Cell ◽  
Functional Divergence ◽  
Ectopic Expression ◽  
Viral Gene ◽  
Wild Type ◽  
Viral Glycoprotein

ABSTRACT The human cytomegalovirus (HCMV) endoplasmic reticulum (ER)-resident glycoprotein UL148 is posited to play roles in immune evasion and regulation of viral cell tropism. UL148 prevents cell surface presentation of the immune cell costimulatory ligand CD58 while promoting maturation and virion incorporation of glycoprotein O, a receptor binding subunit for an envelope glycoprotein complex involved in entry. Meanwhile, UL148 activates the unfolded protein response (UPR) and causes large-scale reorganization of the ER. In order to determine whether the seemingly disparate effects of UL148 are related or discrete, we generated six charged cluster-to-alanine (CCTA) mutants within the UL148 ectodomain and compared them to wild-type UL148, both in the context of infection studies using recombinant viruses and in ectopic expression experiments, assaying for effects on ER remodeling and CD58 surface presentation. Two mutants, targeting charged clusters spanning residues 79 to 83 (CC3) and 133 to 136 (CC4), retained the potential to impede CD58 surface presentation. Of the six mutants, only CC3 retained the capacity to reorganize the ER, but it showed a partial phenotype. Wild-type UL148 accumulates in a detergent-insoluble form during infection. However, all six CCTA mutants were fully soluble, which implies a relationship between insolubility and organelle remodeling. Additionally, we found that the chimpanzee cytomegalovirus UL148 homolog suppresses surface presentation of CD58 but fails to reorganize the ER, while the homolog from rhesus cytomegalovirus shows neither activity. Collectively, our findings illustrate various degrees of functional divergence between homologous primate cytomegalovirus immunevasins and suggest that the capacity to cause ER reorganization is unique to HCMV UL148. IMPORTANCE In myriad examples, viral gene products cause striking effects on cells, such as activation of stress responses. It can be challenging to decipher how such effects contribute to the biological roles of the proteins. The HCMV glycoprotein UL148 retains CD58 within the ER, thereby preventing it from reaching the cell surface, where it functions to stimulate cell-mediated antiviral responses. Intriguingly, UL148 also triggers the formation of large, ER-derived membranous structures and activates the UPR, a set of signaling pathways involved in adaptation to ER stress. We demonstrate that the potential of UL148 to reorganize the ER and to retain CD58 are separable by mutagenesis and, possibly, by evolution, since chimpanzee cytomegalovirus UL148 retains CD58 but does not remodel the ER. Our findings imply that ER reorganization contributes to other roles of UL148, such as modulation of alternative viral glycoprotein complexes that govern the virus’ ability to infect different cell types.

scholarly journals Determinants of thetrans-Dominant Negative Effect of Truncated Forms of the CCR5 Chemokine Receptor

2001 ◽  
Vol 276 (50) ◽  
pp. 46975-46982 ◽  
Author(s):  
Maurice Chelli ◽  
Marc Alizon
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Surface ◽  
Chemokine Receptor ◽  
Transmembrane Domains ◽  
Viral Envelope ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Wild Type ◽  
Ccr5 Chemokine Receptor ◽  
Hiv 1

The human immunodeficiency virus, type 1 (HIV-1) entry process is triggered by interaction between the viral envelope and a seven membrane-spanning domain receptor at the cell surface, usually the CCR5 chemokine receptor. Different naturally occurring mutations in theCCR5gene abolish receptor function, the most frequent being a 32-nucleotide deletion resulting in a truncated protein (Δ32) lacking the last three transmembrane domains (TM5–7). This mutant is retained in the endoplasmic reticulum and exerts atrans-dominant negative (TDN) effect on the wild type, preventing its exit from this compartment. This TDN effect is often considered as evidence for the oligomerization of CCR5 during transport to the cell surface. Here we use a genetic approach to define the structural determinants of the TDN effect of the Δ32 mutant. It was abolished by certain deletions and by mutations of cysteine residues preventing formation of a disulfide link between the first and second extracellular loops, suggesting that conformation of Δ32 is important for its interaction with CCR5. To circumvent this problem, we used chimeric forms of the Δ32 and wild type CCR5, consisting in substitutions with homologous domains from the mouse CCR5. All chimeric full-length receptors were expressed at the cell surface and were functional for interaction with HIV-1 or with a chemokine ligand, when assayed. The TDN effect was only observed if both the TM3 domain in CCR5 and the TM4 domain in Δ32 were from human origin, whereas the rest of the proteins could be from either origin. This suggests that the TDN effect involves some form of interaction between these transmembrane domains. Alternatively, but less likely to us, substitutions in TM4 could affect the conformation of CCR5 in the endoplasmic reticulum but not at the cell surface. However that may be, it seems that the TDN effect of the Δ32 mutant has no bearing to the issue of CCR5 dimerization and to its possible role in the processing of the receptor to the cell surface.

A possible role for stable microtubules in intracellular transport from the endoplasmic reticulum to the Golgi apparatus

1994 ◽  
Vol 107 (5) ◽  
pp. 1321-1331 ◽  
Author(s):  
M. Mizuno ◽  
S.J. Singer
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Transfer Process ◽  
Intracellular Transport ◽  
Early Stage ◽  
Critical Role ◽  
Treatment Protocol ◽  
Secretory Proteins ◽  
Random Diffusion

The intracellular transport of secretory proteins involves at an early stage the formation of vesicles from transitional elements of the endoplasmic reticulum (ER) containing these proteins and the transfer of these vesicles to the cis-face of the Golgi apparatus. We propose that the latter transfer process does not occur by random diffusion, but is instead mediated by tracking along stable microtubules. To test this proposal, we have carried out double immunoelectron microscopic labeling experiments on frozen sections of HepG2 hepatoma cells secreting the protein human serum albumin (HSA). By a cycloheximide treatment protocol, the stage during which the transfer of newly synthesized HSA from the ER to the Golgi apparatus occurs in vivo was determined. Sections of the cells were then double immunolabeled using primary antibodies to HSA and to glu-tubulin, the latter specifically detecting stable microtubules. We observed a significantly high frequency of HSA-containing structures between the ER and the Golgi apparatus with which stable microtubules were closely associated. These results support the proposal that stable microtubules may play a critical role in directing the transfer process from the ER to the Golgi apparatus.

scholarly journals Localization of T25 glycoprotein in wild-type and Thy 1- mutant cells by immunofluorescence and immunoelectron microscopy.

1978 ◽  
Vol 147 (5) ◽  
pp. 1348-1354 ◽  
Author(s):  
L Y Bourguignon ◽  
R Hyman ◽  
I Trowbridge ◽  
S J Singer
Keyword(s):  
Cell Surface ◽  
Cell Line ◽  
Immunoelectron Microscopy ◽  
Mutant Cell ◽  
Wild Type Cell ◽  
Wild Type ◽  
Mutant Cell Line ◽  
Mutant Derivative ◽  
Mutant Cells

The wild-type BW5147 (Thy 1+) cell line and its Thy 1- mutant derivative BW5147 (Thy 1-a) were examined by immunofluorescence and immunoelectron microscopy for the presence of T25, the glycoprotein which bears the Thy 1 alloantigen. The wild-type cell had T25 predominantly localized on the cell surface. In the mutant cell line, T25 accumulated intracellularly and was present in a clustered distribution throughout the cytoplasm. T25 was not present on the surface of the mutant cell line in significant amount.

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