scholarly journals Lysosome-associated protein transmembrane 4α (LAPTM4α) requires two tandemly arranged tyrosine-based signals for sorting to lysosomes

2002 ◽  
Vol 365 (3) ◽  
pp. 721-730 ◽  
Author(s):  
Douglas L. HOGUE ◽  
Colin NASH ◽  
Victor LING ◽  
Tom C. HOBMAN
Keyword(s):  
Cultured Cells ◽  
Chemotherapeutic Agents ◽  
P Glycoprotein ◽  
C Terminus ◽  
Sorting Signals ◽  
Suitable Target ◽  
Membrane Spanning ◽  
High Level ◽  
Membrane Spanning Domains ◽  
Intracellular Compartmentalization

Lysosome-associated protein transmembrane 4α (LAPTM4α) and homologues comprise a family of conserved proteins, which are found in mammals, insects and nematodes. LAPTM4α functions to regulate the intracellular compartmentalization of amphipathic solutes and possibly the sensitivity of cells toward anthracyclines, antibiotics, ionophores, nucleobases and organic cations. This is similar to the multidrug-resistance phenotype exhibited by cells synthesizing high levels of P-glycoprotein. Accordingly, it is possible that LAPTM4α may be a suitable target for development of novel chemotherapeutic agents. LAPTM4α contains four putative membrane-spanning domains and a 55 amino acid C-terminal region that faces the cytoplasm. Localization of LAPTM4α to endosomes and lysosomes appears to be tightly controlled as transient high-level expression of LAPTM4α in cultured cells resulted in no detectable protein on the cell surface. Mutagenic analysis of the C-terminus of LAPTM4α indicated that two tandomly arranged tyrosine-containing motifs in the cytoplasmic domain are required for efficient localization of LAPTM4α to vesicles containing the lysosomal marker lysosomal glycoprotein 120. Although a number of membrane proteins that localize to endosomes/lysosomes contain more than one independently functioning sorting signal, to our knowledge, LAPTM4α is the first example of a membrane protein that requires two tandemly arranged tyrosine-based sorting signals for efficient localization in these compartments.

scholarly journals Analysis of the interactions between the C-terminal cytoplasmic domains of KCNQ1 and KCNE1 channel subunits

2010 ◽  
Vol 428 (1) ◽  
pp. 75-84 ◽  
Author(s):  
Renjian Zheng ◽  
Keith Thompson ◽  
Edmond Obeng-Gyimah ◽  
Dana Alessi ◽  
Jerri Chen ◽  
...  
Keyword(s):  
Cultured Cells ◽  
Proximal Region ◽  
Physical Interaction ◽  
Interaction Domain ◽  
Α Subunit ◽  
C Terminus ◽  
Tetramerization Domain ◽  
K Current ◽  
Membrane Spanning ◽  
Lqt Syndrome

Ion channel subunits encoded by KCNQ1 and KCNE1 produce the slowly activating K+ current (IKs) that plays a central role in myocardial repolarization. The KCNQ1 α-subunit and the KCNE1 β-subunit assemble with their membrane-spanning segments interacting, resulting in transformation of channel activation kinetics. We recently reported a functional interaction involving C-terminal portions of the two subunits with ensuing regulation of channel deactivation. In the present study, we provide evidence characterizing a physical interaction between the KCNQ1-CT (KCNE1 C-terminus) and the KCNE1-CT (KCNE1 C-terminus). When expressed in cultured cells, the KCNE1-CT co-localized with KCNQ1, co-immunoprecipitated with KCNQ1 and perturbed deactivation kinetics of the KCNQ1 currents. Purified KCNQ1-CT and KCNE1-CT physically interacted in pull-down experiments, indicating a direct association. Deletion analysis of KCNQ1-CT indicated that the KCNE1-CT binds to a KCNQ1 region just after the last transmembrane segment, but N-terminal to the tetramerization domain. SPR (surface plasmon resonance) corroborated the pull-down results, showing that the most proximal region (KCNQ1 amino acids 349–438) contributed most to the bimolecular interaction with a dissociation constant of ~4 μM. LQT (long QT) mutants of the KCNE1-CT, D76N and W87F, retained binding to the KCNQ1-CT with comparable affinity, indicating that these disease-causing mutations do not alter channel behaviour by disruption of the association. Several LQT mutations involving the KCNQ1-CT, however, showed various effects on KCNQ1/KCNE1 association. Our results indicate that the KCNQ1-CT and the KCNE1-CT comprise an independent interaction domain that may play a role in IKs channel regulation that is potentially affected in some LQTS (LQT syndrome) mutations.

N-glycosylation analysis of the human Tweety family of putative chloride ion channels supports a penta-spanning membrane arrangement: impact of N-glycosylation on cellular processing of Tweety homologue 2 (TTYH2)

2008 ◽  
Vol 412 (1) ◽  
pp. 45-55 ◽  
Author(s):  
Yaowu He ◽  
Andrew J. Ramsay ◽  
Melanie L. Hunt ◽  
Astrid K. Whitbread ◽  
Stephen A. Myers ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Chloride Ion ◽  
Glycosylation Site ◽  
C Terminus ◽  
Cl Channels ◽  
Cellular Processing ◽  
Site Mutagenesis ◽  
Membrane Spanning ◽  
Chloride Ion Channels ◽  
Membrane Spanning Domains

The Tweety proteins are a family of recently identified putative Cl− channels predicted to be modified by N-glycosylation and, controversially, to contain five or six membrane-spanning domains, leading to the contentious proposal that members of this family do not share the same topology at the plasma membrane. In humans, three family members have been identified, designated TTYH1 (Tweety homologue 1), TTYH2 and TTYH3. To gain greater insight into the arrangement of membrane-spanning domains and cellular processing of Tweety proteins, in the present study we have examined the sequence homology, hydrophobicity and N-glycan content of members of this family and performed N-glycosylation site-mutagenesis studies on TTYH2 and TTYH3. Based on these observations we propose a structure for Tweety family proteins which incorporates five membrane-spanning domains with a topology at the cell surface in which the N-terminus is located extracellularly and the C-terminus cytoplasmically. Our results also suggest that N-glycosylation is important, but not essential, in the processing of members of the Tweety family with results indicating that, although incomplete N-glycosylation mediates reduced expression and increased ubiquitination of TTYH2, N-glycosylation is not the determining factor for TTYH2 trafficking to the plasma membrane. This information will be important for the characterization of Tweety family proteins in normal physiology and disease.

scholarly journals GIT1, a Gene Encoding a Novel Transporter for Glycerophosphoinositol in Saccharomyces cerevisiae

Genetics ◽  
1998 ◽  
Vol 149 (4) ◽  
pp. 1707-1715 ◽  
Author(s):  
J L Patton-Vogt ◽  
S A Henry
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Regulatory Gene ◽  
Sugar Transport ◽  
Open Reading Frame ◽  
Gene Encoding ◽  
Reading Frame ◽  
Membrane Spanning ◽  
Membrane Spanning Domains ◽  
Uptake And Metabolism ◽  
Cells Cultured

Abstract Phosphatidylinositol catabolism in Saccharomyces cerevisiae cells cultured in media containing inositol results in the release of glycerophosphoinositol (GroPIns) into the medium. As the extracellular concentration of inositol decreases with growth, the released GroPIns is transported back into the cell. Exploiting the ability of the inositol auxotroph, ino1, to use exogenous GroPIns as an inositol source, we have isolated mutants (Git−) defective in the uptake and metabolism of GroPIns. One mutant was found to be affected in the gene encoding the transcription factor, SPT7. Mutants of the positive regulatory gene INO2, but not of its partner, INO4, also have the Git− phenotype. Another mutant was complemented by a single open reading frame (ORF) termed GIT1 (glycerophosphoinositol). This ORF consists of 1556 bp predicted to encode a polypeptide of 518 amino acids and 57.3 kD. The predicted Git1p has similarity to a variety of S. cerevisiae transporters, including a phosphate transporter (Pho84p), and both inositol transporters (Itr1p and Itr2p). Furthermore, Git1p contains a sugar transport motif and 12 potential membrane-spanning domains. Transport assays performed on a git1 mutant together with the above evidence indicate that the GIT1 gene encodes a permease involved in the uptake of GroPIns.

scholarly journals Structure and expression of human IgG FcRII(CD32). Functional heterogeneity is encoded by the alternatively spliced products of multiple genes.

1989 ◽  
Vol 170 (4) ◽  
pp. 1369-1385 ◽  
Author(s):  
D G Brooks ◽  
W Q Qiu ◽  
A D Luster ◽  
J V Ravetch
Keyword(s):  
Structural Heterogeneity ◽  
Amino Acid Sequences ◽  
Cdna Clones ◽  
Membrane Glycoproteins ◽  
Igg Binding ◽  
Genes Expression ◽  
Alternatively Spliced ◽  
Membrane Spanning ◽  
High Degree ◽  
Membrane Spanning Domains

The structural heterogeneity of the human low affinity receptor for IgG, FcRII(CD32), has been elucidated through the isolation, characterization, and expression of cDNA clones derived from myeloid and lymphoid RNA. These clones predict amino acid sequences consistent with integral membrane glycoproteins with single membrane spanning domains. The extracellular domains display sequence homology to other Fc gamma Rs and members of the Ig supergene family. A minimum of three genes (Fc gamma RIIa, IIa', and Fc gamma RIIb) encode these transcripts, which demonstrate highly related extracellular and membrane spanning domains. IIa/IIa' differ substantially in the intracytoplasmic domain from IIb. Alternative splicing of the IIb gene generates further heterogeneity in both NH2- and COOH-terminal domains of the predicted proteins. Comparison to the murine homologues of these molecules reveals a high degree of conservation between the products of one of these genes, Fc gamma RIIb, and the murine beta gene in primary sequence, splicing pattern, and tissue distribution. In contrast, the sequence of IIa' indicates its relationship to the beta-like genes, with mutation giving rise to a novel cytoplasmic domain, while IIa is a chimera of both alpha- and beta-like genes. Expression of these cDNA molecules by transfection results in the appearance of IgG binding molecules that bear the epitopes defined by the FcRII(CD32) mAbs previously described.

NHE3 contains a cleavable signal peptide followed by 11 membrane spanning domains: New insights into topology

2000 ◽  
Vol 118 (4) ◽  
pp. A871
Author(s):  
Mirza Zizak ◽  
M E Cavet ◽  
D. Bayle ◽  
C M Tse ◽  
S. Hallen ◽  
...  
Keyword(s):  
Signal Peptide ◽  
Membrane Spanning ◽  
Membrane Spanning Domains

scholarly journals Synthesis and Biological Evaluation of New 3,4-diarylmaleimides as Enhancers (modulators) of Doxorubicin Cytotoxic Activity on Cultured Tumor Cells from a Real Case of Breast Cancer

2017 ◽  
Vol 61 (1) ◽  
Author(s):  
Jessica R. Gutierrez-Cano ◽  
Pradip D. Nahide ◽  
Velayudham Ramadoss ◽  
Yuvraj Satkar ◽  
Rafael Ortiz-Alvarado ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cultured Cells ◽  
Biological Evaluation ◽  
Doxorubicin Treatment ◽  
P Glycoprotein ◽  
Molecular Action ◽  
Mortality Effect ◽  
Kinase Phosphorylation ◽  
Synthesis And Biological Evaluation ◽  
Cultured Tumor Cells

<p>A series of new 3,4-diarylmaleimides were synthesized in an optimized and efficient lineal sequence of three steps, starting from commercial maleimide. The biological evaluation of these compounds as enhancers (activity modulators) in the co-administration with doxorubicin treatment in breast cancer cells directly obtained from a patient, were essayed. The cancerous tissue BT026-512N was provided by the National Institute of Cancerology (INCAN) of México. This tissue was obtained by biopsy from a patient diagnosed with stage IIB ductal breast cancer. The results obtained in the assays, show decreased cell viability on the cultured cells for all of the maleimides synthesized in combinatorial administration with doxorubicin. The highest mortality effect was determined for maleimides <strong>9</strong> and <strong>29</strong> increased in close to three times the effect compared with treatment using only doxorubicin. Based on previous functionalized maleimides core reports and Molinspiration chemoinformatic analysis, these results could possibly point out to the Pg-p glycoprotein as bio-molecular action target of maleimides by kinase phosphorylation-inhibition, although more experimental data is necessary.</p>

Reversal of chemoresistance in malignant gliomas by calcium antagonists: correlation with the expression of multidrug-resistant p-glycoprotein

1994 ◽  
Vol 81 (4) ◽  
pp. 587-594 ◽  
Author(s):  
Jurgen Carl Walther Kiwit ◽  
Anja Hertel ◽  
Alexander E. Matuschek
Keyword(s):  
Calcium Antagonists ◽  
Malignant Gliomas ◽  
Multidrug Resistant ◽  
Chemotherapeutic Agents ◽  
Positive Staining ◽  
Glycoprotein P ◽  
Content Type ◽  
P Glycoprotein ◽  
Multiple Drugs

✓ Resistance to multiple drugs is often observed in malignant gliomas. The authors used a microtiter tetrazolium test to analyze primary in vitro chemoresistance and chemosensitivity of 15 early cultures of human malignant glioma exposed to 50 µg/ml (1,4-amino-2-methyl-5-pyrimidinyl)-methyl-3-(2-chloroethyl)-3-nitrosourea (ACNU), 50 µg/ml cisplatin, 1 µg/ml vincristine, or combinations of these chemotherapeutic agents. Primary chemoresistance was observed in 87% of tumors for ACNU, in 87% for cisplatin, and in 83% for vincristine. All tumors were examined for expression of multidrug-resistant p-glycoprotein, a transport protein of 170,000 D, by means of immunohistochemical staining with the JSB-1 antibody on paraffinized tumor sections. Eight of 15 specimens (53%) showed positive staining for the monoclonal antibody. Primary chemoresistance was overcome by addition of the calcium antagonists verapamil or nimodipine to the cultures if the original tumor expressed p-glycoprotein (p < 0.01 for verapamil, p < 0.05 for nimodipine). In tumors not expressing p-glycoprotein, addition of calcium antagonists to the cell cultures did not influence primary chemoresistance. It is concluded from these data that addition of calcium antagonists to the adjuvant chemotherapy of malignant gliomas might overcome primary chemoresistance in tumors expressing the multidrugresistant phenotype.

scholarly journals Molecular cloning, characterization, subcellular localization and dynamics of p23, the mammalian KDEL receptor.

1993 ◽  
Vol 120 (2) ◽  
pp. 325-338 ◽  
Author(s):  
B L Tang ◽  
S H Wong ◽  
X L Qi ◽  
S H Low ◽  
W Hong
Keyword(s):  
Golgi Apparatus ◽  
Brefeldin A ◽  
Cell Types ◽  
In Vitro Translation ◽  
Tubular Structures ◽  
Human Sequence ◽  
Intermediate Compartment ◽  
Membrane Spanning ◽  
Membrane Spanning Domains

We have isolated a cDNA clone (mERD2) for the mammalian (bovine) homologue of the yeast ERD2 gene, which codes for the yeast HDEL receptor. The deduced amino acid sequence bears extensive homology to its yeast counterpart and is almost identical to a previously described human sequence. The sequence predicts a very hydrophobic protein with multiple membrane spanning domains, as confirmed by analysis of the in vitro translation product. The protein encoded by mERD2 (p23) has widespread occurrence, being present in all the cell types examined. p23 was localized to the cis-side of the Golgi apparatus and to a spotty intermediate compartment which mediates ER to Golgi transport. A majority of the intracellular staining could be accumulated in the intermediate compartment by a low temperature (15 degrees C) or brefeldin A. During recovery from these treatments, the spotty intermediate compartment staining of p23 was shifted to the perinuclear staining of the Golgi apparatus and tubular structures marked by p23 were observed. These tubular structures may serve to mediate transport between the intermediate compartment and the Golgi apparatus.

scholarly journals Coupled Translocation Events Generate Topological Heterogeneity at the Endoplasmic Reticulum Membrane

1998 ◽  
Vol 9 (9) ◽  
pp. 2681-2697 ◽  
Author(s):  
Kenneth Moss ◽  
Andrew Helm ◽  
Yun Lu ◽  
Alvina Bragin ◽  
William R. Skach
Keyword(s):  
Endoplasmic Reticulum ◽  
Structural Features ◽  
Endoplasmic Reticulum Membrane ◽  
Type I ◽  
Type Ii ◽  
Hydrophobic Residues ◽  
P Glycoprotein ◽  
C Terminus ◽  
Signal Anchor ◽  
Hydrophilic Residues

Topogenic determinants that direct protein topology at the endoplasmic reticulum membrane usually function with high fidelity to establish a uniform topological orientation for any given polypeptide. Here we show, however, that through the coupling of sequential translocation events, native topogenic determinants are capable of generating two alternate transmembrane structures at the endoplasmic reticulum membrane. Using defined chimeric and epitope-tagged full-length proteins, we found that topogenic activities of two C-trans (type II) signal anchor sequences, encoded within the seventh and eighth transmembrane (TM) segments of human P-glycoprotein were directly coupled by an inefficient stop transfer (ST) sequence (TM7b) contained within the C-terminus half of TM7. Remarkably, these activities enabled TM7 to achieve both a single- and a double-spanning TM topology with nearly equal efficiency. In addition, ST and C-trans signal anchor activities encoded by TM8 were tightly linked to the weak ST activity, and hence topological fate, of TM7b. This interaction enabled TM8 to span the membrane in either a type I or a type II orientation. Pleiotropic structural features contributing to this unusual topogenic behavior included 1) a short, flexible peptide loop connecting TM7a and TM7b, 2) hydrophobic residues within TM7b, and 3) hydrophilic residues between TM7b and TM8.

Sign in / Sign up

Export Citation Format

Share Document