scholarly journals Structural Determinants Critical for Localization and Signaling within the Seventh Transmembrane Domain of the Type 1 Corticotropin Releasing Hormone Receptor: Lessons from the Receptor Variant R1d

2008 ◽  
Vol 22 (11) ◽  
pp. 2505-2519 ◽  
Author(s):  
Danijela Markovic ◽  
Hendrik Lehnert ◽  
Michael A. Levine ◽  
Dimitris K. Grammatopoulos
Keyword(s):  
Plasma Membrane ◽  
Transmembrane Domain ◽  
Intracellular Localization ◽  
Receptor Expression ◽  
Site Directed Mutagenesis ◽  
Functional Responses ◽  
Membrane Expression ◽  
Recombinant Receptors ◽  
C Terminus

Abstract The type 1 CRH receptor (CRH-R1) plays a fundamental role in homeostatic adaptation to stressful stimuli. CRH-R1 gene activity is regulated through alternative splicing and generation of various CRH-R1 mRNA variants. One such variant is the CRH-R1d, which has 14 amino acids missing from the putative seventh transmembrane domain due to exon 13 deletion, a splicing event common to other members of the B1 family of G protein-coupled receptors. In this study, using overexpression of recombinant receptors in human embryonic kidney 293 and myometrial cells, we showed by confocal microscopy that in contrast to CRH-R1α, the R1d variant is primarily retained in the cytoplasm, although some cell membrane expression is also evident. Use of antibodies against the CRH-R1 C terminus in nonpermeabilized cells showed that membrane-expressed CRH-R1d contains an extracellular C terminus. Interestingly, treatment of CRH-R1d-expressing cells with CRH (100 nM) for 45–60 min elicited functional responses associated with a significant reduction of plasma membrane receptor expression, redistribution of intracellular receptors, and increased receptor degradation. Site-directed mutagenesis studies identified the cassette G356-F358 within transmembrane domain 7 as crucial for CRH-R1α stability to the plasma membrane because deletion of this cassette caused substantial intracellular localization of CRH-R1 α. Most importantly, coexpression studies between CRH-R1d and CRH-R2β demonstrated that the CRH-R2β could partially rescue CRH-R1d membrane expression, and this was associated with a significant attenuation of urocotrin II-induced cAMP production and ERK1/2 and p38MAPK activation, suggesting that CRH-R1d might specifically induce heterologous impairment of CRH-R2 signaling responses. This mechanism appears to involve accelerated CRH-R2β endocytosis.

Identification of a di-leucine motif within the C terminus domain of the Menkes disease protein that mediates endocytosis from the plasma membrane

1999 ◽  
Vol 112 (11) ◽  
pp. 1721-1732 ◽  
Author(s):  
M.J. Francis ◽  
E.E. Jones ◽  
E.R. Levy ◽  
R.L. Martin ◽  
S. Ponnambalam ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Golgi Apparatus ◽  
Transmembrane Domain ◽  
Menkes Disease ◽  
Cytoplasmic Domain ◽  
Endocytic Pathway ◽  
Site Directed Mutagenesis ◽  
Trans Golgi Network ◽  
C Terminus ◽  
Golgi Network

The protein encoded by the Menkes disease gene (MNK) is localised to the Golgi apparatus and cycles between the trans-Golgi network and the plasma membrane in cultured cells on addition and removal of copper to the growth medium. This suggests that MNK protein contains active signals that are involved in the retention of the protein to the trans-Golgi network and retrieval of the protein from the plasma membrane. Previous studies have identified a signal involved in Golgi retention within transmembrane domain 3 of MNK. To identify a motif sufficient for retrieval of MNK from the plasma membrane, we analysed the cytoplasmic domain, downstream of transmembrane domain 7 and 8. Chimeric constructs containing this cytoplasmic domain fused to the reporter molecule CD8 localised the retrieval signal(s) to 62 amino acids at the C terminus. Further studies were performed on putative internalisation motifs, using site-directed mutagenesis, protein expression, chemical treatment and immunofluorescence. We observed that a di-leucine motif (L1487L1488) was essential for rapid internalisation of chimeric CD8 proteins and the full-length Menkes cDNA from the plasma membrane. We suggest that this motif mediates the retrieval of MNK from the plasma membrane into the endocytic pathway, via the recycling endosomes, but is not sufficient on its own to return the protein to the Golgi apparatus. These studies provide a basis with which to identify other motifs important in the sorting and delivery of MNK from the plasma membrane to the Golgi apparatus.

Conserved residues F316 and G476 in the concentrative nucleoside transporter 1 (hCNT1) affect guanosine sensitivity and membrane expression, respectively

2005 ◽  
Vol 288 (1) ◽  
pp. C39-C45 ◽  
Author(s):  
Yurong Lai ◽  
Eun-Woo Lee ◽  
Carl C. Ton ◽  
Shashi Vijay ◽  
Huixia Zhang ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Fluorescent Protein ◽  
Transmembrane Domain ◽  
Mdck Cells ◽  
Site Directed Mutagenesis ◽  
Nucleoside Transporter ◽  
Wild Type ◽  
Membrane Expression ◽  
Concentrative Nucleoside Transporter ◽  
Green Fluorescent

The functional significance of two highly conserved amino acid residues, F316 [putative transmembrane domain (TM)7] and G476 (putative TM11), in the concentrative nucleoside transporter hCNT1 (SLC28A1) was examined by performing site-directed mutagenesis. Conservative mutations at these positions (F316A, F316Y, G476A, and G476L) were generated and expressed in Madin-Darby canine kidney (MDCK) cells as fusion polypeptides with green fluorescent protein (GFP). Unlike wild-type hCNT1, G476A-GFP and G476L-GFP were not expressed in the plasma membrane in undifferentiated or differentiated MDCK cells and had no functional activity. Like wild-type hCNT1, F316A-GFP and F316Y-GFP were expressed in the plasma membrane of undifferentiated MDCK cells and in the apical membrane of differentiated MDCK cells. Remarkably, transport of [3H]uridine by F316Y-GFP or F316A-GFP was highly sensitive to inhibition by guanosine. Furthermore, genotyping of exon 11 of hCNT1 (TM7) in a panel of 260 anonymous human DNA samples revealed a novel F316H variant (TT>CA; 1/260). When expressed in MDCK cells, [3H]uridine transport by F316H was also found to be sensitive to inhibition by guanosine (IC50 = 148 μM). The effect of the F316H mutation resembles the N4 type nucleoside transporter phenotype previously reported to be present in human kidneys. We suggest that the N4 transport system is a naturally occurring variant of hCNT1, perhaps at the F316 position. Collectively, our data show that G476 is important for correct membrane targeting, folding, and/or intracellular processing of hCNT1. In addition, we have discovered that hCNT1 displays natural variation at position F316 and that the variant F316H confers on the transporter an unusual sensitivity to inhibition by guanosine.

scholarly journals Counterregulation of clathrin-mediated endocytosis by the actin and microtubular cytoskeleton in human neutrophils

2009 ◽  
Vol 296 (4) ◽  
pp. C857-C867 ◽  
Author(s):  
Silvia M. Uriarte ◽  
Neelakshi R. Jog ◽  
Gregory C. Luerman ◽  
Samrath Bhimani ◽  
Richard A. Ward ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Actin Cytoskeleton ◽  
Human Neutrophils ◽  
Functional Responses ◽  
Membrane Expression ◽  
Granule Exocytosis ◽  
Plasma Membrane Expression ◽  
Microtubular Cytoskeleton ◽  
Latrunculin A ◽  
Azurophil Granule

We have recently reported that disruption of the actin cytoskeleton enhanced N-formylmethionyl-leucyl-phenylalanine (fMLP)-stimulated granule exocytosis in human neutrophils but decreased plasma membrane expression of complement receptor 1 (CR1), a marker of secretory vesicles. The present study was initiated to determine if reduced CR1 expression was due to fMLP-stimulated endocytosis, to determine the mechanism of this endocytosis, and to examine its impact on neutrophil functional responses. Stimulation of neutrophils with fMLP or ionomycin in the presence of latrunculin A resulted in the uptake of Alexa fluor 488-labeled albumin and transferrin and reduced plasma membrane expression of CR1. These effects were prevented by preincubation of the cells with sucrose, chlorpromazine, or monodansylcadaverine (MDC), inhibitors of clathrin-mediated endocytosis. Sucrose, chlorpromazine, and MDC also significantly inhibited fMLP- and ionomycin-stimulated specific and azurophil granule exocytosis. Disruption of microtubules with nocodazole inhibited endocytosis and azurophil granule exocytosis stimulated by fMLP in the presence of latrunculin A. Pharmacological inhibition of phosphatidylinositol 3-kinase, ERK1/2, and PKC significantly reduced fMLP-stimulated transferrin uptake in the presence of latrunculin A. Blockade of clathrin-mediated endocytosis had no significant effect on fMLP-stimulated phosphorylation of ERK1/2 in neutrophils pretreated with latrunculin A. From these data, we conclude that the actin cytoskeleton functions to limit microtubule-dependent, clathrin-mediated endocytosis in stimulated human neutrophils. The limitation of clathrin-mediated endocytosis by actin regulates the extent of both specific and azurophilic granule exocytosis.

Roles of the cytoplasmic and transmembrane domains of syntaxins in intracellular localization and trafficking

2001 ◽  
Vol 114 (17) ◽  
pp. 3115-3124 ◽  
Author(s):  
Kazuo Kasai ◽  
Kimio Akagawa
Keyword(s):  
Plasma Membrane ◽  
Transmembrane Domain ◽  
Intracellular Localization ◽  
Transmembrane Domains ◽  
Cytoplasmic Domains ◽  
Immunofluorescence Analysis ◽  
Attachment Protein ◽  
Transport Pathways ◽  
Sensitive Factor ◽  
Protein Receptors

Syntaxins are target-soluble N-ethylmaleimide-sensitive factor-attachment protein receptors (t-SNAREs) involved in docking and fusion of vesicles in exocytosis and endocytosis. Many syntaxin isoforms have been isolated, and each one displays a distinct intracellular localization pattern. However, the signals that drive the specific intracellular localization of syntaxins are poorly understood. In this study, we used indirect immunofluorescence analysis to examine the localization of syntaxin chimeras, each containing a syntaxin transmembrane domain fused to a cytoplasmic domain derived from a different syntaxin. We show that the cytoplasmic domains of syntaxins 5, 6, 7 and 8 have important effects on intracellular localization. We also demonstrate that the transmembrane domain of syntaxin 5 is sufficient to localize the chimera to the compartment expected for wild-type syntaxin 5. Additionally, we find that syntaxins 6, 7 and 8, but not syntaxin 5, are present at the plasma membrane, and that these syntaxins cycle through the plasma membrane by virtue of their cytoplasmic domains. Finally, we find that di-leucine-based motifs in the cytoplasmic domains of syntaxins 7 and 8 are necessary for their intracellular localization and trafficking via distinct transport pathways. Combined, these results suggest that both the cytoplasmic and the transmembrane domains play important roles in intracellular localization and trafficking of syntaxins.

scholarly journals N-glycosylation requirements for the AT1a angiotensin II receptor delivery to the plasma membrane

1999 ◽  
Vol 339 (2) ◽  
pp. 397-405 ◽  
Author(s):  
Benoit DESLAURIERS ◽  
Cecilia PONCE ◽  
Colette LOMBARD ◽  
Renée LARGUIER ◽  
Jean-Claude BONNAFOUS ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Angiotensin Ii ◽  
Cellular Localization ◽  
Inositol Phosphate ◽  
Glycosylation Site ◽  
Receptor Expression ◽  
Site Directed Mutagenesis ◽  
Pharmacological Properties ◽  
Peptide Epitope ◽  
Glycosylation Sites

The purpose of this work was to investigate the role of N-glycosylation in the expression and pharmacological properties of the the rat AT1a angiotensin II (AII) receptor. Glycosylation-site suppression was carried out by site-directed mutagenesis (Asn → Gln) of Asn176 and Asn188 (located on the second extracellular loop) and by the removal of Asn4 at the N-terminal end combined with the replacement of the first four amino acids by a 10 amino acid peptide epitope (c-Myc). We generated seven possible N-glycosylation-site-defective mutants, all tagged at their C-terminal ends with the c-Myc epitope. This double-tagging strategy, associated with photoaffinity labelling, allowed evaluation of the molecular masses and immunocytochemical cellular localization of the various receptors transiently expressed in COS-7 cells. We showed that: (i) each of the three N-glycosylation sites are utilized in COS-7 cells; (ii) the mutant with three defective N-glycosylation sites was not (or was very inefficiently) expressed at the plasma membrane and accumulated inside the cell at the perinuclear zone; (iii) the preservation of two sites allowed normal receptor delivery to the plasma membrane, the presence of only Asn176 ensuring a behaviour similar to that of the wild-type receptor; and (iv) all expressed receptors displayed unchanged pharmacological properties (Kd for 125I-sarcosine1-AII; sarcosine1-AII-induced inositol phosphate production). These results demonstrate that N-glycosylation is required for the AT1 receptor expression. They are discussed in the light of current knowledge of membrane-protein maturation and future prospects of receptor overexpression for structural studies.

scholarly journals Expression and Differential Intracellular Localization of Two Major Forms of Human 8-Oxoguanine DNA Glycosylase Encoded by Alternatively Spliced OGG1 mRNAs

1999 ◽  
Vol 10 (5) ◽  
pp. 1637-1652 ◽  
Author(s):  
Kenichi Nishioka ◽  
Toshio Ohtsubo ◽  
Hisanobu Oda ◽  
Toshiyuki Fujiwara ◽  
Dongchon Kang ◽  
...  
Keyword(s):  
Intracellular Localization ◽  
Nuclear Extract ◽  
Dna Glycosylase ◽  
Mitochondrial Targeting ◽  
Electron Microscopic ◽  
C Terminus ◽  
Alternatively Spliced ◽  
Targeting Signal ◽  
Processed Form

We identified seven alternatively spliced forms of human 8-oxoguanine DNA glycosylase (OGG1) mRNAs, classified into two types based on their last exons (type 1 with exon 7: 1a and 1b; type 2 with exon 8: 2a to 2e). Types 1a and 2a mRNAs are major in human tissues. Seven mRNAs are expected to encode different polypeptides (OGG1–1a to 2e) that share their N terminus with the common mitochondrial targeting signal, and each possesses a unique C terminus. A 36-kDa polypeptide, corresponding to OGG1–1a recognized only by antibodies against the region containing helix-hairpin-helix-PVD motif, was copurified from the nuclear extract with an activity introducing a nick into DNA containing 8-oxoguanine. A 40-kDa polypeptide corresponding to a processed form of OGG1–2a was detected in their mitochondria using antibodies against its C terminus. Electron microscopic immunocytochemistry and subfractionation of the mitochondria revealed that OGG1–2a locates on the inner membrane of mitochondria. Deletion mutant analyses revealed that the unique C terminus of OGG1–2a and its mitochondrial targeting signal are essential for mitochondrial localization and that nuclear localization of OGG1–1a depends on the NLS at its C terminus.

A nematode gene required for sperm vesicle fusion

1997 ◽  
Vol 110 (9) ◽  
pp. 1073-1081 ◽  
Author(s):  
W.E. Achanzar ◽  
S. Ward
Keyword(s):  
Plasma Membrane ◽  
Transmembrane Domain ◽  
Sequence Similarity ◽  
Biological Significance ◽  
Single Amino Acid ◽  
Viral Fusion ◽  
Missense Mutations ◽  
Direct Role ◽  
C Terminus ◽  
Human Proteins

During maturation of spermatids to motile spermatozoa in Caenorhabditis elegans, large vesicles called membranous organelles (MOs) fuse with the spermatid plasma membrane. Mutations in the gene fer-1 cause abnormal spermatozoa in which the MOs do not fuse, although they abut the plasma membrane normally. Here we describe the fer-1 gene, which we found to be approximately 8.6 kb in length and to encode a 6.2 kb transcript whose expression is limited to the primary spermatocytes, the cells in which the MOs form. fer-1 is predicted to encode a 235 kDa protein which is highly charged except for a putative transmembrane domain near the C terminus. We identified the mutations associated with five fer-1 alleles, all of which are missense mutations causing single amino acid changes. FER-1 is not similar to any characterized proteins in sequence databases, nor does it contain known functional motifs other than the predicted transmembrane domain. The C-terminal transmembrane domain makes FER-1 resemble some viral fusion proteins, suggesting it may play a direct role in MO-plasma membrane fusion. FER-1 does show significant sequence similarity to several predicted human proteins of unknown function. Two of the identified fer-1 mutations are located in regions of similarity between FER-1 and two of these predicted proteins. This strengthens the biological significance of these similarities and suggests these regions of similarity represent functionally important domains of FER-1 and the human proteins.

Lenalidomide Upregulates Erythropoietin Receptor Expression in Hematopoietic Progenitors by Modulating Receptor Turnover

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2382-2382
Author(s):  
Ashley A Basiorka ◽  
Kathy McGraw ◽  
Justine Clark ◽  
Gisela Caceres ◽  
Joe Johnson ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Actin Polymerization ◽  
Drug Exposure ◽  
Rho Gtpase ◽  
Erythropoietin Receptor ◽  
Receptor Expression ◽  
Janus Kinase ◽  
Erythroid Progenitors ◽  
Epor Expression

Abstract Abstract 2382 The erythropoietin receptor (EpoR) is a type 1 cytokine receptor and key effector of Epo signaling. Maturation and shuttling of receptor to the plasma membrane is dependent on its association with Janus Kinase 2 (JAK2). Upon ligand binding, EpoR homo-dimerizes to activate JAK2, which in turn phosphorylates tyrosine residues on the receptor cytoplasmic tail permitting recruitment and phosphorylation of the signal transduction and activator of transcription (STAT)-5. Lenalidomide (LEN), a second generation immunomodulatory drug (IMiD), promotes the expansion of primitive erythroid progenitors in vitro and induces the expression of genes involved in erythroid lineage commitment (JCI 2008;118(1):248-58; PLoS medicine 2008;5(2):e35). In del(5q) clones, LEN is cytotoxic by inhibiting the haplodeficient serine/threonine phosphatase PP2A (Wei S, et. al PNAS 2009). To investigate the mechanism by which LEN promotes erythropoiesis, we investigated its action on EpoR cellular dynamics. Using the human erythroleukemia cell line, UT-7, treatment with LEN elicited concentration dependent upregulation of EpoR in whole cell lysates demonstrable by immunoblot (IB) analysis at concentrations ranging from 0.1–10μM, reaching a 2-fold increase vs. controls. Treatment with pomalidomide, an IMiD with superior erythropoietic activity, displayed greater potency, increasing cellular EpoR at concentrations as low as 1nM; whereas thalidomide was ineffective. Upregulation of EpoR was apparent as early as 1 hour (h) after LEN exposure, reaching maximal receptor induction at 8h. Isolation of membrane and cystosolic fractions followed by IB showed that EpoR was upregulated predominantly in the membrane fraction, increasing 4.3-fold. IB of detergent insoluble membrane raft (MR) fractions showed that LEN promoted lipid raft assembly and F-actin polymerization accompanied by translocation of EpoR into MR microdomains with JAK2, STAT-5 and Lyn signal intermediates. Pretreatment with the Rho-GTPase inhibitor, Y27632, suppressed LEN effects on MR and F-actin. Immunoprecipitation (IP) of EpoR showed that JAK2 co-precipitated with the receptor in LEN treated cells, suggesting drug induced release of pre-assembled EpoR/JAK2 complexes from golgi and endosomal compartments. Q-PCR showed no changes in EpoR gene mRNA expression up to 24 hours after drug exposure, supporting post translational drug effects. To confirm that the observed changes in EpoR dynamics in UT-7 cells extends to normal erythroid progenitors, we assessed EpoR expression and MR assembly (cholera toxin B labeled GM1) by fluorescence microscopy in CD71+ erythroid precursors from three normal bone marrow donors. Expression of EpoR increased a mean of 17% (range:0-68%) after 1h LEN [1μM] exposure (p=0.003), accompanied by an increase in MR aggregates. Because the LEN inhibitable target PP2A is a key regulator of Rho-GTPase guided receptor trafficking and AP-1 directed formation of MR targeted clathrin-coated vesicles, we examined its effects on EpoR induction. Pretreatment of UT-7 cells with the PP2A activator FTY720 antagonized the upregulation of EpoR by LEN, whereas treatment with the PP2A inhibitor cantharadin [0.5μM] induced EpoR protein expression. Inhibition of dynamin activity, a PP2A sensitive GTPase involved in type 1 receptor recycling from membrane to endosomes, recapitulated the effects of cantharadin, inducing EpoR expression. In summary, these findings indicate that LEN modulates EpoR equilibrium, mediated in part through its inhibitory effect on PP2A. LEN induces rapid translocation of mature EpoR/JAK2 complexes to the plasma membrane while promoting the assembly of MRs with signaling competent receptor platforms to optimize cytokine signal integrity. Disclosures: List: Celgene: Consultancy.

scholarly journals The Trafficking Protein GABARAP Binds to and Enhances Plasma Membrane Expression and Function of the Angiotensin II Type 1 Receptor

2008 ◽  
Vol 102 (12) ◽  
pp. 1539-1547 ◽  
Author(s):  
Julia L. Cook ◽  
Richard N. Re ◽  
Dawn L. deHaro ◽  
Jennifer M. Abadie ◽  
Michelle Peters ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Angiotensin Ii ◽  
Membrane Expression ◽  
Plasma Membrane Expression ◽  
And Function

scholarly journals Role of the N-terminal transmembrane domain in the endo-lysosomal targeting and function of the human ABCB6 protein

2015 ◽  
Vol 467 (1) ◽  
pp. 127-139 ◽  
Author(s):  
Katalin Kiss ◽  
Nora Kucsma ◽  
Anna Brozik ◽  
Gabor E. Tusnady ◽  
Ptissam Bergam ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Transmembrane Domain ◽  
Intracellular Localization ◽  
Atp Binding ◽  
Multivesicular Bodies ◽  
Molecular Dissection ◽  
Terminal Domain ◽  
Lysosomal Targeting ◽  
And Function

The intracellular localization of ATP-binding cassette, sub family B (ABCB) 6 is a matter of debate. We show that ABCB6 is internalized from the plasma membrane to multivesicular bodies and lysosomes. Molecular dissection of the ABCB6 protein reveals a role of its N-terminal domain in targeting.

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