Roles of the transmembrane domain and the cytoplasmic domain of Fc?RI? in immunoglobulin E-mediated up-regulation of surface Fc?RI expression

Despite the known importance of the transmembrane domain (TMD) of syndecan receptors in cell adhesion and signaling, the molecular basis for syndecan TMD function remains unknown. Using in vivo invertebrate models, we found that mammalian syndecan-2 rescued both the guidance defects in C. elegans hermaphrodite-specific neurons and the impaired development of the midline axons of Drosophila caused by the loss of endogenous syndecan. These compensatory effects, however, were reduced significantly when syndecan-2 dimerization-defective TMD mutants were introduced. To further investigate the role of the TMD, we generated a chimera, 2eTPC, comprising the TMD of syndecan-2 linked to the cytoplasmic domain of platelet-derived growth factor receptor (PDGFR). This chimera exhibited SDS-resistant dimer formation that was lost in the corresponding dimerization-defective syndecan-2 TMD mutant, 2eT(GL)PC. Moreover, 2eTPC specifically enhanced Tyr 579 and Tyr 857 phosphorylation in the PDGFR cytoplasmic domain, while the TMD mutant failed to support such phosphorylation. Finally, 2eTPC, but not 2eT(GL)PC, induced phosphorylation of Src and PI3 kinase (known downstream effectors of Tyr 579 phosphorylation) and promoted Src-mediated migration of NIH3T3 cells. Taken together, these data suggest that the TMD of a syndecan-2 specifically regulates receptor cytoplasmic domain function and subsequent downstream signaling events controlling cell behavior.

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Gating Motions of KirBac1.1 Cytoplasmic Domain with Respect to Transmembrane Domain Revealed by FRET

Biophysical Journal ◽

10.1016/j.bpj.2012.11.739 ◽

2013 ◽

Vol 104 (2) ◽

pp. 129a

Author(s):

Shizhen Wang ◽

Sarah Heyman ◽

Decha Enkvetchakul ◽

Colin G. Nichols

Keyword(s):

Transmembrane Domain ◽

Cytoplasmic Domain

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

Journal of Cell Science ◽

10.1242/jcs.112.11.1721 ◽

1999 ◽

Vol 112 (11) ◽

pp. 1721-1732 ◽

Cited By ~ 2

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.

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Functional regions of the mouse interleukin-10 receptor cytoplasmic domain.

Molecular and Cellular Biology ◽

10.1128/mcb.15.9.5043 ◽

1995 ◽

Vol 15 (9) ◽

pp. 5043-5053 ◽

Cited By ~ 81

Author(s):

A S Ho ◽

S H Wei ◽

A L Mui ◽

A Miyajima ◽

K W Moore

Keyword(s):

Transmembrane Domain ◽

Cytoplasmic Domain ◽

Interleukin 10 ◽

Proliferation Assay ◽

Wild Type ◽

Cell Surface Antigens ◽

Functional Regions ◽

Heat Stable ◽

C Terminus ◽

Heat Stable Antigen

The functions of wild-type and mutant mouse interleukin-10 receptors (mIL-10R) expressed in murine Ba/F3 cells were studied. As observed previously, IL-10 stimulates proliferation of IL-10R-expressing Ba/F3 cells. Accumulation of viable cells in the proliferation assay is to a significant extent balanced by concomitant cell death. Moreover, growth in IL-10 also induces a previously unrecognized response, differentiation of the cells, as evidenced both by formation of large clusters of cells in cultures with IL-10 and by induction or enhancement of expression of several cell surface antigens, including CD32/16, CD2, LECAM-1 (v-selectin), and heat-stable antigen. Two distinct functional regions near the C terminus of the mIL-10R cytoplasmic domain which mediate proliferation were identified; one of these regions also mediates the differentiation response. A third region proximal to the transmembrane domain was identified; removal of this region renders the cell 10- to 100-fold more sensitive to IL-10 in the proliferation assay. In cells expressing both wild-type and mutant IL-10R, stimulation with IL-10 leads to tyrosine phosphorylation of the kinases JAK1 and TYK2 but not JAK2 or JAK3 under the conditions tested.

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Mutants of the membrane-binding region of Semliki Forest virus E2 protein. II. Topology and membrane binding.

The Journal of Cell Biology ◽

10.1083/jcb.102.3.902 ◽

1986 ◽

Vol 102 (3) ◽

pp. 902-910 ◽

Cited By ~ 21

Author(s):

D F Cutler ◽

P Melancon ◽

H Garoff

Keyword(s):

Cell Membranes ◽

Semliki Forest Virus ◽

Transmembrane Domain ◽

Membrane Binding ◽

Cytoplasmic Domain ◽

Wild Type ◽

Hydrophobic Domain ◽

E2 Protein ◽

Amino Terminal ◽

Charge Cluster

The p62/E2 protein of Semliki Forest virus (SFV) is a typical transmembrane glycoprotein, with an amino-terminal lumenal domain, a transmembrane (hydrophobic) domain, and a carboxy-terminal cytoplasmic domain (or tail). Our hypothesis has been that the membrane-binding polypeptide region (membrane anchor) of this protein consists of both the transmembrane domain and the adjacent positively charged peptide, Arg-Ser-Lys, which is part of the cytoplasmic domain. We have investigated three anchor mutants of the p62 protein with respect to both their disposition and their stability in cell membranes. The construction of the three mutants has been described (Cutler, D.F., and H. Garoff, J. Cell Biol., 102:889-901). They are as follows: A1, changing the basic charge cluster from Arg-Ser-Lys(+2) to Gly-Ser-Glu(-1); A2, replacing an Ala in the middle of the hydrophobic stretch with a Glu; A3, changing the charge cluster from Arg-Ser-Lys(+2) to Gly-Ser-Met(0). All three mutants retain the transmembrane configuration of the wild-type p62. In a cell homogenate they have a cytoplasmic domain that is accessible to protease. In living cells an anti-peptide antibody specific for the cytoplasmic tail of p62 reacts with the tails of both wild-type and mutant p62s following its introduction into the cytoplasm. All three mutant proteins have Triton X-114 binding properties similar to the wild-type p62. However, when the membranes of cells expressing the three mutants or the wild-type p62 protein are washed with sodium carbonate, pH 11.5, three to four times as much mutant protein as wild-type p62 is released from the membranes. Thus the stability in cell membranes of the three mutant p62 proteins is significantly reduced.

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Identification of CD20 Mutations in Malignant Lymphoma: Can They Be Predictors of Response to Rituximab?.

Blood ◽

10.1182/blood.v106.11.288.288 ◽

2005 ◽

Vol 106 (11) ◽

pp. 288-288 ◽

Cited By ~ 3

Author(s):

Yasuhito Terui ◽

Takuma Sakurai ◽

Yuji Mishima ◽

Yuko Mishima ◽

Natsuhiko Sugimura ◽

...

Keyword(s):

Flow Cytometry ◽

Malignant Lymphoma ◽

Laser Scanning ◽

Transmembrane Domain ◽

Point Mutations ◽

K562 Cells ◽

Cytoplasmic Domain ◽

Isolated Cells ◽

Mammalian Expression ◽

Cd20 Expression

Abstract Purpose: Although rituximab is commonly used as induction and maintenance therapy for CD20+ malignant lymphoma, some patients become refractory to treatment and the mechanism of resistance is unclear. The aim of this study was to investigate the relationship between CD20 mutations and rituximab resistance. Methods: To investigate whether CD20 mutations affect the response to rituximab, fresh CD19+ lymphoma cells were isolated from the lymph nodes, peripheral blood or bone marrow of 48 patients with NHL using magnetic activated cell sorting (MACS). CD19+/CD20+ cells were subsequently sorted by flow cytometry. RNA was prepared from the isolated cells and RT-PCR was performed. The resulting PCR products were sequenced, subcloned into the mammalian expression vector pTARGET, transfected into K562 cells and CD20 expression was examined by flow cytometry and laser scanning confocal microscopy. Results: In all 48 patients, overall response rate (CR+CRu+PR) to rituximab was 93.8% (45/48), but two cases became PD after PR. DNA sequence analysis revealed that point mutations were mostly observed in two CD20 domains - the third transmembrane domain and the C-terminal cytoplasmic domain. One patient had point mutations in the transmembrane domain, three cases showed point mutations in the C-terminal cytoplasmic domain and six cases had non-specific CD20 mutations, which did not affect CD20 expression. Thirty-eight patients showed no mutations of CD20 gene. CD20 expression was very weak in patients with point mutations in the C-terminal cytoplasmic domain, whereas expression was increased in patients with point mutations in the transmembrane domain. Conclusions: This study suggests that point mutations in CD20 may cause rituximab resistance and identification of CD20 mutations upon diagnosis may help to predict a patient’s response to rituximab.

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Assessing the Biochemistry of Kindlin-3 In Human Platelets

Blood ◽

10.1182/blood.v116.21.2011.2011 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2011-2011

Author(s):

Craig N. Streu ◽

David Thomas Moore ◽

Paul C. Billings ◽

Patrik Nygren ◽

Karen P. Fong ◽

...

Keyword(s):

Lipid Bilayers ◽

Transmembrane Domain ◽

Conflicts Of Interest ◽

Short Form ◽

Cytoplasmic Domain ◽

Human Platelets ◽

Resonance Spectroscopy ◽

Ph Domain ◽

Biochemical Basis ◽

And Function

Abstract Abstract 2011 Although both talin and kindlin-3 binding to the β3 cytoplasmic domain are required for agonist-induced αIIbβ3 activation in platelets, the biochemical basis for this dual requirement is not clear. Recent NMR and hydrogen-deuterium exchange studies of disulfide-stabilized complexes containing the full cytoplasmic domains of αIIb and β3 dispersed in detergent micelles or lipid bilayers revealed that the β3 cytoplasmic domain consists of three helices: a stable proximal helix contiguous with the transmembrane domain and two distal dynamic amphiphilic helices whose fluctuations allow interaction of the helices with lipid bilayers or cytoplasmic proteins. These results suggest a cooperative model for talin and kindlin-3 binding to β3 with the talin and kindlin-3 binding sites kinetically- and thermodynamically-linked. Whether there is a preferred temporal sequence for kindlin-3 versus talin binding to β3 during physiological αIIbβ3 activation in platelets is not known, but the greater mobility of the kindlin-3 binding site suggests it might have a kinetic advantage over talin, assuming both are present in appropriate forms for binding. Much is known about the structure and function of talin, but substantially less is known about kindlin-3 in part because it has not been possible to express the complete molecule in bacterial expression systems. To address this issue, we have examined kindlin-3 expression and function in human platelets. Two kindlin-3 isoforms have been identified, a long form (Mr ∼ 76 kDa; accession: NM_178443) and a short form (Mr ∼75 kDa accession: NM_031471), that differ by the presence of 4 residues (RIPR; residues 360–363) in the PH domain of the long isoform. Using highly purified platelet and leukocyte RNA and RT PCR, we found that kindlin-3 expressed in platelets and leukocytes consist almost entirely of the 663 amino acid short isoform. Kindlin-3 present in platelet extracts is functionally active and spontaneously binds to the β3 cytoplasmic domain in pull down assays. Moreover, because kindlin-3 binding is abrogated by the β3 mutations S752P and T759A, but not T759F, the in vitro binding of kindlin-3 does not appear to be phosphorylation dependent. Further, surface plasmon resonance spectroscopy suggests that the PH domain of kindlin-3 partially drives membrane binding in the presence of phospholipids. Treatment of washed human platelets with the PAR1-activating peptide TRAP-6 (SFLLRNP) resulted in the rapid incorporation of kindlin-3 present in the platelet cytosol into the platelet cytoskeleton. We also found that kindlin-3 is present in dense fractions when platelets extracts were fractionated on sucrose gradients. Likewise, immunofluorescent images of platelets adherent to fibrinogen and platelet immuno-electron microscopy detected the presence of kindlin-3 in vesicular structures. Finally, using out-dated human platelets as starting material, we purified kindlin-3 to near homogeneity by the sequential use of ion-exchange and gel-filtration chromatography. These results provide a foundation for understanding the unique role that kindlin-3 plays in regulating the activity of platelet αIIbβ3. Disclosures: No relevant conflicts of interest to declare.

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Point mutation of C-terminal region of CD20 molecule predicts rituximab-induced complement-dependent cytotoxicity and clinical response to rituximab in non-Hodgkin’s lymphoma

Journal of Clinical Oncology ◽

10.1200/jco.2006.24.18_suppl.7563 ◽

2006 ◽

Vol 24 (18_suppl) ◽

pp. 7563-7563 ◽

Cited By ~ 2

Author(s):

Y. Terui ◽

Y. Mishima ◽

M. Yokoyama ◽

K. Hatake ◽

...

Keyword(s):

Flow Cytometry ◽

Laser Scanning ◽

Transmembrane Domain ◽

Point Mutations ◽

K562 Cells ◽

Cytoplasmic Domain ◽

Laser Scanning Confocal Microscopy ◽

Cytoplasmic Domains ◽

Isolated Cells ◽

Cd20 Expression

7563 Background: Although rituximab is commonly used as induction and maintenance therapy for malignant lymphoma, some patients become refractory to treatment and the mechanism of resistance is unclear. The aim of this study was to investigate the relationship between CD20 mutations and rituximab resistance. Methods: To investigate whether CD20 mutations affect response to rituximab, fresh CD19+ lymphoma cells were isolated from the lymph nodes, or bone marrow of 68 patients with NHL. The cells were subsequently sorted by flow cytometry. RNA was prepared from the isolated cells and RT-PCR was performed. The PCR products were sequenced, subcloned into an expression vector pTARGET, transfected into K562 cells. CD20 expression was examined by flow cytometry and laser scanning confocal microscopy. Results: In all 68 patients, overall response rate (CR+CRu+PR) to rituximab was 91.2% (62/68), but t four cases became PD after PR. DNA sequence analysis revealed that point mutations were mostly observed in three CD20 domains - extracellular/cytoplasmic domains, the third transmembrane domain and the C-terminal cytoplasmic domain. Two cases had point mutations in extracellular/cytoplasmic domains, one patient had point mutations in the transmembrane domain, four cases showed point mutations in the C-terminal cytoplasmic domain and six cases had non-specific CD20 mutations, which did not affect CD20 expression. 56 patients showed no mutations of CD20 gene. CD20 expression was very weak in patients with point mutations in the C-terminal cytoplasmic domain, whereas expression was increased in patients with point mutations in the transmembrane domain. Conclusions: Point mutations in CD20 may cause rituximab resistance and identification of CD20 mutations upon diagnosis may help to predict a patient’s response to rituximab. No significant financial relationships to disclose.

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The function of the transmembrane and cytoplasmic domains of pneumococcal penicillin-binding proteins 2x and 2b extends beyond that of simple anchoring devices

Microbiology ◽

10.1099/mic.0.078535-0 ◽

2014 ◽

Vol 160 (8) ◽

pp. 1585-1598 ◽

Cited By ~ 7

Author(s):

Kari Helene Berg ◽

Daniel Straume ◽

Leiv Sigve Håvarstein

Keyword(s):

Binding Proteins ◽

Transmembrane Domain ◽

Mutational Analysis ◽

Cytoplasmic Domain ◽

Loss Of Function ◽

Penicillin Binding Proteins ◽

Catalytic Domains ◽

Complex Process ◽

Cytoplasmic Tails ◽

Cross Links

The biosynthesis of cell-wall peptidoglycan is a complex process that involves six different penicillin-binding proteins (PBPs) in Streptococcus pneumoniae. Two of these, PBP2x and PBP2b, are monofunctional transpeptidases that catalyse the formation of peptide cross-links between adjacent glycan strands. Both of them are bitopic membrane proteins with a small cytoplasmic and a large extracellular domain. PBP2x and PBP2b are essential for septal and peripheral peptidoglycan synthesis, respectively. Although several studies have investigated the properties of their extracellular catalytic domains, it is not known whether the role of their N-terminal non-catalytic domains extends beyond that of being simple anchoring devices. We therefore decided to use reciprocal domain swapping and mutational analysis to gain more information about the biological function of the membrane anchors and cytoplasmic tails of PBP2x and PBP2b. In the case of PBP2x both domains are essential, but neither the membrane anchor nor the cytoplasmic domain of PBP2x appear to serve as major localization signals. Instead, our results suggest that they are involved in interactions with other components of the divisome. Mutations of conserved amino acids in the cytoplasmic domain of PBP2x resulted in loss of function, underlining the importance of this region. The cytoplasmic domain of PBP2b could be swapped with the corresponding domain from PBP2x, whereas replacement of the PBP2b transmembrane domain with the corresponding PBP2x domain gave rise to slow-growing cells with grossly abnormal morphology. When both domains were exchanged simultaneously the cells were no longer viable.

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Phorbol 12-Myristate 13-Acetate-Induced Release of the Colony-Stimulating Factor 1 Receptor Cytoplasmic Domain into the Cytosol Involves Two Separate Cleavage Events

Molecular and Cellular Biology ◽

10.1128/mcb.24.1.454-464.2004 ◽

2004 ◽

Vol 24 (1) ◽

pp. 454-464 ◽

Cited By ~ 48

Author(s):

Kevin Wilhelmsen ◽

Peter van der Geer

Keyword(s):

Protein Tyrosine Kinase ◽

Transmembrane Domain ◽

Interleukin 2 ◽

Extracellular Domain ◽

Cytoplasmic Domain ◽

Proteolytic Processing ◽

Colony Stimulating Factor ◽

Chimeric Receptors ◽

Regulated Intramembrane Proteolysis ◽

Stimulating Factor

ABSTRACT The colony-stimulating factor 1 (CSF-1) receptor is a protein-tyrosine kinase that regulates cell division, differentiation, and development. In response to phorbol 12-myristate 13-acetate (PMA), the CSF-1 receptor is subject to proteolytic processing. Use of chimeric receptors indicates that the CSF-1 receptor is cleaved at least two times, once in the extracellular domain and once in the transmembrane domain. Cleavage in the extracellular domain results in ectodomain shedding while the cytoplasmic domain remains associated with the membrane. Intramembrane cleavage depends on the sequence of the transmembrane domain and results in the release of the cytoplasmic domain. This process can be blocked by γ-secretase inhibitors. The cytoplasmic domain localizes partially to the nucleus, displays limited stability, and is degraded by the proteosome. CSF-1 receptors are continuously subject to down-modulation and regulated intramembrane proteolysis (RIP). RIP is stimulated by granulocyte-macrophage-CSF, CSF-1, interleukin-2 (IL-2), IL-4, lipopolysaccharide, and PMA and may provide the CSF-1 receptor with an additional mechanism for signal transduction.

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