scholarly journals Membrane proximal cleavage of L-selectin: identification of the cleavage site and a 6-kD transmembrane peptide fragment of L-selectin.

1994 ◽  
Vol 125 (2) ◽  
pp. 461-470 ◽  
Author(s):  
J Kahn ◽  
R H Ingraham ◽  
F Shirley ◽  
G I Migaki ◽  
T K Kishimoto
Keyword(s):  
Cleavage Site ◽  
Cytoplasmic Domain ◽  
Proximal Region ◽  
Soluble Form ◽  
Activated Neutrophils ◽  
Blood Neutrophils ◽  
Integral Process ◽  
Transmembrane Fragment ◽  
Membrane Proximal Region ◽  
Polyclonal Serum

Rapid downregulation of L-selectin expression occurs in response to leukocyte activation, and it has been speculated to be an integral process in the adhesion cascade leading to neutrophil recruitment to sites of inflammation. It has previously been proposed that L-selectin is proteolytically cleaved from the cell surface; however, the nature of the cleavage site has been unknown. We have produced polyclonal antisera against the extracellular domain and against the cytoplasmic domain of L-selectin. Both antisera immunoprecipitate the intact form of L-selectin from metabolically labeled phytohemagglutinin-stimulated lymphoblasts and peripheral blood neutrophils. In addition, the anti-cytoplasmic domain serum, but not the antiectodomain serum, immunoprecipitate a 6-kD species from PMA activated lymphoblasts and formylmethionylleucylphenylalanine-activated neutrophils. Conversely, the antiectodomain serum but not the anti-cytoplasmic domain serum immunoprecipitate a 68-kD soluble form of L-selectin from the supernatant of PMA-activated lymphoblasts. The appearance of the 6-kD species on activated cells correlated with the disappearance of the intact form of L-selectin and the appearance of the soluble form of L-selectin. A third polyclonal serum generated against the membrane proximal region of the ectodomain also reacted with the 6-kD species, indicating that this is a transmembrane peptide of L-selectin. That the 6-kD species is derived from L-selectin was confirmed by immunoprecipitation of the 6-kD species from L-selectin transfectants but not from mock transfectants. Radiochemical sequence analysis defined a cleavage site between Lys321 and Ser322, which would predict a transmembrane fragment consistent in size with the observed 6-kD fragment. A Ser-Phe-Ser motif adjacent to the cleavage site is conserved between human, mouse, and rat L-selectin, and a related motif is found proximal to transmembrane domains of other downregulated proteins, such as ACE, CD16-II, and TNF-RII, suggesting the possibility of a common recognition motif.

scholarly journals Fc receptor endocytosis is controlled by a cytoplasmic domain determinant that actively prevents coated pit localization.

1992 ◽  
Vol 116 (4) ◽  
pp. 875-888 ◽  
Author(s):  
H M Miettinen ◽  
K Matter ◽  
W Hunziker ◽  
J K Rose ◽  
I Mellman
Keyword(s):  
Cytoplasmic Domain ◽  
Insoluble Fraction ◽  
Fc Receptor ◽  
Proximal Region ◽  
Wild Type ◽  
Coated Pits ◽  
Tyrosine Residues ◽  
Receptor Endocytosis ◽  
Alternative Mrna Splicing ◽  
Membrane Proximal Region

Macrophages and B-lymphocytes express two major isoforms of Fc receptor (FcRII-B2 and FcRII-B1) that exhibit distinct capacities for endocytosis. This difference in function reflects the presence of an in-frame insertion of 47 amino acids in the cytoplasmic domain of the lymphocyte isoform (FcRII-B1) due to alternative mRNA splicing. By expressing wild type and mutant FcRII cDNAs in fibroblasts, we have now examined the mechanism by which the insertion acts to prevent coated pit localization and endocytosis. We first identified the region of the FcRII-B2 cytoplasmic domain that is required for rapid internalization. Using a biochemical assay for endocytosis and an immuno-EM assay to determine coated pit localization directly, we found that the distal half of the cytoplasmic domain, particularly a region including residues 18-31, as needed for coated pit-mediated endocytosis. Elimination of the tyrosine residues at position 26 and 43, separately or together, had little effect on coated pit localization and a partial effect on endocytosis of ligand. Since the FcRII-B1 insertion occurs in the membrane-proximal region of the cytoplasmic domain (residue 6) not required for internalization, it is unlikely to act by physically disrupting the coated pit localization determinant. In fact, the insertion was found to prevent endocytosis irrespective of its position in the cytoplasmic tail and appeared to selectively exclude the receptor from coated regions. Moreover, receptors bearing the insertion exhibited a temperature- and ligand-dependent association with a detergent-insoluble fraction and with actin filaments, perhaps in part explaining the inability of FcRII-B1 to enter coated pits.

scholarly journals Characterization of the Rapid Proteolytic Shedding of Murine L-Selectin

2001 ◽  
Vol 8 (3-4) ◽  
pp. 267-277 ◽  
Author(s):  
Li-Chao Zhao ◽  
John B. Edgar ◽  
Morris O. Dailey
Keyword(s):  
Cleavage Site ◽  
Point Mutations ◽  
Proximal Region ◽  
Sequence Specificity ◽  
Site Analysis ◽  
Maximal Induction ◽  
A Site ◽  
Proteolytic Shedding ◽  
Membrane Proximal Region

The structural requirements for L-selectin shedding were studied in murine leukocytes. Upon activation, L-selectin on both lymphocytes and neutrophils undergoes cleavage by a membrane metalloprotease, resulting in the generation of a soluble ectodomain and a membrane- retained 6 kD fragment. Radiochemical sequencing demonstrated a cleavage site in the membrane-proximal region (MPR) between R321 and S322, which is homologous to the human site. Although intact neutrophil L-selectin is larger, it is cleaved at the same, or very close, site. Analysis of several transfectants expressing L-selectin point mutations and chimeric constructs suggest that, like human shedding, the proteolytic process has relatively loose sequence specificity for the substrate site. In addition, some constructs are susceptible to slow constitutive cleavage, but their shedding does not increase upon PMA stimulation, showing that basal and activated shedding are separable processes. Insertion of the 15 amino acid MPR into murine B7.2 conferred upon this molecule susceptibility to constitutive shedding. PMA stimulation results in little or no acceleration of down regulation of this molecule. These results suggest that recognition of both the membrane-proximal cleavage site and of a site distant from the MPR are required for maximal induction of L-selectin shedding.

scholarly journals The membrane proximal region of the cytoplasmic domain of the growth hormone receptor is involved in the activation of Stat 3

FEBS Letters ◽  
1995 ◽  
Vol 369 (2-3) ◽  
pp. 169-172 ◽  
Author(s):  
Athanassia Sotiropoulos ◽  
Soraya Moutoussamy ◽  
Nadine Binart ◽  
Paul A. Kelly ◽  
Joëlle Finidori
Keyword(s):  
Growth Hormone ◽  
Hormone Receptor ◽  
Growth Hormone Receptor ◽  
Cytoplasmic Domain ◽  
Proximal Region ◽  
Membrane Proximal Region

scholarly journals The Conserved Membrane-proximal Region of an Integrin Cytoplasmic Domain Specifies Ligand Binding Affinity

1995 ◽  
Vol 270 (21) ◽  
pp. 12411-12417 ◽  
Author(s):  
Paul E. Hughes ◽  
Timothy E. O'Toole ◽  
Jari Ylänne ◽  
Sanford J. Shattil ◽  
Mark H. Ginsberg
Keyword(s):  
Ligand Binding ◽  
Binding Affinity ◽  
Cytoplasmic Domain ◽  
Proximal Region ◽  
Membrane Proximal Region

scholarly journals Talin-dependent integrin activation is required for fibrin clot retraction by platelets

Blood ◽  
2011 ◽  
Vol 117 (5) ◽  
pp. 1719-1722 ◽  
Author(s):  
Jacob R. Haling ◽  
Susan J. Monkley ◽  
David R. Critchley ◽  
Brian G. Petrich
Keyword(s):  
Actin Cytoskeleton ◽  
Cytoplasmic Domain ◽  
Fibrin Clot ◽  
Proximal Region ◽  
Integrin Activation ◽  
Clot Retraction ◽  
Genetic Deletion ◽  
Membrane Proximal Region ◽  
First Time ◽  
Fibrin Clots

Abstract Talin functions both as a regulator of integrin affinity and as an important mechanical link between integrins and the cytoskeleton. Using genetic deletion of talin, we show for the first time that the capacity of talin to activate integrins is required for fibrin clot retraction by platelets. To further dissect which talin functions are required for this process, we tested clot retraction in platelets expressing a talin1(L325R) mutant that binds to integrins, but exhibits impaired integrin activation ascribable to disruption of the interaction between talin and the membrane-proximal region (MPR) in the β-integrin cytoplasmic domain. Talin-deficient and talin1(L325R) platelets were defective in retracting fibrin clots. However, the defect in clot retraction in talin1(L325R) platelets, but not talin-deficient platelets, was rescued by extrinsically activating integrins with manganese, thereby proving that integrin activation is required and showing that talin1(L325R) can form functional links to the actin cytoskeleton.

Functional Differentiation Signals Mediated by Distinct Regions of the Cytoplasmic Domain of the Granulocyte Colony-Stimulating Factor Receptor

Blood ◽  
1999 ◽  
Vol 93 (11) ◽  
pp. 3774-3784 ◽  
Author(s):  
Debbie C. Koay ◽  
Alan C. Sartorelli
Keyword(s):  
Functional Differentiation ◽  
Cytoplasmic Domain ◽  
Proximal Region ◽  
Functional Markers ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Amino Terminal ◽  
Efficient Induction ◽  
Membrane Proximal Region ◽  
Stimulating Factor

Abstract Granulocyte colony-stimulating factor receptor (G-CSFR) regulates the proliferation and differentiation of neutrophilic progenitor cells through interaction with its cytokine. Exposure of WEHI-3B D+ myelomonocytic leukemia and myeloid LGM-1 cells overexpressing the G-CSFR to G-CSF resulted in induction of differentiation as measured by (1) the ability to reduce nitroblue tetrazolium (NBT), (2) the expression of Mac-I antigen, and (3) the expression of FcγII/III receptor. Mutational analyses indicated that distinct regions of the cytoplasmic domain were critical for efficient induction of each functional marker. The membrane proximal region containing homology sequences of boxes 1 and 2 was important for the activation of all three functional markers of mature neutrophils. Induction of the capacities to express Mac-I antigen or FcγII/III receptor also required additional sequences in the membrane proximal region between amino acids 70 and 100 and may be dependent on the phosphorylation of Tyr703. The findings suggest that distinct sequences within the amino-terminal region of the cytoplasmic domain of the receptor are sufficient to induce these functional markers of differentiation, and receptor tyrosine phosphorylation may be necessary.

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