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.

Structural requirements of the interleukin-6 signal transducer gp130 for its interaction with Janus kinase 1: the receptor is crucial for kinase activation

2001 ◽  
Vol 361 (1) ◽  
pp. 105-111 ◽  
Author(s):  
Claude HAAN ◽  
Peter C. HEINRICH ◽  
Iris BEHRMANN
Keyword(s):  
Point Mutations ◽  
Proximal Region ◽  
Janus Kinase ◽  
Signal Transducer ◽  
Kinase Activation ◽  
Gp130 Receptor ◽  
Janus Kinase 1 ◽  
Receptor Chain ◽  
Common Signal ◽  
Membrane Proximal Region

We analysed the interaction of gp130, the common signal-transducing receptor chain of interleukin (IL)-6 type cytokines, with Jak1, the Janus family kinase which is crucial for signal transduction of this group of cytokines. With a truncated chimaeric IL-5Rβ–gp130 receptor expressed in COS-7 cells, we show that the membrane-proximal 69 amino acids are sufficient to mediate Jak1 binding and activation. Deletion of box2 drastically reduced binding of endogenous, but not of overexpressed, Jak1. Several point mutations in the membrane-proximal region of gp130 (W652A, P671/P672A, F676A, Y683F, where W, A, P, F and Y are tryptophan, alanine, proline, phenylalanine and tyrosine) did not affect Jak1 association. However, stimulation of chimaeric receptors with the mutations P671/P672A and F676A in the interbox1/box2 region resulted in a reduced activation of STAT (signal transducer and activator of transcription) transcription factors. Most importantly, signalling by the receptor with the box1 mutation W652A was totally abrogated. Although this mutation did not affect Jak1 association, stimulation-dependent phosphorylation of Jak1 was prevented. The W652 mutation acts dominantly, since no signalling occured even when only a single cytoplasmic chain of a gp130 dimer contained the mutation. Our data demonstrate that the mere proximity of Jaks in an activated receptor complex is not sufficient to mediate their activation. Rather, it seems that parts of the receptor, including the box1 region, are involved in positioning Jaks correctly so that ligand-induced receptor dimerization and reorientation can lead to their mutual activation and subsequently to downstream signalling events.

scholarly journals Investigating the conformational response of the Sortilin receptor upon binding endogenous peptide- and protein ligands by HDX-MS

2018 ◽  
Author(s):  
Esben Trabjerg ◽  
Nadia Abu-Asad ◽  
Ziqian Wan ◽  
Fredrik Kartberg ◽  
Søren Christensen ◽  
...  
Keyword(s):  
Binding Sites ◽  
Proximal Region ◽  
Binding Region ◽  
Exchange Mass Spectrometry ◽  
Protein Ligands ◽  
Hydrogen Deuterium ◽  
Deuterium Exchange Mass Spectrometry ◽  
Hdx Ms ◽  
Membrane Proximal Region

AbstractSortilin is a multifunctional transmembrane neuronal receptor involved in sorting of neurotrophic factors and apoptosis signalling. So far, structural characterization of Sortilin and its endogenous ligands has been limited to crystallographic studies of Sortilin in complex with the neuropeptide Neurotensin. Here, we use hydrogen/deuterium exchange mass spectrometry to investigate the conformational response of Sortilin to binding biological ligands including the peptides Neurotensin and the Sortilin propeptide and the proteins Progranulin and pro-Nerve growth factor-β. The results show that the ligands employ two binding sites inside the cavity of the β-propeller of Sortilin. However, ligands have distinct differences in their conformational impact on the receptor. Interestingly, the protein ligands induce conformational stabilization in a remote membrane-proximal domain, hinting at an unknown conformational link between the ligand binding region and this membrane-proximal region of Sortilin. Our findings improves our molecular understanding of Sortilin and how it mediates diverse ligand-dependent functions important in neurobiology.

Family-wide characterization of matrix metalloproteinases from Arabidopsis thaliana reveals their distinct proteolytic activity and cleavage site specificity

2013 ◽  
Vol 457 (2) ◽  
pp. 335-346 ◽  
Author(s):  
Giada Marino ◽  
Pitter F. Huesgen ◽  
Ulrich Eckhard ◽  
Christopher M. Overall ◽  
Wolfgang P. Schröder ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Cleavage Site ◽  
Biochemical Properties ◽  
Site Specificity ◽  
Sequence Specificity ◽  
Cleavage Sites ◽  
Protease Cleavage ◽  
Protease Cleavage Sites ◽  
Proteomic Identification

The five recombinant MMP-like proteins of Arabidopsis thaliana have specific biochemical properties. Detailed analysis of their sequence specificity using proteomic identification of protease cleavage sites revealed cleavage profiles similar to human MMPs.

scholarly journals Structural requirements regulate endoproteolytic release of the L-selectin (CD62L) adhesion receptor from the cell surface of leukocytes.

1995 ◽  
Vol 182 (2) ◽  
pp. 519-530 ◽  
Author(s):  
A Chen ◽  
P Engel ◽  
T F Tedder
Keyword(s):  
Amino Acid ◽  
Cell Surface ◽  
Cell Activation ◽  
Proximal Region ◽  
Sequence Specificity ◽  
Lymphocyte Migration ◽  
Structural Requirements ◽  
Amino Acid Region ◽  
Extracellular Region ◽  
Membrane Proximal Region

L-selectin mediates leukocyte rolling on vascular endothelium at sites of inflammation and lymphocyte migration to peripheral lymph nodes. L-selectin is rapidly shed from the cell surface after leukocyte activation by a proteolytic mechanism that cleaves the receptor in a membrane proximal extracellular region. This process may allow rapid leukocyte detachment from the endothelial surface before entry into tissues. In this study, the structural requirements for regulation of human L-selectin endoproteolytic release were examined through analysis of chimeric selectin molecules and mutant L-selectin receptors. The use of chimeric selectins and a cytoplasmic tail truncation mutant demonstrated that the extracellular membrane-proximal 15-amino acid region of L-selectin is required for endoproteolytic release. The introduction of alanine-scanning mutations within this membrane-proximal region did not prevent endoproteolytic release, indicating that a specific amino acid motif was not an absolute requirement for cleavage. Furthermore, alterations within the putative primary cleavage site (K283-S284) resulted in either constitutive endoproteolytic release of the receptor or inhibition of cell activation-induced shedding to variable extents. The length of the membrane-proximal region was also critical since truncations of this region completely abolished endoproteolytic release. Thus, release of L-selectin is likely to be regulated by the generation of an appropriate tertiary conformation within the membrane-proximal region of the receptor which allows recognition by a membrane-bound endoprotease with relaxed sequence specificity that cleaves the receptor at a specific distance from the plasma membrane. These observations suggest a generalized protein-processing pathway involved in the endoproteolytic release of specific transmembrane proteins which harbor widely differing primary sequences at or neighboring their cleavage sites.

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 Erythropoietin induces activation of Stat5 through association with specific tyrosines on the receptor that are not required for a mitogenic response.

1996 ◽  
Vol 16 (4) ◽  
pp. 1622-1631 ◽  
Author(s):  
F W Quelle ◽  
D Wang ◽  
T Nosaka ◽  
W E Thierfelder ◽  
D Stravopodis ◽  
...  
Keyword(s):  
Cell Receptor ◽  
Distal Region ◽  
Binding Activity ◽  
Proximal Region ◽  
Erythropoietin Receptor ◽  
Dna Binding Activity ◽  
Gamma Locus ◽  
A Site ◽  
Necessary And Sufficient ◽  
Membrane Proximal Region

The cytoplasmic domain of the erythropoietin receptor (EpoR) contains a membrane-distal region that is dispensable for mitogenesis but is required for the recruitment and tyrosine phosphorylation of a variety of signaling proteins. The membrane-proximal region of 96 amino acids is necessary and sufficient for mitogenesis as well as Jak2 activation, induction of c-fos, c-myc, cis, the T-cell receptor gamma locus (TCR-gamma), and c-pim-1. The studies presented here demonstrate that this region is also necessary and sufficient for the activation of Stat5A and Stat5B. The membrane-proximal domain contains a single tyrosine, Y-343, which when mutated eliminates the ability of the receptor to couple Epo binding to the activation of Stat5. Furthermore, peptide competitions demonstrate that this site, when phosphorylated, can disrupt Stat5 DNA binding activity, consistent with a role of Y-343 as a site of recruitment to the receptor. Cells expressing the truncated, Y343F mutant (a mutant with a Y-to-F alteration at position 343) proliferate in response to Epo in a manner comparable to that of the controls. However, in these cells, Epo stimulation does not induce the appearance of transcripts for cis, TCR-gamma, or c-fos, suggesting a role for Stat5 in their regulation.

scholarly journals Characterization of CRISPR Spacer and Protospacer Sequences in Paenibacillus larvae and Its Bacteriophages

Viruses ◽  
2021 ◽  
Vol 13 (3) ◽  
pp. 459
Author(s):  
Casey Stamereilers ◽  
Simon Wong ◽  
Philippos K. Tsourkas
Keyword(s):  
Comparative Studies ◽  
Point Mutations ◽  
Paenibacillus Larvae ◽  
Bacterial Disease ◽  
American Foulbrood ◽  
Antibiotic Resistant ◽  
Future Studies ◽  
Complete Genomes ◽  
Point Of Reference

The bacterium Paenibacillus larvae is the causative agent of American foulbrood, the most devastating bacterial disease of honeybees. Because P. larvae is antibiotic resistant, phages that infect it are currently used as alternative treatments. However, the acquisition by P. larvae of CRISPR spacer sequences from the phages could be an obstacle to treatment efforts. We searched nine complete genomes of P. larvae strains and identified 714 CRISPR spacer sequences, of which 384 are unique. Of the four epidemiologically important P. larvae strains, three of these have fewer than 20 spacers, while one strain has over 150 spacers. Of the 384 unique spacers, 18 are found as protospacers in the genomes of 49 currently sequenced P. larvae phages. One P. larvae strain does not have any protospacers found in phages, while another has eight. Protospacer distribution in the phages is uneven, with two phages having up to four protospacers, while a third of phages have none. Some phages lack protospacers found in closely related phages due to point mutations, indicating a possible escape mechanism. This study serve a point of reference for future studies on the CRISPR-Cas system in P. larvae as well as for comparative studies of other phage–host systems.

scholarly journals Functional characterization of somatic point mutations of the human estrogen receptor α (hERα) in adenomyosis uteri

2004 ◽  
Vol 10 (12) ◽  
pp. 853-860 ◽  
Author(s):  
Martin K. Oehler ◽  
Holger Greschik ◽  
Dagmar-C. Fischer ◽  
Xiaowen Tong ◽  
Roland Schuele ◽  
...  
Keyword(s):  
Estrogen Receptor ◽  
Functional Characterization ◽  
Point Mutations ◽  
Estrogen Receptor Α ◽  
Human Estrogen Receptor
Sign in / Sign up

Export Citation Format

Share Document