Negatively Charged Amino Acids within the Intraluminal Loop of Ryanodine Receptor Are Involved in the Interaction with Triadin

Junctin is a transmembrane protein of striated muscles, localized at the junctional sarcoplasmic reticulum (j-SR). It is characterized by a luminal C-terminal tail, through which it functionally interacts with calsequestrin and the ryanodine receptor. Interaction with calsequestrin was ascribed to the presence of stretches of charged amino acids. However, the regions able to bind calsequestrin have not been defined in detail. We report here that, in non-muscle cells, junctin and calsequestrin assemble in long linear regions within the endoplasmic reticulum, mirroring the formation of calsequestrin polymers. In differentiating myotubes, the two proteins co-localize at triads, where they assemble with other j-SR proteins. By performing GST pull-down assays with distinct regions of the junctin tail, we identified two KEKE motifs able to bind calsequestrin. In addition, stretches of charged amino acids downstream these motifs were found to be also able to bind calsequestrin and the ryanodine receptor. Deletion of even one of these regions impaired the ability of junctin to localize at the j-SR, suggesting that interaction with other proteins at this site represents a key element in junctin targeting.

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Insulin-stimulated serine and threonine phosphorylation of the human insulin receptor. An assessment of the role of serines 1305/1306 and threonine 1348 by their replacement with neutral or negatively charged amino acids.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)54708-6 ◽

1991 ◽

Vol 266 (32) ◽

pp. 21804-21809

Author(s):

J.M. Tavaré ◽

B. Zhang ◽

L. Ellis ◽

R.A. Roth

Keyword(s):

Amino Acids ◽

Insulin Receptor ◽

Human Insulin ◽

Charged Amino Acids ◽

Human Insulin Receptor

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Epitope Mapping of Monoclonal Antibodies to Calreticulin Reveals That Charged Amino Acids Are Essential for Antibody Binding

Antibodies ◽

10.3390/antib10030031 ◽

2021 ◽

Vol 10 (3) ◽

pp. 31

Author(s):

Ann Christina Bergmann ◽

Cecilie Kyllesbech ◽

Rimantas Slibinskas ◽

Evaldas Ciplys ◽

Peter Højrup ◽

...

Keyword(s):

Amino Acids ◽

Monoclonal Antibodies ◽

Epitope Mapping ◽

Biological Function ◽

Specific Binding ◽

Enzyme Linked Immunosorbent Assay ◽

Potential Therapeutic Target ◽

Charged Amino Acids ◽

Chaperone Protein ◽

Terminal Amino

Calreticulin is a chaperone protein, which is associated with myeloproliferative diseases. In this study, we used resin-bound peptides to characterize two monoclonal antibodies (mAbs) directed to calreticulin, mAb FMC 75 and mAb 16, which both have significantly contributed to understanding the biological function of calreticulin. The antigenicity of the resin-bound peptides was determined by modified enzyme-linked immunosorbent assay. Specific binding was determined to an 8-mer epitope located in the N-terminal (amino acids 34–41) and to a 12-mer peptide located in the C-terminal (amino acids 362–373). Using truncated peptides, the epitopes were identified as TSRWIESK and DEEQRLKEEED for mAb FMC 75 and mAb 16, respectively, where, especially the charged amino acids, were found to have a central role for a stable binding. Further studies indicated that the epitope of mAb FMC 75 is assessable in the oligomeric structure of calreticulin, making this epitope a potential therapeutic target.

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The Superagonistic Activity of Bovine Thyroid-stimulating Hormone (TSH) and the Human TR1401 TSH Analog Is Determined by Specific Amino Acids in the Hinge Region of the Human TSH Receptor

Journal of Biological Chemistry ◽

10.1074/jbc.m109.005710 ◽

2009 ◽

Vol 284 (24) ◽

pp. 16317-16324 ◽

Cited By ~ 27

Author(s):

Sandra Mueller ◽

Gunnar Kleinau ◽

Mariusz W. Szkudlinski ◽

Holger Jaeschke ◽

Gerd Krause ◽

...

Keyword(s):

Amino Acids ◽

Thyroid Stimulating Hormone ◽

Hinge Region ◽

Camp Production ◽

Glycoprotein Hormone ◽

Charged Amino Acids ◽

Bovine Thyroid ◽

Positively Charged ◽

Bovine Tsh ◽

Signaling Activity

Bovine TSH (bTSH) has a higher affinity to the human TSHR (hTSHR) and a higher signaling activity than human TSH (hTSH). The molecular reasons for these phenomena are unknown. Distinct negatively charged residues (Glu297, Glu303, and Asp382) in the hinge region of the hTSHR are known to be important for bTSH binding and signaling. To investigate the potential relevance of these positions for differences between bTSH and hTSH in the interaction to the hTSHR, we determined bTSH- and hTSH-mediated cAMP production of several substitutions at these three hinge residues. To examine specific variations of hTSH, we also investigated the superagonistic hTSH analog TR1401 (TR1401), whose sequence differs from hTSH by four additional positively charged amino acids that are also present in bTSH. To characterize possible interactions between the acidic hTSHR positions Glu297, Glu303, or Asp382 and the additional basic residues of TR1401, we investigated TR1401 binding and signaling properties. Our data reveal increased cAMP signaling of the hTSHR using TR1401 and bTSH compared with hTSH. Whereas Asp382 seems to be important for bTSH- and TR1401-mediated but not for hTSH-mediated signaling, the substitution E297K exhibits a decreased signaling for all three TSH variants. Interestingly, bTSH and TR1401 showed only a slightly different binding pattern. These observations imply that specific residues of the hinge region are mediators of the superagonistic activity of bTSH and TR1401 in contrast to hTSH. Moreover, the simultaneous localization of binding components in the glycoprotein hormone molecule and the receptor hinge region permits important reevaluation of interacting hormone receptor domains.

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Structural basis of adhesive binding by desmocollins and desmogleins

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1606272113 ◽

2016 ◽

Vol 113 (26) ◽

pp. 7160-7165 ◽

Cited By ~ 74

Author(s):

Oliver J. Harrison ◽

Julia Brasch ◽

Gorka Lasso ◽

Phinikoula S. Katsamba ◽

Goran Ahlsen ◽

...

Keyword(s):

Amino Acids ◽

Crystal Structures ◽

Structural Basis ◽

Cellular Interactions ◽

Opposite Charge ◽

Charged Amino Acids ◽

Structural Framework ◽

Classical Cadherins ◽

Coated Bead

Desmosomes are intercellular adhesive junctions that impart strength to vertebrate tissues. Their dense, ordered intercellular attachments are formed by desmogleins (Dsgs) and desmocollins (Dscs), but the nature of trans-cellular interactions between these specialized cadherins is unclear. Here, using solution biophysics and coated-bead aggregation experiments, we demonstrate family-wise heterophilic specificity: All Dsgs form adhesive dimers with all Dscs, with affinities characteristic of each Dsg:Dsc pair. Crystal structures of ectodomains from Dsg2 and Dsg3 and from Dsc1 and Dsc2 show binding through a strand-swap mechanism similar to that of homophilic classical cadherins. However, conserved charged amino acids inhibit Dsg:Dsg and Dsc:Dsc interactions by same-charge repulsion and promote heterophilic Dsg:Dsc interactions through opposite-charge attraction. These findings show that Dsg:Dsc heterodimers represent the fundamental adhesive unit of desmosomes and provide a structural framework for understanding desmosome assembly.

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Negatively-charged amino acids at the peptide-binding pocket of HLA-DPB1 alleles are associated with susceptibility to anti-topoisomerase I-positive systemic sclerosis

Human Immunology ◽

10.1016/j.humimm.2016.05.012 ◽

2016 ◽

Vol 77 (7) ◽

pp. 550-554 ◽

Cited By ~ 2

Author(s):

Jeong Seok Lee ◽

Jin Kyun Park ◽

Heung Jae Kim ◽

Hyung Ki Lee ◽

Yeong Wook Song ◽

...

Keyword(s):

Amino Acids ◽

Systemic Sclerosis ◽

Topoisomerase I ◽

Peptide Binding ◽

Binding Pocket ◽

Charged Amino Acids

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Interaction of wild-type signal sequences and their charged variants with model and natural membranes

Biochemical Journal ◽

10.1042/bj2930043 ◽

1993 ◽

Vol 293 (1) ◽

pp. 43-49 ◽

Cited By ~ 4

Author(s):

N M Rao ◽

R Nagaraj

Keyword(s):

Amino Acids ◽

Synthetic Peptides ◽

Model Membranes ◽

Signal Peptides ◽

Wild Type ◽

Signal Sequences ◽

Hydrophobic Region ◽

Phospholipases A2 ◽

Charged Amino Acids

The interaction of synthetic peptides corresponding to wild-type signal sequences, and their mutants having charged amino acids in the hydrophobic region, with model and natural membranes has been studied. At high peptide concentrations, i.e. low lipid/peptide ratios, the signal peptides cause release of carboxyfluorescein (CF) from model membranes with lipid compositions corresponding to those of translocation-competent as well as translocation-incompetent membranes. Interestingly, mutant sequences, which were non-functional in vivo, caused considerable release of CF compared with the wild-type sequences. Both wild-type and mutant signal sequences perturb model membranes even at lipid/peptide ratios of 1000:1, as indicated by the activities of phospholipases A2, C and D. These studies indicate that such mutant signals are non-functional not because of their inability to interact with membranes, but due to defective targeting to the membrane. The signal peptides inhibit phospholipase C activity in microsomes, uncouple oxidative phosphorylation in mitochondria and increase K+ efflux from erythrocytes, and one of the mutant sequences is a potent degranulator of the mast cells. Both wild-type and mutant signal sequences have the ability to perturb vesicles of various lipid compositions. With respect to natural membranes, the peptides do not show any bias towards translocation-competent membranes.

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