scholarly journals Peptide Binding Sites of Connexin Proteins

Chemistry ◽  
2020 ◽  
Vol 2 (3) ◽  
pp. 662-673
Author(s):  
Ágnes Simon ◽  
Csaba Magyar ◽  
László Héja ◽  
Julianna Kardos
Keyword(s):  
Molecular Mechanics ◽  
Plasma Membranes ◽  
Specific Binding ◽  
Peptide Inhibitors ◽  
Peptide Mimetics ◽  
Binding Interactions ◽  
Lack Of Information ◽  
Subtype Specificity ◽  
High Resolution Structure ◽  
Connexin Proteins

Intercellular gap junction (GJ) contacts formed by the coupling of connexin (Cx) hemichannels (HCs) embedded into the plasma membranes of neighboring cells play significant role in the development, signaling and malfunctions of mammalian tissues. Understanding and targeting GJ functions, however, calls for finding valid Cx subtype-specific inhibitors. We conjecture the lack of information about binding interactions between the GJ interface forming extracellular EL1 and EL2 loops and peptide mimetics designed to specifically inhibit Cx43HC coupling to Cx43GJ. Here, we explore active spots at the GJ interface using known peptide inhibitors that mimic various segments of EL1 and EL2. Binding interactions of these peptide inhibitors and the non-peptide inhibitor quinine has been modelled in combination with the use of blind docking molecular mechanics (MM). The neuron-specific Cx36HC and astrocyte-specific Cx43HC subtypes were modelled with a template derived from the high-resolution structure of Cx26GJ. GJ-coupled and free Cx36HC and Cx43HC models were obtained by dissection of GJs (GJ-coupled) followed by 50 ns molecular dynamics (free). Molecular mechanics (MM) calculations were performed by the docking of inhibitors, explicitly the designed Cx43 EL1 or EL2 loop sequence mimetics (GAP26, P5 or P180–195, GAP27, Peptide5, respectively) and the Cx36 subtype-specific quinine into the model structures. In order to explore specific binding interactions between inhibitors and CxHC subtypes, MM/Generalized Born Surface Area (MM/GBSA) ΔGbind values for representative conformers of peptide mimetics and quinine were evaluated by mapping the binding surface of Cx36HC and Cx43HC for all inhibitors. Quinine specifically contacts Cx36 EL1 residues V54-C55-N56-T57-L58, P60 and N63. Blocking the vestibule by the side of Cx36HC entry, quinine explicitly interacts with the non-conserved V54, L58, N63 residues of Cx36 EL1. In addition, our work challenges the predicted specificity of peptide mimetics, showing that the docking site of peptides is unrelated to the location of the sequence they mimic. Binding features, such as unaffected EL2 residues and the lack of Cx43 subtype-specificity of peptide mimetics, suggest critical roles for peptide stringency and dimension, possibly pertaining to the Cx subtype-specificity of peptide inhibitors.

Characteristic features of specific binding of pentapeptide HFPGP labeled at the C-terminal proline residue to rat forebrain plasma membranes

2014 ◽  
Vol 456 (1) ◽  
pp. 101-103 ◽  
Author(s):  
T. V. Vyunova ◽  
L. A. Andreeva ◽  
K. V. Shevchenko ◽  
V. P. Shevchenko ◽  
M. Yu. Bobrov ◽  
...  
Keyword(s):  
Plasma Membranes ◽  
Specific Binding ◽  
Proline Residue ◽  
Rat Forebrain ◽  
Characteristic Features

scholarly journals Photoaffinity labelling of a nitrobenzylthioinosine-binding polypeptide from cultured Novikoff hepatoma cells

1986 ◽  
Vol 236 (3) ◽  
pp. 665-670 ◽  
Author(s):  
W P Gati ◽  
J A Belt ◽  
E S Jakobs ◽  
J D Young ◽  
S M Jarvis ◽  
...  
Keyword(s):  
Gel Electrophoresis ◽  
Plasma Membranes ◽  
Specific Binding ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Hepatoma Cells ◽  
Nucleoside Transport ◽  
Facilitated Diffusion ◽  
Animal Cells ◽  
Novikoff Hepatoma

Site-specific binding of nitrobenzylthioinosine (NBMPR) to plasma membranes of some animal cells results in the inhibition of the facilitated diffusion of nucleosides. The present study showed that nucleoside transport in Novikoff UA rat hepatoma cells is insensitive to site-saturating concentrations of NBMPR. Equilibrium binding experiments demonstrated the presence of high-affinity sites for NBMPR in a membrane-enriched fraction from these cells. In the presence of uridine or dipyridamole, specific binding of NBMPR at these sites was inhibited. When Novikoff UA membranes were covalently labelled with [3H]NBMPR by using photoaffinity techniques, specifically bound radioactivity was incorporated exclusively into a polypeptide(s) with an apparent Mr of 72,000-80,000, determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Covalent labelling of this polypeptide was abolished in the presence of excess nitrobenzylthioguanosine (NBTGR) and reduced in the presence of adenosine, uridine or dipyridamole. The apparent Mr of the NBMPR-binding polypeptide in Novikoff UA cells is significantly higher than that reported for corresponding polypeptides in other cell types (Mr 45,000-66,000). When membrane-enriched preparations from S49 mouse lymphoma cells were photolabelled and mixed with labelled NovikoffUA membrane-enriched preparations, gel electrophoresis resolved the NBMPR-binding polypeptides from the two preparations.

Ontogeny of epidermal growth factor receptor tyrosine kinase in rat liver

1991 ◽  
Vol 260 (2) ◽  
pp. G290-G298 ◽  
Author(s):  
B. K. De ◽  
T. L. Brown ◽  
F. J. Suchy
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Tyrosine Kinase ◽  
Rat Liver ◽  
Plasma Membranes ◽  
Egf Receptor ◽  
Specific Binding ◽  
Liver Plasma Membranes ◽  
Epidermal Growth ◽  
Autokinase Activity

The binding of epidermal growth factor (EGF) to its receptor and the activity of the receptor intrinsic protein-tyrosine kinase were studied during the ontogeny of rat liver. The number of EGF receptors during pre- and postnatal development was first compared in crude liver plasma membranes using 1) specific binding of 125I-labeled EGF and 2) immunoblot analysis using any antireceptor polyclonal rabbit antibody. Both methods detected the expression of the EGF receptor in fetal rat liver on day 17 of gestation, but in an amount markedly less than the adult. Within 24 h, there was a more than twofold increase in EGF binding to plasma membranes as well as a marked increase in receptor immunoreactivity. However, after birth, there was a precipitous drop in receptor number to less than 20% of the adult level by the end of the first postnatal day (P less than 0.001). Next, the presence of EGF-stimulated tyrosine kinase activity (autophosphorylation) was determined during the same stages of development. Electrophoresis of membranes phosphorylated in the presence or absence of EGF followed by autoradiography demonstrated autokinase activity stimulated by EGF in day 18 and 19 fetal liver plasma membranes, but not in membranes on day 17 of gestation. Similar to the pattern observed with EGF binding, there was a decrease in autokinase activity in early neonatal plasma membranes followed by an increase to near adult levels by 7 days postnatally. Quantitation of the amount of 32P radioactivity associated with the EGF receptor bands in each age group, correlated with the degree of autophosphorylation assessed by autoradiography.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Interaction of Bacillus thuringiensis Cry1 and Vip3A Proteins with Spodoptera frugiperda Midgut Binding Sites

2009 ◽  
Vol 75 (7) ◽  
pp. 2236-2237 ◽  
Author(s):  
Janete A. D. Sena ◽  
Carmen Sara Hernández-Rodríguez ◽  
Juan Ferré
Keyword(s):  
Bacillus Thuringiensis ◽  
Spodoptera Frugiperda ◽  
Binding Sites ◽  
Specific Binding ◽  
Binding Assays ◽  
Binding Interactions ◽  
Specific Binding Sites

ABSTRACT Vip3Aa, Vip3Af, Cry1Ab, and Cry1Fa were tested for their toxicities and binding interactions. Vip3A proteins were more toxic than Cry1 proteins. Binding assays showed independent specific binding sites for Cry1 and Vip3A proteins. Cry1Ab and Cry1Fa competed for the same binding sites, whereas Vip3Aa competed for those of Vip3Af.

Processing of receptor-bound somatostatin: internalization and degradation by pancreatic acini

1987 ◽  
Vol 252 (4) ◽  
pp. G535-G542 ◽  
Author(s):  
N. Viguerie ◽  
J. P. Esteve ◽  
C. Susini ◽  
N. Vaysse ◽  
A. Ribet
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Specific Binding ◽  
Specific Radioactivity ◽  
Pancreatic Acinar Cells ◽  
Nuclear Fraction ◽  
Pancreatic Acini ◽  
Specific Binding Sites ◽  
Membrane Bound ◽  
Membrane Level

We have previously demonstrated the presence of specific binding sites for somatostatin on plasma membranes from pancreatic acinar cells. In the present study we attempted to characterize the fate of receptor-bound 125I-[Tyr11]somatostatin. Internalization of somatostatin was rapid (reaching a plateau at 20% of the cell-associated specific radioactivity) and temperature dependent. To follow the processing of bound somatostatin, acini were incubated with 125I-[Tyr11]somatostatin at 5 degrees C during 16 h then, after washing, incubated at 37 degrees C for 90 min in fresh medium. Surface-bound somatostatin decreased rapidly, whereas radioactivity increased in the cell interior and the incubation medium. Intracellular and membrane-bound radioactivity was mainly intact 125I-[Tyr11]somatostatin. Degradation occurred at the plasma membrane level and led to iodotyrosine production. After 15 min of incubation, 15% of the initially surface-bound 125I-[Tyr11]somatostatin was compartmentalized within the cell, mainly in the microsomal fraction. After 30 min, a significant increase in radioactivity appeared in the nuclear fraction. These results indicate that the major part of somatostatin cellular degradation takes place at the plasma membrane level. Within the cell, somatostatin is routed to the nucleus via particular fractions sedimenting with microsomal vesicles.

Ontogeny of hepatic insulin and glucagon receptors and adenylate cyclase in rabbit

1983 ◽  
Vol 244 (6) ◽  
pp. E624-E631
Author(s):  
S. Ganguli ◽  
M. K. Sinha ◽  
B. Sterman ◽  
P. Harris ◽  
M. A. Sperling
Keyword(s):  
Adenylate Cyclase ◽  
Cholera Toxin ◽  
Plasma Membranes ◽  
Specific Binding ◽  
Regulatory Protein ◽  
Late Gestation ◽  
Camp Production ◽  
Hormonal Activation ◽  
Glucagon Receptors ◽  
Significant Increment

In rabbit liver plasma membranes (LPM), specific binding of 125I-insulin rapidly increased in late gestation and peaked at birth, declining thereafter. In contrast, 125I-glucagon binding was lowest in late gestation, somewhat higher at birth, and increased by 48 h although only to 20-25% of adult. These changes in binding were due to changing numbers of receptors involving predominantly high affinity sites for insulin and low affinity sites for glucagon, with only minor changes in affinity. Despite measurable glucagon receptors by birth, fetal LPM produced no increment above basal in cAMP production with maximal doses of glucagon (10(-6) M), prostaglandin E1 (10(-4) M), or epinephrine (10(-4) M). Near birth only NaF (10 mM) produced a modest but significant increment in cAMP. By 2 h postbirth, all stimuli evoked significant increments in cAMP production that increased progressively but was still only 15-20% of adult at 48 h. Furthermore, although specific binding of cholera toxin was greater in fetal LPM (11 +/- 1 vs. 6 +/- 1%), cholera toxin-stimulated cAMP production increased by only 12-26% above basal in the fetus compared with 220% in adult. Markers of membrane purity including 5'-nucleotidase, phosphodiesterase, and insulin or glucagon degradation were not different in fetus and adult. We conclude that receptors and components of the adenylate cyclase complex mature independently; initial coupling occurs between the G/F regulatory protein and the catalytic unit (NaF but not hormonal activation) followed within hours of birth by coupling to the hormone receptor.

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