Amino acids in the cloned mouse kappa receptor that are necessary for high affinity agonist binding but not antagonist binding

Abstract It is fundamentally important to define how agonist-receptor interaction differs from antagonist-receptor interaction. The V1a vasopressin receptor (V1aR) is a member of the neurohypophysial hormone subfamily of G protein-coupled receptors. Using alanine-scanning mutagenesis of the N-terminal juxtamembrane segment of the V1aR, we now establish that Glu54 (1.35) is critical for arginine vasopressin binding. The mutant [E54A]V1aR exhibited decreased arginine vasopressin affinity (1700-fold) and disrupted signaling, but antagonist binding was unaffected. Mutation of Glu54 had an almost identical pharmacological effect as mutation of Arg46, raising the possibility that agonist binding required a mutual interaction between Glu54 and Arg46. The role of these two charged residues was investigated by 1) substituting Glu54; 2) inserting additional Glu/Arg in transmembrane helix (TM) 1; 3) repositioning the Glu/Arg in TM1; and 4) characterizing the reciprocal mutant [R46E/E54R]V1aR. We conclude that 1) the positive/negative charges need to be precisely positioned in this N terminus/TM1 segment; and 2) Glu54 and Arg46 function independently, providing two discrete epitopes required for high-affinity agonist binding and signaling. This study explains why Glu and Arg, part of an -R(X3)L/V(X3)E(X3)L- motif, are conserved at these loci throughout this G protein-coupled receptor subfamily and provides molecular insight into key differences between agonist and antagonist binding requirements.

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Identification of Four Amino Acids in the Gastrin-releasing Peptide Receptor That Are Required for High Affinity Agonist Binding

Journal of Biological Chemistry ◽

10.1074/jbc.272.28.17405 ◽

1997 ◽

Vol 272 (28) ◽

pp. 17405-17409 ◽

Cited By ~ 36

Author(s):

Mark Akeson ◽

Eduardo Sainz ◽

Samuel A. Mantey ◽

Robert T. Jensen ◽

James F. Battey

Keyword(s):

Amino Acids ◽

Agonist Binding ◽

Peptide Receptor ◽

Gastrin Releasing Peptide ◽

High Affinity

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Molecular docking studies of tea (Thea sinensis Linn.) polyphenols inhibition pattern with Rat P-glycoprotein

Physical Sciences Reviews ◽

10.1515/psr-2018-0165 ◽

2020 ◽

Vol 0 (0) ◽

Author(s):

Babar Ali ◽

Qazi Mohammad Sajid Jamal ◽

Showkat R. Mir ◽

Saiba Shams ◽

Mohammad Amjad Kamal

Keyword(s):

Amino Acids ◽

Molecular Docking ◽

Binding Energy ◽

Oral Administration ◽

Docking Studies ◽

Vital Role ◽

Glycoprotein P ◽

High Affinity ◽

P Glycoprotein ◽

Inhibition Pattern

AbstractSince 3000 B.C., evergreen plant Thea sinensis (Theaceae) is used both as a social and medicinal beverage. Leaves of T. sinensis contain amino acids, vitamins, caffeine, polysaccharides and polyphenols. Most of the natural medicinal actions of tea are due to the availability and abundance of polyphenols mainly catechins. It has also been stated that some catechins were absorbed more rapidly than other compounds after the oral administration of tea and could increase the bio-enhancing activities of anticancer drugs by inhibiting P-glycoprotein (P-gp). The results of the molecular docking showed that polyphenols bind easily to the active P-gp site. All compounds exhibited fluctuating binding affinity ranged from −11.67 to −8.36 kcal/mol. Observed binding energy required for theaflavin to bind to P-gp was lowest (−11.67 kcal/mol). The obtained data that supports all the selected polyphenols inhibited P-gp and therefore may enhance the bioavailability of drugs. This study may play a vital role in finding hotspots in P-gp and eventually may be proved useful in designing compounds with high affinity and specificity to the protein.

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Unique uncoupling of the frog erythrocyte adenylate cyclase system by manganese. Loss of hormone and guanine nucleotide-sensitive enzyme activities without loss of nucleotide-sensitive, high affinity agonist binding.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(17)30125-4 ◽

1979 ◽

Vol 254 (8) ◽

pp. 2677-2683

Author(s):

L.E. Limbird ◽

A.R. Hickey ◽

R.J. Lefkowitz

Keyword(s):

Adenylate Cyclase ◽

Enzyme Activities ◽

Adenylate Cyclase System ◽

Guanine Nucleotide ◽

Agonist Binding ◽

High Affinity

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Human Rev1 relies on insert-2 to promote selective binding and accurate replication of stabilized G-quadruplex motifs

Nucleic Acids Research ◽

10.1093/nar/gkab041 ◽

2021 ◽

Author(s):

Amit Ketkar ◽

Lane Smith ◽

Callie Johnson ◽

Alyssa Richey ◽

Makayla Berry ◽

...

Keyword(s):

Amino Acids ◽

Mutation Frequency ◽

Control Sequence ◽

Wild Type ◽

Binding Properties ◽

High Affinity ◽

G4 Dna ◽

G Quadruplex ◽

Forward Mutagenesis

Abstract We previously reported that human Rev1 (hRev1) bound to a parallel-stranded G-quadruplex (G4) from the c-MYC promoter with high affinity. We have extended those results to include other G4 motifs, finding that hRev1 exhibited stronger affinity for parallel-stranded G4 than either anti-parallel or hybrid folds. Amino acids in the αE helix of insert-2 were identified as being important for G4 binding. Mutating E466 and Y470 to alanine selectively perturbed G4 binding affinity. The E466K mutant restored wild-type G4 binding properties. Using a forward mutagenesis assay, we discovered that loss of hRev1 increased G4 mutation frequency >200-fold compared to the control sequence. Base substitutions and deletions occurred around and within the G4 motif. Pyridostatin (PDS) exacerbated this effect, as the mutation frequency increased >700-fold over control and deletions upstream of the G4 site more than doubled. Mutagenic replication of G4 DNA (±PDS) was partially rescued by wild-type and E466K hRev1. The E466A or Y470A mutants failed to suppress the PDS-induced increase in G4 mutation frequency. These findings have implications for the role of insert-2, a motif conserved in vertebrates but not yeast or plants, in Rev1-mediated suppression of mutagenesis during G4 replication.

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