Kinetic evidence for tandemly arranged ligand binding sites in melanocortin 4 receptor complexes

Kopanchuk, Sergei; Veikšina, Santa; Mutulis, Felikss; Mutule, Ilze; Yahorava, Sviatlana; Mandrika, Ilona; Petrovska, Ramona; Rinken, Ago; Wikberg, Jarl E. S.

doi:10.1016/j.neuint.2006.04.006

Cited by 31 publications

(39 citation statements)

References 21 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The studies with muscarinic receptors, where the cooperative regulation of the antagonist binding was abolished by the treatment of membranes with high ionic strength [34] or by the solubilization of the receptors [35] gives also hints about the role of receptor heteromerization on the ligand binding kinetics. The model of the interrerlated binding sites as been proposed by Jakubik and coworkers as ''Tandem Two-site Model of Ligand Binding to Muscarinic Acetylcholine Receptor'' [36], has been generalized to receptor oligomers for melanocortin receptors [37]. Unfortunately we could not obtain systematic kinetic data for [ 3 H]WAY100635 binding due to its faster association at higher radioligand concentrations and more detail kinetic mechanism of its binding remains to be clarified.…”

Section: Resultsmentioning

confidence: 94%

“…If using higher concentrations [ 3 H]WAY100635, the radioligand binding is very fast and it would overshot the competitors binding, but its release from the receptors is very slow. This would lead to underestimation of competitors' potencies, as it has been described for melanocortin receptors, where the potencies differ more than 1000 times [37]. Thus the competitive binding experiments using [ 3 H]WAY100635 are challenged, but it seems to be an excellent tool for receptor number determination, since it binds almost irreversibly, with very high affinity and relatively low level of non-specific binding.…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Characteristics of Binding of [3H]WAY100635 to Rat Hippocampal Membranes

Parkel

Rinken

2006

Neurochem Res

Self Cite

View full text Add to dashboard Cite

Kinetic analysis of binding of [(3)H][N-[2-[4-(2-[O-methyl-(3)H]methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)cyclohexane carboxamide ([(3)H]WAY100635) to 5-HT(1A) receptors in rat hippocampal membranes has revealed complex regulation mechanism for this radioligand. Saturation binding experiments revealed that [(3)H]WAY100635 binds to a single class of receptors with very high apparent affinity (K (D) = 87 +/- 4 pM, B (max) = 15.1 +/- 0.2 fmol/mg protein). The binding was almost irreversible, as the dissociation rate constant obtained k (off) = (7.8 +/- 1.1) x 10(-3) min(-1), means that equilibrium with this radioligand cannot be achieved before 7.5 h incubation at 25 degrees C. Systematic association kinetic studies of [(3)H]WAY100635 binding revealed sharp reaction acceleration at higher radioligand concentration, proposing mechanism of positive cooperativity. The affinities of antagonists determined from competition with [(3)H]WAY100635 did not coincide with their abilities to inhibit 5-HT-dependent activation of [(35)S]GTPgammaS binding probably due to the ligand's kinetic peculiarities. Thus, [(3)H]WAY100635 appears to be an excellent tool for determining receptor binding sites, but its applicability in equilibrium studies is strongly limited.

show abstract

Section: Resultsmentioning

confidence: 94%

Section: Resultsmentioning

confidence: 99%

Characteristics of Binding of [3H]WAY100635 to Rat Hippocampal Membranes

Parkel

Rinken

2006

Neurochem Res

Self Cite

View full text Add to dashboard Cite

show abstract

“…Mutations of single amino acids are thought to disrupt the dimerization interface by changing the hydrophobicity of the helices. Recently, calculation of ligand binding kinetics suggested existence of two tandemly arranged binding sites in a MC4R oligomer [16]. To fully understand MC4R function, it is of profound importance to know how interaction of wild type/wild type or wild type/mutant MC4R occurs.…”

Section: A Heterozygous Mutation In the Third Transmembrane Domain Camentioning

confidence: 99%

A Heterozygous Mutation in the Third Transmembrane Domain Causes a Dominant-Negative Effect on Signalling Capability of the MC4R

Tarnow

Rediger²,

Brumm³

et al. 2008

Obes Facts

View full text Add to dashboard Cite

Background: Heterozygous MC4R mutation is the most frequent cause of monogenic obesity. For most MC4R mutations a gene dosage effect seems to be the underlying mechanism. However, a dominant negative effect of a heterozygous MC4R mutation was recently identified, pointing to an additional mechanism of MC4R inactivation. Methods: The complete loss-of-function mutation (Ser136Phe), identified in a cohort of obese Austrian patients, was characterized for cell surface expression, signal transduction and ligand binding properties. Co-transfection studies tested for a dominant negative effect. Dimerization was investigated by a sandwich ELISA and by fluorescence resonance energy transfer (FRET) approach. Potential intramolecular interactions of Ser136 were studied by homologous receptor modelling based on the crystal structure of the β2-adrenergic receptor. Results: The Ser136Phe mutation showed a dominant negative effect. The sandwich ELISA and FRET approach demonstrated dimerization of mutant and wild type receptor. Receptor modelling revealed an essential function of Ser136 at transmembrane helix 3 (TMH3) for establishing H-bonds between TMH2, TMH3, and TMH7. The mutation Ser136Phe most likely disrupts this network and leads to an incompetent helix-helix arrangement in the mutated receptor. Conclusion: Identification of dominant negative MC4R mutations is important to fully understand receptor function and to determine receptor regions that are involved in MC4R dimer activation.

show abstract

“…It enabled making the receptor sample and assay environment "cleaner" for the binding studies, so acceptable data of MCR ligand-binding affinities became available. After revealing allosteric interactions between binding sites of MCR, 3 models of tandemly arranged ligandbinding sites 4 and ago-allosteric modulation 5 were proposed. Implementation of a fluorescence anisotropy/intensity assay with a dye-labeled NDP-α-MSH for MC 4 R allowed monitoring of ligand binding in real time 6 and revealed complexities in the binding processes, which have a big impact on the apparent affinities.…”

Section: Introductionmentioning

confidence: 99%