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Orthosteric binding of ?-Da1a, a natural peptide of snake venom interacting selectively with the ?1A-adrenoceptor.

ABSTRACT: ?-Da1a is a three-finger fold toxin from green mamba venom that is highly selective for the ?1A-adrenoceptor. This toxin has atypical pharmacological properties, including incomplete inhibition of (3)H-prazosin or (125)I-HEAT binding and insurmountable antagonist action. We aimed to clarify its mode of action at the ?1A-adrenoceptor. The affinity (pKi 9.26) and selectivity of ?-Da1a for the ?1A-adrenoceptor were confirmed by comparing binding to human adrenoceptors expressed in eukaryotic cells. Equilibrium and kinetic binding experiments were used to demonstrate that ?-Da1a, prazosin and HEAT compete at the ?1A-adrenoceptor. ?-Da1a did not affect the dissociation kinetics of (3)H-prazosin or (125)I-HEAT, and the IC50 of ?-Da1a, determined by competition experiments, increased linearly with the concentration of radioligands used, while the residual binding by ?-Da1a remained stable. The effect of ?-Da1a on agonist-stimulated Ca(2+) release was insurmountable in the presence of phenethylamine- or imidazoline-type agonists. Ten mutations in the orthosteric binding pocket of the ?1A-adrenoceptor were evaluated for alterations in ?-Da1a affinity. The D106(3.32)A and the S188(5.42)A/S192(5.46)A receptor mutations reduced toxin affinity moderately (6 and 7.6 times, respectively), while the F86(2.64)A, F288(6.51)A and F312(7.39)A mutations diminished it dramatically by 18- to 93-fold. In addition, residue F86(2.64) was identified as a key interaction point for (125)I-HEAT, as the variant F86(2.64)A induced a 23-fold reduction in HEAT affinity. Unlike the M1 muscarinic acetylcholine receptor toxin MT7, ?-Da1a interacts with the human ?1A-adrenoceptor orthosteric pocket and shares receptor interaction points with antagonist (F86(2.64), F288(6.51) and F312(7.39)) and agonist (F288(6.51) and F312(7.39)) ligands. Its selectivity for the ?1A-adrenoceptor may result, at least partly, from its interaction with the residue F86(2.64), which appears to be important also for HEAT binding.

SUBMITTER: Maiga A

PROVIDER: S-EPMC3723878 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Orthosteric binding of ρ-Da1a, a natural peptide of snake venom interacting selectively with the α1A-adrenoceptor.

Maïga Arhamatoulaye A Merlin Jon J Marcon Elodie E Rouget Céline C Larregola Maud M Gilquin Bernard B Fruchart-Gaillard Carole C Lajeunesse Evelyne E Marchetti Charles C Lorphelin Alain A Bellanger Laurent L Summers Roger J RJ Hutchinson Dana S DS Evans Bronwyn A BA Servent Denis D Gilles Nicolas N

PloS one 20130725 7

ρ-Da1a is a three-finger fold toxin from green mamba venom that is highly selective for the α1A-adrenoceptor. This toxin has atypical pharmacological properties, including incomplete inhibition of (3)H-prazosin or (125)I-HEAT binding and insurmountable antagonist action. We aimed to clarify its mode of action at the α1A-adrenoceptor. The affinity (pKi 9.26) and selectivity of ρ-Da1a for the α1A-adrenoceptor were confirmed by comparing binding to human adrenoceptors expressed in eukaryotic cells. ...[more]

PMID: 23935897

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Project description:The ?1A-AR is thought to couple predominantly to the G?q/PLC pathway and lead to phosphoinositide hydrolysis and calcium mobilization, although certain agonists acting at this receptor have been reported to trigger activation of arachidonic acid formation and MAPK pathways. For several G protein-coupled receptors (GPCRs) agonists can manifest a bias for activation of particular effector signaling output, i.e., not all agonists of a given GPCR generate responses through utilization of the same signaling cascade(s). Previous work with G?q coupling-defective variants of ?1A-AR, as well as a combination of Ca2+ channel blockers, uncovered cross-talk between ?1A-AR and ?2-AR that leads to potentiation of a G?q-independent signaling cascade in response to ?1A-AR activation. We hypothesized that molecules exist that act as biased agonists to selectively activate this pathway. In this report, isoproterenol (Iso), typically viewed as ?-AR-selective agonist, was examined with respect to activation of ?1A-AR. ?1A-AR selective antagonists were used to specifically block Iso evoked signaling in different cellular backgrounds and confirm its action at ?1A-AR. Iso induced signaling at ?1A-AR was further interrogated by probing steps along the G?q /PLC, G?s and MAPK/ERK pathways. In HEK-293/EBNA cells transiently transduced with ?1A-AR, and CHO_?1A-AR stable cells, Iso evoked low potency ERK activity as well as Ca2+ mobilization that could be blocked by ?1A-AR selective antagonists. The kinetics of Iso induced Ca2+ transients differed from typical G?q- mediated Ca2+ mobilization, lacking both the fast IP3R mediated response and the sustained phase of Ca2+ re-entry. Moreover, no inositol phosphate (IP) accumulation could be detected in either cell line after stimulation with Iso, but activation was accompanied by receptor internalization. Data are presented that indicate that Iso represents a novel type of ?1A-AR partial agonist with signaling bias toward MAPK/ERK signaling cascade that is likely independent of coupling to G?q.

Dataset Information

Orthosteric binding of ?-Da1a, a natural peptide of snake venom interacting selectively with the ?1A-adrenoceptor.

Publications

Orthosteric binding of ρ-Da1a, a natural peptide of snake venom interacting selectively with the α1A-adrenoceptor.

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