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NK1 receptor fused to beta-arrestin displays a single-component, high-affinity molecular phenotype
Lene Martini1, Hanne Hastrup, Birgitte Holst
1Laboratory for Molecular Pharmacology, Department of Pharmacology, The Panum Institute, University of Copenhagen, Denmark.
Abstract:
Arrestins are cytosolic proteins that, upon stimulation of seven transmembrane (7TM) receptors, terminate signaling by binding to the receptor, displacing the G protein and targeting the receptor to clathrin-coated pits. Fusion of beta-arrestin1 to the C-terminal end of the neurokinin NK1 receptor resulted in a chimeric protein that was expressed to some extent on the cell surface but also accumulated in transferrin-labeled recycling endosomes independently of agonist stimulation. As expected, the fusion protein was almost totally silenced with respect to agonist-induced signaling through the normal Gq/G11 and Gs pathways. The NK1-beta-arrestin1 fusion construct bound nonpeptide antagonists with increased affinity but surprisingly also bound two types of agonists, substance P and neurokinin A, with high, normal affinity. In the wild-type NK1 receptor, neurokinin A (NKA) competes for binding against substance P and especially against antagonists with up to 1000-fold lower apparent affinity than determined in functional assays and in homologous binding assays. When the NK1 receptor was closely fused to G proteins, this phenomenon was eliminated among agonists, but the agonists still competed with low affinity against antagonists. In contrast, in the NK1-beta-arrestin1 fusion protein, all ligands bound with similar affinity independent of the choice of radioligand and with Hill coefficients near unity. We conclude that the NK1 receptor in complex with arrestin is in a high-affinity, stable, agonist-binding form probably best suited to structural analysis and that the receptor can display binding properties that are nearly theoretically ideal when it is forced to complex with only a single intracellular protein partner.
Insights
Fusion of beta-arrestin1 to the neurokinin NK1 receptor created a stable complex. This NK1-beta-arrestin1 fusion protein exhibits ideal binding properties, making it suitable for structural analysis.
Area of Science:
- Molecular pharmacology
- Cell biology
- Structural biology
Background:
- Arrestins are key regulators of seven transmembrane (7TM) receptor signaling, terminating downstream pathways.
- Neurokinin NK1 receptor (NK1R) mediates signaling via G proteins and is regulated by arrestins.
- Understanding receptor-arrestin interactions is crucial for drug development and signaling pathway elucidation.
Purpose of the Study:
- To investigate the functional and binding properties of a chimeric NK1 receptor fused to beta-arrestin1.
- To determine if constitutive arrestin binding alters NK1 receptor ligand affinity and signaling.
- To assess the potential of the NK1-beta-arrestin1 fusion for structural studies.
Main Methods:
- Construction and expression of a chimeric NK1 receptor fused to beta-arrestin1.
- Cell surface expression analysis and localization studies using transferrin.
- Agonist and antagonist binding assays to determine ligand affinities and kinetics.
- Functional signaling assays to assess G protein-mediated pathways (Gq/G11 and Gs).
Main Results:
- The NK1-beta-arrestin1 fusion protein was expressed on the cell surface and in recycling endosomes.
- The fusion protein was functionally silenced for G protein-mediated signaling.
- Unlike wild-type NK1R, the fusion protein exhibited similar high affinities for agonists (substance P, neurokinin A) and antagonists, with Hill coefficients near unity.
- This indicates a stable, high-affinity agonist-binding conformation upon arrestin complexation.
Conclusions:
- Constitutive fusion of beta-arrestin1 to the NK1 receptor stabilizes an agonist-bound conformation.
- The NK1-beta-arrestin1 complex displays near-ideal binding properties, simplifying ligand affinity determination.
- This stabilized receptor-arrestin complex is a promising candidate for high-resolution structural analysis.