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The orthosteric agonist neurokinin A (NKA) interacts with the tachykinin NK2 receptors (NK2Rs) via an apparent sequential binding process, which stabilizes the receptor in at least two different active conformations (A1L and A2L). The A1L conformation exhibits fast NKA dissociation kinetics and triggers intracellular calcium elevation; the A2L conformation exhibits slow NKA dissociation kinetics and triggers cAMP production. The new compound LPI805 is a partial and noncompetitive inhibitor of NKA binding to NK2Rs. Analysis of NKA dissociation in the presence of LPI805 suggests that LPI805 decreases the number of NKA-NK2R complexes in A2L conformation while increasing those in the A1L conformation. Analysis of signaling pathways of NK2Rs shows that LPI805 dramatically inhibits the NKA-induced cAMP response while slightly enhancing the NKA-induced calcium response. Analysis of NKA association kinetics reveals that LPI805 promotes strong and specific destabilization of the NKA-NK2R complexes in the A2L conformation whereas access of NKA to the A1L conformations is unchanged. Thus, to our knowledge, LPI805 is the first example of a conformation-specific allosteric antagonist of a G-protein-coupled receptor. This work establishes the use of allosteric modulators in order to promote functional selectivity on certain agonist-receptor interactions.

A functional fluorescent neurokinin NK2 receptor, EGFP-NK2, was previously used to follow, by fluorescence resonance energy transfer measurements in living cells, the binding of its fluorescently labeled agonist, bodipy-neurokinin A (NKA). Local agonist application suggested that the activation and desensitization of the NK2 receptors were compartmentalized at the level of the plasma membrane. In this study, fluorescence recovery after photobleaching experiments are carried out at variable observation radius (vrFRAP) to probe EGFP-NK2 receptor mobility and confinement. Experiments are carried out at 20 degrees C to maintain the number of receptors constant at the cell surface during recordings. In the absence of agonist, 35% EGFP-NK2 receptors diffuse within domains of 420 +/- 80 nm in radius with the remaining 65% of receptors able to diffuse with a long range lateral diffusion coefficient between the domains. When cells are incubated with a saturating concentration of NKA, 30% EGFP-NK2 receptors become immobilized in small domains characterized by a radius equal to 170 +/- 50 nm. Biochemical experiments show that the confinement of EGFP-NK2 receptor is not due to its association with rafts at any given time. Colocalization of the receptor with beta-arrestin and transferrin supports that the small domains, containing 30% of activated EGFP-NK2, correspond to clathrin-coated pre-pits. The similar amount of confined EGFP-NK2 receptors found before and after activation (30-35%) is discussed in term of putative transient interactions of the receptors with preexisting scaffolds of signaling molecules.

By combining real time measurements of agonist binding, by fluorescence resonance energy transfer, and of subsequent responses, we proposed previously that the neurokinin NK2 receptor preexists in equilibrium between three states: inactive, calcium-triggering, and cAMP-producing. Thr(24) and Phe(26) of the NK2 receptor extracellular domain are considered to interact with neuropeptide agonists based on the reduction of affinity when they are substituted by alanine. Using fluorescence resonance energy transfer, we now quantify the binding kinetics of two Texas Red-modified neurokinin A agonists to the fluorescent wild-type (Y-NK2wt) and the mutant (Y-NK2mut) receptor carrying Thr(24) --> Ala and Phe(26) --> Ala mutations. TR1-neurokinin A binds with a fast component and a slow component to the Y-NK2wt receptor and triggers both a calcium and a cAMP response. In contrast, on the mutant receptor, it binds in a single fast step with a lower apparent affinity and activates only the calcium response. Another agonist, TRC4-neurokinin A, binds to both wild-type and mutant receptors in a single fast step, with similar affinities and kinetics and promotes only calcium signaling. Kinetic modeling of ligand binding and receptor interconversions is carried out to analyze phenotypic changes in terms of binding alterations or changes in the transitions between conformational states. We show that the binding and response properties of the Y-NK2mut receptor are best described according to a phenotype where a reduction of the transition between the inactive and the active states occurs.

International audience; Neurokinins (substance P, neurokinin A and neurokinin B) and the neurokinin receptors, the NK1 and NK3 receptors, are largely expressed in the nucleus of the solitary tract (NST) where they are involved in the central regulation of visceral function. Studying the mechanisms that control neurokinin release can provide valuable information concerning the control of autonomic functions subserved by the NST. Glutamate is the principal excitatory neurotransmitter in the NST and the main neurotransmitter of afferent vagal fibers. Neurokinins and glutamate may interact within the NST. In the present study, we have examined the contribution of the N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) subtypes of glutamate receptors on the release of the endogenous neurokinins in the NST. We used internalization of the NK1 or NK3 receptor as an index of endogenous neurokinin release assessed by immunocytochemical visualization of the NK1 or NK3 receptor endocytosis. Experiments were performed in vitro using rat brainstem slices. A first series of experiments were done in order to validate our in vitro preparation. Application of substance P, neurokinin A or neurokinin B induced dose-dependent internalization of NK1 and NK3 receptor. This was blocked by the endocytosis inhibitor, phenylarzine oxide. The NK1 receptor antagonist SR140333 blocked internalization of NK1 receptor induced by the three neurokinins. In addition, the internalization NK1 or NK3 receptor was reversible. These results demonstrate that internalization and recycling mechanisms of NK1 or NK3 receptor were preserved in in vitro brainstem slices. Application of NMDA or AMPA induced internalization of NK1 receptor. This was blocked by the application of SR140333 suggesting that NK1 receptor internalization is due to the binding of endogenous neurokinin released under the effects of NMDA and AMPA. Application of NMDA or AMPA had no effect on NK3 receptor. Application of tetrodotoxin blocked NK1 receptor internalization induced by NMDA, demonstrating that the release of neurokinins is dependent of axon potential propagation. This result excludes the hypothesis of a release on neurokinins via pre-synaptic NMDA receptors located on neurokinin-containing axon terminals. NMDA or AMPA may directly induce neurokinin release in the NST by acting on receptors located on the cell bodies and dendrites of neurokinin-containing neurons. Release of neurokinins may also be the result of a general activation of neuron networks of the NST by NMDA or AMPA. To conclude, our results suggest that glutamate, through activation of post-synaptic NMDA and AMPA receptors, contributes to neurokinin signaling in the NST.