Mutations in the extracellular amino-terminal domain of the NK2 neurokinin receptor abolish cAMP signaling but preserve intracellular calcium responses.

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.