CHARACTERIZATION OF A FLUORESCENT SUBSTANCE-P ANALOG

We describe the development and characterization of substance P labele d at Lys(3) with fluorescein ([fluorescein Lys(3)]SP) as a fluorescent probe for the neurokinin 1 (NK1) receptor. [fluorescein Lys(3)]SP is an agonist at the human NK1 receptor, with an affinity for both the hi gh-affinity and low-affinity binding states of the receptor approximat ely 6-fold lower than that of substance P. Binding of the probe to the human NK1 receptor expressed in Sf9 insect cells was observed directl y by monitoring either a decrease in fluorescence intensity or an incr ease in anisotropy of the [fluorescein Lys(3)]SP. Detection by anisotr opy gave the larger signal and thus was used to characterize the inter action of [fluorescein Lys(3)]SP with the receptor. The anisotropy of the bound ligand was 0.17, compared to 0.04 for the free ligand. The f luorescence was quenched by about 15% upon binding to the receptor. Bo und [fluorescein Lys(3)]SP was displaced by unlabeled SP and by the qu inuclidine antagonist L-703,606. As expected for an agonist, binding w as also reduced by the addition of the nonhydrolyzable guanine nucleot ide analog GppNHp. [fluorescein Lys(3)]SP should provide a useful stru ctural and kinetic probe for the NK1 receptor.