title: Characterization of a fluorescent substance P analog.
abstract: We describe the development and characterization of substance P labeled at Lys3 with fluorescein ([fluorescein Lys3]SP) as a fluorescent probe for the neurokinin 1 (NK1) receptor. [fluorescein Lys3]SP is an agonist at the human NK1 receptor, with an affinity for both the high-affinity and low-affinity binding states of the receptor approximately 6-fold lower than that of substance P. Binding of the probe to the human NK1 receptor expressed in Sf9 insect cells was observed directly by monitoring either a decrease in fluorescence intensity or an increase in anisotropy of the [fluorescein Lys3]SP. Detection by anisotropy gave the larger signal and thus was used to characterize the interaction of [fluorescein Lys3]SP with the receptor. The anisotropy of the bound ligand was 0.17, compared to 0.04 for the free ligand. The fluorescence was quenched by about 15% upon binding to the receptor. Bound [fluorescein Lys3]SP was displaced by unlabeled SP and by the quinuclidine antagonist L-703,606. As expected for an agonist, binding was also reduced by the addition of the nonhydrolyzable guanine nucleotide analog GppNHp. [fluorescein Lys3]SP should provide a useful structural and kinetic probe for the NK1 receptor.
authors.affiliation: Department of Molecular Pharmacology and Biochemistry, Merck Research Laboratories, Rahway, New Jersey 07065.
authors.forename: M R
authors.initials: MR
authors.lastname: Tota
authors.forename: S
authors.initials: S
authors.lastname: Daniel
authors.forename: A
authors.initials: A
authors.lastname: Sirotina
authors.forename: K E
authors.initials: KE
authors.lastname: Mazina
authors.forename: T M
authors.initials: TM
authors.lastname: Fong
authors.forename: J
authors.initials: J
authors.lastname: Longmore
authors.forename: C D
authors.initials: CD
authors.lastname: Strader
mesh_terms: D000595:Amino Acid Sequence; D016880:Anisotropy; D001667:Binding, Competitive; D002021:Buffers; D019793:Fluorescein; D005452:Fluoresceins / Q000737:chemistry*; D005456:Fluorescent Dyes; D005786:Gene Expression Regulation / Q000235:genetics; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007457:Iodine Radioisotopes; D008239:Lysine / Q000737:chemistry*; D008969:Molecular Sequence Data; D064729:Neurokinin-1 Receptor Antagonists; D011812:Quinuclidines / Q000737:chemistry / Q000378:metabolism; D018040:Receptors, Neurokinin-1 / Q000096:biosynthesis / Q000235:genetics; D012016:Reference Values; D013050:Spectrometry, Fluorescence; D013373:Substance P / Q000031:analogs & derivatives*; D013696:Temperature
chemical_list: D002021:Buffers; D005452:Fluoresceins; D005456:Fluorescent Dyes; D007457:Iodine Radioisotopes; D064729:Neurokinin-1 Receptor Antagonists; D011812:Quinuclidines; D018040:Receptors, Neurokinin-1; C076851:L 703606; D013373:Substance P; D008239:Lysine; D019793:Fluorescein