title: Photoaffinity labeling of pituitary GnRH receptors: significance of the position of photolabel on the ligand.
abstract: Photoreactive derivatives of GnRH and its analogues were prepared by incorporation of the 2-nitro-4(5)-azidophenylsulfenyl [2,4(5)-NAPS] group into amino acid residues at positions 1, 3, 6, or 8 of the decapeptide sequence. The modification of Trp3 by the 2,4-NAPS group led to a complete loss of the luteinizing hormone (LH) releasing as well as LH-release-inhibiting activity of the peptide. The [D-Lys(2,4-NAPS)]6 analogue was a very potent agonist that, after covalent attachment by photoaffinity labeling, caused prolonged LH secretion at a submaximal rate. [Orn(2,4-NAPS)]8-GnRH, a full agonist with a relative potency of 7% of GnRH, after photoaffinity labeling caused prolonged maximal LH release from cultured pituitary cells. In contrast, [Orn(2,5-NAPS)]8-GnRH, although being equipotent with the 2,4-NAPS isomer in terms of LH releasing ability, was unable to cause prolonged LH release after photoaffinity labeling. Thus, [Orn(2,4-NAPS)]8-GnRH is a very effective photolabeling ligand of the functionally significant pituitary GnRH receptor. Based on this compound, a pituitary peptidase resistant derivative, D-Phe6,[Orn(2,4-NAPS)]8-GnRH-(1-9)-ethylamide, was synthesized. This derivative showed high-affinity binding to pituitary membranes with a Kd comparable to those of other GnRH analogues. A radioiodinated form of this peptide was used for pituitary GnRH-receptor labeling. This derivative labeled 59- and 57-kDa proteins in rat and 58- and 56-kDa proteins in bovine pituitary membrane preparations, respectively. This peptide also labeled pituitary GnRH receptors in the solubilized state and therefore appears to be a suitable ligand for the isolation and further characterization of the receptor.
authors.affiliation: Department of Developmental Biology, Genentech, Inc., South San Francisco, California 94080.
authors.forename: K
authors.initials: K
authors.lastname: Nikolics
authors.forename: E
authors.initials: E
authors.lastname: Szönyi
authors.forename: J
authors.initials: J
authors.lastname: Ramachandran
mesh_terms: D000345:Affinity Labels / Q000138:chemical synthesis*; D000818:Animals; D001667:Binding, Competitive; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D007987:Gonadotropin-Releasing Hormone / Q000031:analogs & derivatives* / Q000138:chemical synthesis*; D007202:Indicators and Reagents; D007700:Kinetics; D008024:Ligands; D008970:Molecular Weight; D010777:Photochemistry; D010903:Pituitary Gland, Anterior / Q000378:metabolism*; D051381:Rats; D011966:Receptors, LHRH / Q000302:isolation & purification / Q000378:metabolism*; D013329:Structure-Activity Relationship
chemical_list: D000345:Affinity Labels; D007202:Indicators and Reagents; D008024:Ligands; D011966:Receptors, LHRH; D007987:Gonadotropin-Releasing Hormone