title: Strategies for positioning fluorescent probes and crosslinkers on formyl peptide ligands.
abstract: Chemoattractant receptors represent a major subset of the G-protein coupled receptor (GPCR) family. One of the best characterized, the N-formyl peptide receptor (FPR), participates in host defense responses of neutrophils. The features of the ligand which regulate its interaction with the FPR are well-known. By manipulating these features we have developed new ligands to probe structural and mechanistic aspects of the peptide-receptor interaction. Three ligand groups have been developed: 1) ligands containing a Lys residue located in positions 2 through 7 that can be conjugated to FITC (N-formyl-Met1-Lys2-Phe3-Phe4, N-formyl-Met1-Leu2-Lys3-Phe4, N-formyl-Met1-Leu2-Phe3-Lys4, N-formyl-Met1-Leu2-Phe3-Phe4-Lys5, N-formyl-nLeu1-Leu2-Phe3-nLeu4-Tyr5-Lys6 and N-formyl-Met1-Leu2-Phe3-Phe4-Gly5-Gly6-Lys7; 2) fluorescent pentapeptide ligands (N-formyl-Met-X-Phe-Phe-Lys(FITC) where X = Leu, Ala, Val or Gly); and 3) small crosslinking ligands where the photoaffinity crosslinker 4-azidosalicylic acid (ASA) was conjugated to Lys in positions 3 and 4 and p-benzoyl-phenylalanine (Bpa) was located in position 2 in N-formyl-Met1-Bpa2-Phe3-Tyr4. The peptides were characterized according to activity and affinity in human neutrophils and cell lines transfected with FPR. All of the peptides were agonists, with parallel affinity and activity. In the first group, the peptide activity decreases as Lys is placed closer to the N-formyl group and the activity is improved by 1-3 orders of magnitude by conjugation with FITC. In the second group, the dissociation rate of the peptide from the receptor increases as position 2 is replaced by aliphatic amino acids with smaller alkyl groups. In the third group, crosslinking ligands remain biologically active, display nM affinity and covalently label the FPR.
authors.affiliation: Cancer Center and Pathology, University of New Mexico Health Sciences Center, Albuquerque, USA.
authors.forename: J C
authors.initials: JC
authors.lastname: Vilven
authors.forename: M
authors.initials: M
authors.lastname: Domalewski
authors.forename: E R
authors.initials: ER
authors.lastname: Prossnitz
authors.forename: R D
authors.initials: RD
authors.lastname: Ye
authors.forename: N
authors.initials: N
authors.lastname: Muthukumaraswamy
authors.forename: R B
authors.initials: RB
authors.lastname: Harris
authors.forename: R J
authors.initials: RJ
authors.lastname: Freer
authors.forename: L A
authors.initials: LA
authors.lastname: Sklar
mesh_terms: D000818:Animals; D001386:Azides / Q000737:chemistry; D001667:Binding, Competitive; D002460:Cell Line; D003432:Cross-Linking Reagents / Q000737:chemistry*; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism* / Q000528:radiation effects; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D066298:In Vitro Techniques; D008024:Ligands; D008027:Light; D051379:Mice; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism* / Q000528:radiation effects; D009504:Neutrophils / Q000378:metabolism; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic / Q000096:biosynthesis / Q000378:metabolism*; D018000:Receptors, Peptide / Q000096:biosynthesis / Q000378:metabolism*; D012459:Salicylates / Q000737:chemistry; D013329:Structure-Activity Relationship
chemical_list: D001386:Azides; D003432:Cross-Linking Reagents; D005456:Fluorescent Dyes; D008024:Ligands; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic; D018000:Receptors, Peptide; D012459:Salicylates; D009240:N-Formylmethionine Leucyl-Phenylalanine; C063873:4-azidosalicylic acid; D016650:Fluorescein-5-isothiocyanate