Synthesis of Chemotactic Peptide Analogs with a Photoaffinity Cross-Linker as New Probes for Analysis of Receptor-Ligand Interaction

Shibue, Mitsukuni; Yoshiki, Masahiro; Miyazaki, Masaya; Fujita, Ichiro; Osada, Shinji; Yasuda, Seiji; Nakamura, Hiroyuki; Hamasaki, Yuhei; Kondo, Michio; Maeda, Hideaki; Kodama, Hiroaki; Shimizu, Hazime

doi:10.2174/0929866033479031

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“…Early studies on the angiotensin II receptor (45) and recent work on chemotactic peptide analogues (46) and the integrin receptor (47) showed that biotin can be used as a reporting tag instead of radioactive isotopes. However, most of these studies were hampered by the low affinity of the ligand analogues due to incorporation of biotin as a reporter tag.…”

Section: Discussionmentioning

confidence: 99%

Identification of Ligand Binding Regions of the Saccharomyces cerevisiae α-Factor Pheromone Receptor by Photoaffinity Cross-Linking

et al. 2004

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Analogues of alpha-factor, Saccharomyces cerevisiae tridecapeptide mating pheromone (H-Trp-His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr-OH), containing p-benzoylphenylalanine (Bpa), a photoactivatable group, and biotin as a tag, were synthesized using solid-phase methodologies on a p-benzyloxybenzyl alcohol polystyrene resin. Bpa was inserted at positions 1, 3, 5, 8, and 13 of alpha-factor to generate a set of cross-linkable analogues spanning the pheromone. The biological activity (growth arrest assay) and binding affinities of all analogues for the alpha-factor receptor (Ste2p) were determined. Two of the analogues that were tested, Bpa(1) and Bpa(5), showed 3-4-fold lower affinity than the alpha-factor, whereas Bpa(3) and Bpa(13) had 7-12-fold lower affinities. Bpa(8) competed poorly with [(3)H]-alpha-factor for Ste2p. All of the analogues tested except Bpa(8) had detectable halos in the growth arrest assay, indicating that these analogues are alpha-factor agonists. Cross-linking studies demonstrated that [Bpa(1)]-alpha-factor, [Bpa(3)]-alpha-factor, [Bpa(5)]-alpha-factor, and [Bpa(13)]-alpha-factor were cross-linked to Ste2p; the biotin tag on the pheromone was detected by a NeutrAvidin-HRP conjugate on Western blots. Digestion of Bpa(1), Bpa(3), and Bpa(13) cross-linked receptors with chemical and enzymatic reagents suggested that the N-terminus of the pheromone interacts with a binding domain consisting of residues from the extracellular ends of TM5-TM7 and portions of EL2 and EL3 close to these TMs and that there is a direct interaction between the position 13 side chain and a region of Ste2p (F55-R58) at the extracellular end of TM1. The results further define the sites of interaction between Ste2p and the alpha-factor, allowing refinement of a model for the pheromone bound to its receptor.

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Section: Discussionmentioning

confidence: 99%