Photoaffinity labeling of the head-activator receptor from hydra

A photoaffinity ligand for the head-activator (HA) receptor from hydra was synthesized using solid-phase peptide synthesis and coupling of two HA peptides over their E-amino groups of Lys7 with succinimidyl esters. The new ligand, Bpa-HA-HA bipeptide, contains one normal HA peptide and another where p-benzoylphenylalanine (Bpa) was added at the amino terminus to allow ultraviolet activation and Tyr11 instead of Phe11 for radioiodination. The I-125-Bpa-HA-HA bipeptide bound with nanomolar affinity to the HA receptor from the multiheaded mutant of Chlorohydra viridissima as measured in a filter assay. After photoaffinity labeling of the hydra membrane fraction, a 200-kDa band was detected using reducing or non-reducing SDS/PAGE and autoradiography. Unlabeled HA derivatives, but no other neuropeptides, inhibited the labeling. Competition experiments with HA-HA homobipeptide in the nanomolar range indicate that predominantly the low-affinity and not the high-affinity HA receptor was photolabeled. Further evidence that the labeled molecule is the HA receptor comes from specific photoaffinity labeling with a second ultraviolet-activatable ligand containing p-nitrophenylalanine. The HA receptor could be functionally solubilized with Triton X-100 or Chaps. In the solubilizate the 200-kDa HA receptor was photolabeled specifically by both ligands. Liquid-phase isoelectric focussing of the solubilizate indicated a pi of about 5.4 of the photolabeled molecule. After chemical deglycosylation with trifluoromethanesulfonic acid, the apparent molecular mass of the labeled molecule was decreased to 180 kDa, indicating that the receptor is glycosylated.