Novel high-affinity photoactivatable antagonists of corticotropin-releasing factor (CRF) - Photoaffinity labeling studies on CRF receptor, type 1 (CRFR1)

Novel photoactivatable antagonists of human/rat corticotropin-releasing fac tor (h/rCRF) have been synthesized and characterized. The N-terminal amino acid d-phenylalanine in astressin {cyclo(30-33) [d-Phe12, Nle21,38, Glu30, Lys33]h/rCRF-(12-41)}, a potent CRF peptide antagonist, was replaced by a p henyldiazirine, the 4-(1-azi-2,2,2-trifluoroethyl)benzoyl (ATB) residue. Ad ditionally, His32 of astressin was substituted by either alanine or tyrosin e for specific radioactive labeling with I-125 at either His13 or Tyr32, re spectively. The photoactivatable CRF antagonists were tested for their abil ity to displace I-125-labeled Tyr0 ovine CRF ([I-125-labeled Tyr0]oCRF) in binding experiments and to inhibit oCRF-stimulated adenylate cyclase activi ty in human embryonic kidney (HEK) 293 cells, permanently transfected with cDNA coding for rat CRF receptor, type 1 (rCRFR1) or human Y-79 retinoblast oma cells known to carry endogenous functional human CRFR1 (hCRFR1). ATB-cy clo(30-33)[Nle21,38, Glu30, Ala32, Lys33]h/rCRF-(13-41) (compound 1) was fo und to bind with higher affinity to rat or human CRFR1 when compared with A TB-cyclo(30-33)[Nle21,38, Glu30, Tyr32, Lys33]h/rCRF-(13-41) (compound 2) a nd exhibited higher inhibition of oCRF-stimulated cAMP accumulation in HEK 293 cells stably transfected with cDNA coding for rCRFR1 (HEK-rCRFR1 cells) or Y-79 cells. A highly glycosylated, 66-kDa protein was identified with S DS/PAGE, when the radioactively iodinated compounds 1 or 2 were covalently linked to rCRFR1. The specificity of the photoactivatable I-125-labeled CRF antagonists was demonstrated with SDS/PAGE by the finding that these analo gs could be displaced from the receptor by their corresponding nonlabeled f orm, but not other unrelated peptides such as vasoactive intestinal peptide . The observed molecular size of the receptor was in agreement with the siz e of CRFR1 found in rat pituitary (66 kDa), but was significantly larger th an the size of CRFR1 found in rat cerebellum and olfactory bulb (53 kDa).