title: Development of a selective photoactivatable antagonist for corticotropin-releasing factor receptor, type 2 (CRF2).
abstract: A novel photoactivatable analog of antisauvagine-30 (aSvg-30), a specific antagonist for corticotropin-releasing factor (CRF) receptor, type 2 (CRF2), has been synthesized and characterized. The N-terminal amino-acid d-Phe in aSvg-30 [d-Phe11,His12]Svg(11-40) was replaced by a phenyldiazirine, the 4-(1-azi-2,2,2-trifluoroethyl)benzoyl (ATB) residue. The photoactivatable aSvg-30 analog ATB-[His12]Svg was tested for its ability to displace [125I-Tyr0]oCRF or [125I-Tyr0]Svg from membrane homogenates of human embryonic kidney (HEK) 293 cells stably transfected with cDNA coding for rat CRF receptor, type 1 (rCRF1) or mouse CRF receptor, type 2beta (mCRF2beta). Furthermore, the ability of ATB-[His12]Svg(12-40) to inhibit oCRF- or Svg-stimulated cAMP production of transfected HEK 293 cells expressing either rCRF1 (HEK-rCRF1 cells) or mCRF2beta (HEK-mCRF2beta cells) was determined. Unlike astressin and photo astressin, ATB-[His12]Svg(12-40) showed high selective binding to mCRF2beta (Ki = 3.1 +/- 0.2 nm) but not the rCRF1 receptor (Ki = 142.5 +/- 22.3 nm) and decreased Svg-stimulated cAMP activity in mCRF2beta-expressing cells in a similar fashion as aSvg-30. A 66-kDa protein was identified by SDS/PAGE, when the radioactively iodinated analog of ATB-[His12]Svg(12-40) was covalently linked to mCRF2beta receptor. The specificity of the photoactivatable 125I-labeled CRF2beta antagonist was demonstrated with SDS/PAGE by the finding that this analog could be displaced from the receptor by antisauvagine-30, but not other unrelated peptides such as vasoactive intestinal peptide (VIP).
authors.affiliation: WITA Proteomics AG, Teltow/Berlin, Germany.
authors.forename: Ines
authors.initials: I
authors.lastname: Bonk
authors.forename: Andreas
authors.initials: A
authors.lastname: Rühmann
mesh_terms: D000595:Amino Acid Sequence; D029845:Amphibian Proteins; D000818:Animals; D001388:Aziridines / Q000737:chemistry* / Q000494:pharmacology; D001389:Azirines / Q000737:chemistry; D001565:Benzoates; D001667:Binding, Competitive / Q000187:drug effects / Q000502:physiology; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D003346:Corticotropin-Releasing Hormone / Q000737:chemistry / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D006801:Humans; D007457:Iodine Radioisotopes; D007668:Kidney / Q000737:chemistry / Q000166:cytology / Q000378:metabolism; D008024:Ligands; D051379:Mice; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry* / Q000494:pharmacology; D036361:Peptide Hormones; D010455:Peptides / Q000737:chemistry / Q000494:pharmacology; D010777:Photochemistry; D051381:Rats; D018019:Receptors, Corticotropin-Releasing Hormone / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity / Q000502:physiology; D014162:Transfection; D000099052:CRF Receptor, Type 1
chemical_list: D029845:Amphibian Proteins; D001388:Aziridines; D001389:Azirines; D001565:Benzoates; C101531:CRF receptor type 2; D007457:Iodine Radioisotopes; D008024:Ligands; D010446:Peptide Fragments; D036361:Peptide Hormones; D010455:Peptides; D018019:Receptors, Corticotropin-Releasing Hormone; C116607:antisauvagine 30; C469233:sauvagine (11-40), 4-(1-azi-2,2,2-trifluoroethyl)benzoyl-His(12)-; C104416:astressin; D000099052:CRF Receptor, Type 1; C025727:sauvagine; C051320:4-(3-trifluoromethyldiazirino)benzoic acid; D003346:Corticotropin-Releasing Hormone; D000242:Cyclic AMP