TAILIEUCHUNG - Báo cáo Y học: Development of a selective photoactivatable antagonist for corticotropin-releasing factor receptor, type 2 (CRF2)

Anovel photoactivatable analog of antisauvagine-30 (aSvg-30), a specific antagonist for corticotropin-releasing factor (CRF) receptor, type 2 (CRF2), has been synthesized and characterized. TheN-terminal amino-acidD-Phe in aSvg-30 [D-Phe11,His12]Svg(11)40) was replacedbyaphenyldiazirine, the 4-(1-azi-2,2,2-trifluoroethyl)benzoyl (ATB) residue. The photoactivatable aSvg-30 analog ATB-[His12]Svg was tested for its abilityto displace [ 125 I-Tyr0]oCRF or [ 125 I-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,. | Eur. J. Biochem. 269 5288-5294 2002 FEBS 2002 doi Development of a selective photoactivatable antagonist for corticotropin-releasing factor receptor type 2 CRF2 Ines Bonk1 and Andreas Riihmann2 1WITA Proteomics AG Teltow Berlin Germany 2Institute for Molecular Biosciences The University of Queensland St Lucia Australia 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_4 was replaced by a phenyldi 11 100 . 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 2b mCRF2b . Furthermore the ability of ATB- Hi1l2 Svg to inhibit oCRF- or Svg-stimulated cAMP production of transfected HEK 293 cells expressing either rCRF1 HEK-rCRF1 cells or mCRF2b HEK-mCRF2b cells was determined. Unlike astressin and photo astressin ATB- His12 Svg 12_40 showed high selective binding to mCRF2b Ki nM but not the rCRF1 receptor Ki nM and decreased Svg-stimulated cAMP activityin mCRF 2b-expressing cells in a similar fashion as aSvg-30. A 66-kDa protein was identified by sDS PAGE. when the adiioaciively iodillased nnalog ơi ATB- His12 Svg 12_40 was covalently nkkdl to mCRF2p receptor. The specificity of the photoac 1111146 125I-labeled CRF2b antagonist was demonstrated with SDS PAGE by the finding that this analog could be displaced from the receptor byantisauvagine-30 but not other unrelated peptides such as vasoactive intestinal peptide VIP . Keywords photoaffinity laeengg nnSisaupvgike-30 forti-cotropin-releasing factor CRF receptor CRF antagonist human embryonic

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