Direct Identification of the Agonist Binding Site in the Human Brain CholecystokininB Receptor

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• 作者：Jonas Anders ; Martin Blü ; ggel ; Helmut E. Meyer ; Ronald Kü ; hne ; Antonius M. ter Laak ; Elzbieta Kojro ; and Falk Fahrenholz
• 刊名：Biochemistry
• 出版年：1999
• 出版时间：May 11, 1999
• 年：1999
• 卷：38
• 期：19
• 页码：6043 - 6055
• 全文大小：195K
• 年卷期：v.38,no.19(May 11, 1999)
• ISSN：1520-4995

文摘

In investigating the agonist binding site of the human brain cholecystokinin_B receptor (CCK_BR),we employed the direct protein chemical approach using a photoreactive tritiated analogue of sulfatedcholecystokinin octapeptide, which contains the p-benzoylbenzoyl moiety at the N-terminus, followed bypurification of the affinity-labeled receptor to homogeneity. This probe bound specifically, saturably, andwith high affinity (K_D = 1.2 nM) to the CCK_BR and has full agonistic activity. As the starting materialfor receptor purification, we used stably transfected HEK 293 cells overexpressing functional CCK_BR.Covalent labeling of the WGA-lectin-enriched receptor revealed a 70-80 kDa glycoprotein with a proteincore of about 50 kDa. Identification of the agonist binding site was achieved by the application of subsequentchemical and enzymatical cleavage to the purified receptor. A radiolabeled peptide was identified byEdman degradation amino acid sequence analysis combined with MALDI-TOF mass spectrometry. Theposition of the radioactive probe within the identified peptide was determined using combined tandemelectrospray mass spectrometry and peptide mapping. The probe was covalently attached within thesequence L⁵²ELAIRITLY⁶¹ that represents the transition between the N-terminal domain and predictedtransmembrane domain 1. Using this interaction as a constraint to orientate the ligand within the putativereceptor binding site, a model of the CCK-8s-occupied CCK_BR was constructed. The hormone was foundto be placed in a binding pocket built from both extracellular and transmembrane domains of CCK_BRwith its N-terminus mainly interacting with residues Arg⁵⁷ and Tyr⁶¹.