The second extracellular loop of GPCRs determines subtype-selectivity and controls efficacy as evidenced by loop exchange study at A₂ adenosine receptors

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• 作者：Benjamin F. Seibt¹ ; Anke C. Schiedel¹ ; Dominik Thimm ; Sonja Hinz ; Farag F. Sherbiny ; Christa E. Mü ; ller ; ^{christa.mueller@uni-bonn.de}
• 关键词：Mutagenesis ; G protein-coupled receptors ; Receptor activation ; Ligand binding site ; Overlap extension mutagenesis ; EL2 loop exchange
• 刊名：Biochemical Pharmacology
• 出版年：2013
• 出版时间：1 May, 2013
• 年：2013
• 卷：85
• 期：9
• 页码：1317-1329
• 全文大小：2322 K

文摘

The second extracellular loop (EL2) of G protein-coupled receptors (GPCRs), which represent important drug targets, may be involved in ligand recognition and receptor activation. We studied the closely related adenosine receptor (AR) subtypes A_2A and A_2B by exchanging the complete EL2 of the human A_2BAR for the EL2 of the A_2AAR. Furthermore, single amino acid residues (Asp148^45.27, Ser149^45.28, Thr151^45.30, Glu164^45.43, Ser165^45.44, and Val169^45.48) in the EL2 of the A_2BAR were exchanged for alanine. The single mutations did not lead to any major effects, except for the T151A mutant, at which NECA showed considerably increased efficacy. The loop exchange entailed significant effects: The A_2A-selective agonist CGS21680, while being completely inactive at A_2BARs, showed high affinity for the mutant A_2B(EL2-A_2A)AR, and was able to fully activate the receptor. Most strikingly, all agonists investigated (adenosine, NECA, BAY60-6583, CGS21680) showed strongly increased efficacies at the mutant A_2B(EL2-A_2A) as compared to the wt AR. Thus, the EL2 of the A_2BAR appears to have multiple functions: besides its involvement in ligand binding and subtype selectivity it modulates agonist-bound receptor conformations thereby controlling signalling efficacy. This role of the EL2 is likely to extend to other members of the GPCR family, and the EL2 of GPCRs appears to be an attractive target structure for drugs.