Key interactions by conserved polar amino acids located at the transmembrane helical boundaries in Class B GPCRs modulate activation, effector specificity and biased signalling in the glucagon-like peptide-1 receptor

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• 作者：Denise Wootten^a ; ^{denise.wootten@monash.edu" class="auth_mail" title="E-mail the corresponding author} ; Christopher A. Reynolds^b ; Kevin J. Smith^b ; Juan C. Mobarec^b ; Sebastian G.B. Furness^a ; Laurence J. Miller^c ; Arthur Christopoulos^a ; Patrick M. Sexton^a ; ^{patrick.sexton@monash.edu" class="auth_mail" title="E-mail the corresponding author}
• 关键词：cAMP ; 3&prime ; 5&prime ; -cyclic adenosine monophosphate ; CHO ; Chinese hamster ovary ; CRF1R ; corticotrophin releasing factor receptor-1 ; DMEM ; Dulbecco&rsquo ; s modified Eagle medium ; FBS ; fetal bovine serum ; GCGR ; glucagon receptor ; GLP-1 ; glucagon-like peptide-1 ; GPCR ; G protein-coupled receptor ; iCa2+ ; intracellular calcium ; pERK ; extracellular signal-regulated kinase 1 and 2 phosphorylation ; PBS ; phosphate buffered saline ; TM ; transmembrane helix
• 刊名：Biochemical Pharmacology
• 出版年：2016
• 出版时间：15 October 2016
• 年：2016
• 卷：118
• 期：Complete
• 页码：68-87
• 全文大小：5166 K

文摘

Class B GPCRs can activate multiple signalling effectors with the potential to exhibit biased agonism in response to ligand stimulation. Previously, we highlighted key TM domain polar amino acids that were crucial for the function of the GLP-1 receptor, a key therapeutic target for diabetes and obesity. Using a combination of mutagenesis, pharmacological characterisation, mathematical and computational molecular modelling, this study identifies additional highly conserved polar residues located towards the TM helical boundaries of Class B GPCRs that are important for GLP-1 receptor stability and/or controlling signalling specificity and biased agonism. This includes (i) three positively charged residues (R3.30²²⁷, K4.64²⁸⁸, R5.40³¹⁰) located at the extracellular boundaries of TMs 3, 4 and 5 that are predicted in molecular models to stabilise extracellular loop 2, a crucial domain for ligand affinity and receptor activation; (ii) a predicted hydrogen bond network between residues located in TMs 2 (R2.46¹⁷⁶), 6 (R6.37³⁴⁸) and 7 (N7.61⁴⁰⁶ and E7.63⁴⁰⁸) at the cytoplasmic face of the receptor that is important for stabilising the inactive receptor and directing signalling specificity, (iii) residues at the bottom of TM 5 (R5.56³²⁶) and TM6 (K6.35³⁴⁶ and K6.40³⁵¹) that are crucial for receptor activation and downstream signalling; (iv) residues predicted to be involved in stabilisation of TM4 (N2.52¹⁸² and Y3.52²⁵⁰) that also influence cell signalling. Collectively, this work expands our understanding of peptide-mediated signalling by the GLP-1 receptor.