Similarity between class A and class B G-protein-coupled receptors exemplified through calcitonin gene-related peptide receptor modelling and mutagenesis studies

Abstract

Modelling class B G-protein-coupled receptors (GPCRs) using class A GPCR structural templates is difficult due to lack of homology. The plant GPCR, GCR1, has homology to both class A and class B GPCRs. We have used this to generate a class A–class B alignment, and by incorporating maximum lagged correlation of entropy and hydrophobicity into a consensus score, we have been able to align receptor transmembrane regions. We have applied this analysis to generate active and inactive homology models of the class B calcitonin gene-related peptide (CGRP) receptor, and have supported it with site-directed mutagenesis data using 122 CGRP receptor residues and 144 published mutagenesis results on other class B GPCRs. The variation of sequence variability with structure, the analysis of polarity violations, the alignment of group-conserved residues and the mutagenesis results at 27 key positions were particularly informative in distinguishing between the proposed and plausible alternative alignments. Furthermore, we have been able to associate the key molecular features of the class B GPCR signalling machinery with their class A counterparts for the first time. These include the [K/R]KLH motif in intracellular loop 1, [I/L]xxxL and KxxK at the intracellular end of TM5 and TM6, the NPXXY/VAVLY motif on TM7 and small group-conserved residues in TM1, TM2, TM3 and TM7. The equivalent of the class A DRY motif is proposed to involve Arg2.39, His2.43 and Glu3.46, which makes a polar lock with T6.37. These alignments and models provide useful tools for understanding class B GPCR function.

Phase-plate cryo-EM structure of a class B GPCR–G-protein complex

“…Class B GPCRs contain highly conserved TM domain polar residues that have crucial roles in receptor integrity, high affinity agonist interaction and/or receptor activation and downstream signalling 6,7,25,29,36–38 . A central polar network (Asn194 2.60b , Asn233 3.43b , Gln355 6.52b and Gln383 7.49b in the CTR) is present in all inactive structures 5–7 and in our inactive CTR homology model.…”

Do Plants Contain G Protein-Coupled Receptors?

“…The second microswitch has readily identifiable counterparts in both class B (VAVLY 7.53 ) and GCR1 (NSIAY 7.53 ), but the DRY 3.51 motif raises difficulties, since the class B positional equivalent (YLH 3.51 ) is not as important in activation as its class A counterpart and the class B DRY 3.51 functional equivalent, which also involves charged residues, is disjointed, as it is distributed between TM2 and TM3 (Frimurer and Bywater, 1999;Vohra et al, 2013). Supplemental Figure S16 shows that contiguous charged/aromatic residues are also missing from TM3 positions 3.49 to 3.51 in class C, class D, class E, GCR1, and class F GPCRs.…”

Structure of Class B GPCRs: new horizons for drug discovery

“…In CRF1, the equivalent amino acid, Tyr363, was mutated to alanine during the thermostabilization and conformational selection process for the inactive state. This further implicates the mechanistic importance of this residue in activation of Class B, as suggested in the past for GLP-1 (Wootten et al, 2013) and CGRP (Vohra et al, 2013), as well as Class A receptors. Class B receptors appear to lack the classical 'ionic lock' connecting TM3 to TM6, disruption of which has long been considered a hallmark of activation in Class A.…”

“…Class B receptors appear to lack the classical 'ionic lock' connecting TM3 to TM6, disruption of which has long been considered a hallmark of activation in Class A. Instead, for this receptor, class functional analyses (Schipani et al, 1995;Heller et al, 1996;Hjorth et al, 1998;Vohra et al, 2013;Wootten et al, 2013) point to an interaction between His 2.50b/2.43 in TM2 and Glu 3.50b/3.46 in TM3 (residues His155 and Glu209 in CRF1), which serves an analogous function (note that Wootten numbering is used when comparisons are made forthwith within Class B, as denoted by x.xxb followed by Ballesteros-Weinstein numbering) (Ballesteros et al, 1995;Wootten et al, 2013;Hollenstein et al, 2014). Identical residues are found in these positions in the glucagon receptor structure with hydrogen bonds formed at a distance of 3.2 Å in both receptors ( Figure 1G).…”