GPCR activation: a mutagenic spotlight on crystal structures

doi:10.1016/j.tips.2012.11.002

Trends in Pharmacological Sciences

Volume 34, Issue 1, January 2013, Pages 67-84

https://doi.org/10.1016/j.tips.2012.11.002 Get rights and content

The crystal structures of antagonist and agonist complexes of isolated β₂ and β₁ adrenoceptors have recently been supplemented by antagonist structures of M₂ and M₃ muscarinic acetylcholine receptors. Importantly, a structure of an agonist-ligated β₂ adrenoceptor complexed with its cognate G protein has provided the first view of a ternary complex representing the transition state in agonist-mediated G protein activation. This review interprets these G-protein-coupled receptor (GPCR) structures through the focus provided by extensive mutagenesis studies on muscarinic receptors, revealing an activation mechanism that is both modular and dynamic. Specific motifs, based around highly conserved residues, functionalise the seven-transmembrane architecture of these receptors. While exploiting conserved motifs, the ligand binding and signal transduction pathways work around and through water-containing cavities, an emerging feature of GPCR structures. These cavities may have undergone evolutionary selection to adapt GPCRs to particular signalling niches, and may provide targeting opportunities to enhance drug selectivity.

Section snippets

GPCR structure–function relationships

The seven-transmembrane (TM)-domain GPCRs are the largest superfamily of signalling molecules in the human genome. They provide a higher proportion of the actual and prospective targets for drugs used in the clinic (and, notoriously, for illicit recreational purposes) than any other protein family [1].

Our understanding of the structure–function relationships of GPCRs has made several spectacular advances. The greatest was arguably the cloning and sequencing of the classical post-synaptic

A conserved G-protein-binding microdomain

In addition to the ACh counter-ion D3.32, Ala-scanning mutagenesis of the TM domain of the M₁ mAChR identified five amino acids, D2.50, I3.46, R3.50, N7.49, and Y7.53 [designated by the Ballesteros–Weinstein (BW) nomenclature; in extracellular loop (ECL) 2, residues are identified relative to the disulfide-bonded Cys], whose substitution reduced the signalling efficacy of ACh by more than 100-fold (Table 1). Their importance is reflected in the very high conservation of their homologues in the

Functional domains in the receptor conformational change

The very highly conserved residue D2.50 is at the root of the G-protein-binding microdomain that forms in the activated state. This suggested the hypothesis that TM2 might be regarded as the pivot of the activating conformational change. Pursuing this idea, superposition of TM2 between residues 2.38 and 2.65, spanning D2.50, in the inactive- [21] and active-state [35] structures of the β₂AR yielded surprisingly precise superposition of the indole rings of Trp residues in different TM domains of

The receptor domains are functionalised by specific structural motifs

In this section, we show how specific structural motifs mediate the functions of the domains defined above. Of these motifs, two have a stabilising role, six are primarily concerned with regulating signal transduction, and three are specialisations to bind ligands. Some are generated by local sequence elements, whereas others involve inter-domain interactions.

Agonist activation: integration of the components of the conformational change

Activation requires movement of the cytoplasmic terminus of TM3 bearing the G protein contact residue R3.50 towards TM7, and partial unwinding of the C-terminal helix of TM7 to release Y7.53 to stabilise R3.50 and actuate G protein binding. These rearrangements require rupture of the steric clamp that inhibits the outward movement and reorientation of the cytoplasmic end of TM6. The core interactions in the TM6 clamp are highly conserved in all GPCR crystal structures determined so far. It is

Concluding remarks

Comprehensive mutagenesis studies have successfully spotlighted the functionally important residues in GPCR sequences, focusing our attention on particular elements of the crystallographic structures. These complementary approaches, informed by increasingly powerful molecular dynamics calculations, are converging to provide a view of GPCR activation that is both modular and dynamic. The activation of GPCRs may best be modelled by ligand-biased stochastic transitions between different ensembles

Acknowledgements

This work was supported by the Medical Research Council UK, grant-in-aid number MC_U117532184. Structures were visualised and aligned, intramolecular cavities were mapped, and figures were created with Pymol (Schrodinger Inc.). I am grateful to Dr Phil Walker, Division of Molecular Structure, NIMR, for computational assistance.

Glossary

Agonist: ligand that binds to a receptor and stabilises the active state.
Ballesteros–Weinstein (BW) nomenclature: positions of amino acids (single or triple letter code) in TM domain X are indicated relative to the most conserved residue, designated X.50.
First shell: immediate neighbours surrounding a particular set of amino acids
Gq: Gq type heterotrimeric (αβγ subunit) G protein.
Gt: transducin type heterotrimeric (αβγ subunit) G protein.
Gs: Gs type heterotrimeric (αβγ subunit) G protein.
Indel: mutation in

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Disruption of cholinergic signalling via muscarinic receptors is associated with various pathologies, like Alzheimer’s disease or schizophrenia. Selective muscarinic agonists possess therapeutic potential in the treatment of diabetes, pain or Sjögren's syndrome. The orthosteric binding site of all subtypes of the muscarinic receptor is structurally identical, making the development of affinity-based selective agonists virtually impossible. Some agonists, however, are functionally selective; they activate only a subset of receptors or signalling pathways. Others may stabilise specific conformations of the receptor leading to non-uniform modulation of individual signalling pathways (biased agonists). Functionally selective and biased agonists represent a promising approach for selective activation of individual subtypes of muscarinic receptors. In this work we review chemical structures, receptor binding and agonist-specific conformations of currently known functionally selective and biased muscarinic agonists in the context of their intricate intracellular signalling. Further, we take a perspective on the possible use of biased agonists for tissue and organ-specific activation of muscarinic receptors.
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Trends in Pharmacological Sciences

ReviewFeature ReviewGPCR activation: a mutagenic spotlight on crystal structures

Section snippets

GPCR structure–function relationships

A conserved G-protein-binding microdomain

Functional domains in the receptor conformational change

The receptor domains are functionalised by specific structural motifs

Agonist activation: integration of the components of the conformational change

Concluding remarks

Acknowledgements

Glossary

Eur. J. Pharmacol.

Eur. J. Pharmacol.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Eur. J. Pharmacol.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Mol. Biol.

Pharmacol. Ther.

Front. Neuroendocrinol.

Trends Pharmacol. Sci.

Progress in structure based drug design for G protein-coupled receptors

J. Med. Chem.

Evolution of class A G-protein-coupled receptors: implications for molecular modeling

Curr. Med. Chem.

Scanning mutagenesis studies of the M-1 muscarinic acetylcholine receptor

Receptors Channels

The role of the aspartate–arginine–tyrosine triad in the M1 muscarinic receptor: mutations of aspartate 122 and tyrosine 124 decrease receptor expression but do not abolish signalling

Mol. Pharmacol.

Alanine-scanning mutagenesis of transmembrane domain 6 of the M1 muscarinic acetylcholine receptor suggests that Tyr381 plays key roles in receptor function

Mol. Pharmacol.

Scanning mutagenesis identifies amino acid side chains in transmembrane domain 5 of the M1 muscarinic receptor that participate in binding the acetyl methyl group of acetylcholine

Mol. Pharmacol.

Roof and floor of the muscarinic binding pocket: variations in the binding modes of orthosteric ligands

Mol. Pharmacol.

Helix 8 of the m1 muscarinic acetylcholine receptor: scanning mutagenesis delineates a g protein recognition site

Mol. Pharmacol.

Constitutive activity of muscarinic acetylcholine receptors

J. Recept. Signal Transduct. Res.

Constitutive activation of the m5 muscarinic receptor by a series of mutations at the extracellular end of transmembrane 6

Biochemistry

Crystal structure of rhodopsin: a G protein-coupled receptor

Science

High-resolution crystal structure of an engineered human beta2-adrenergic G protein-coupled receptor

Science

Maltose–neopentyl glycol (MNG) amphiphiles for solubilization, stabilization and crystallization of membrane proteins

Nat. Methods

Structure and dynamics of the M3 muscarinic acetylcholine receptor

Nature

Structure of the human M2 muscarinic acetylcholine receptor bound to an antagonist

Nature

Structure and function of an irreversible agonist–β2 adrenoceptor complex

Nature

The structural basis for agonist and partial agonist action on a β1-adrenergic receptor

Nature

Agonist-bound adenosine A2A receptor structures reveal common features of GPCR activation

Nature

New insights for drug design from the X-ray crystallographic structures of G-protein-coupled receptors

Mol. Pharmacol.

Structure of the chemokine receptor CXCR1 in phospholipid bilayers

Nature

Crystal structure of the ligand-free G-protein-coupled receptor opsin

Nature

Crystal structure of opsin in its G-protein-interacting conformation

Nature

Review
Feature Review
GPCR activation: a mutagenic spotlight on crystal structures

Alanine-scanning mutagenesis of transmembrane domain 6 of the M₁ muscarinic acetylcholine receptor suggests that Tyr381 plays key roles in receptor function

Scanning mutagenesis identifies amino acid side chains in transmembrane domain 5 of the M₁ muscarinic receptor that participate in binding the acetyl methyl group of acetylcholine

Structure and function of an irreversible agonist–β₂ adrenoceptor complex

The structural basis for agonist and partial agonist action on a β₁-adrenergic receptor