Helix 8 of the M<sub>1</sub>Muscarinic Acetylcholine Receptor: Scanning Mutagenesis Delineates a G Protein Recognition Site

Kaye, Robert G.; Saldanha, José W.; Lu, Zhi-Liang; Hulme, Edward C.

doi:10.1124/mol.110.070177

Cited by 29 publications

(28 citation statements)

References 41 publications

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“…an appropriate antagonist. A similar pharmacological chaperone effect was also reported for helix 8-defective mutants of the leukotriene B4 type 2 receptor (17) and for the muscarinic M 1 receptor (14), implying a potential therapeutic approach to rescue respective defective receptors.…”

Section: Discussionsupporting

confidence: 73%

“…Therefore, helix 8 is an excellent candidate for the agonist-induced recognition of and interaction with cytosolic binding partners and has been studied extensively in several GPCRs. Studies have not only shown the involvement of helix 8 in G protein activation (11)(12)(13)(14)(15) but also identified hydrophobic residues in helix 8 as essential for the surface expression of the GPCRs investigated (11,16,17). In addition, it has been published only recently that mutation of positively charged residues in helix 8 of the thyrotropin releasing hormone receptor prevented GRK-mediated receptor phosphorylation (18).…”

mentioning

confidence: 99%

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Helix 8 Plays a Crucial Role in Bradykinin B2 Receptor Trafficking and Signaling

Feierler

Wirth

Welte

et al. 2011

Journal of Biological Chemistry

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Upon activation the human bradykinin B 2 receptor (B 2 R) acts as guanine nucleotide exchange factor for the G proteins G q/11 and G i . Thereafter, it gets phosphorylated by G protein-coupled receptor kinases (GRKs) and recruits ␤-arrestins, which block further G protein activation and promote B 2 R internalization via clathrin-coated pits. As for most G protein-coupled receptors of family A, an intracellular helix 8 after transmembrane domain 7 is also predicted for the B 2 R. We show here that disruption of helix 8 in the B 2 R by either C-terminal truncation or just by mutation of a central amino acid (Lys-315) to a helixbreaking proline resulted in strong reduction of surface expression. Interestingly, this malfunction could be overcome by the addition of the membrane-permeable B 2 R antagonist JSM10292, suggesting that helix 8 has a general role for conformational stabilization that can be accounted for by an appropriate antagonist. Intriguingly, an intact helix 8, but not the C terminus with its phosphorylation sites, was indispensable for receptor sequestration and for interaction of the B 2 R with GRK2/3 and ␤-arrestin2 as shown by co-immunoprecipitation. Recruitment of ␤-arrestin1, however, required the presence of the C terminus. Taken together, our results demonstrate that helix 8 of the B 2 R plays a crucial role not only in efficient trafficking to the plasma membrane or the activation of G proteins but also for the interaction of the B 2 R with GRK2/3 and ␤-arrestins. Additional data obtained with chimera of B 2 R with other G protein-coupled receptors of family A suggest that helix 8 might have similar functions in other GPCRs as well.The human bradykinin B 2 receptor (B 2 R) 2 belongs to the family A (rhodopsin/␤-adrenergic-like) of G protein-coupled receptors (GPCRs). The B 2 R is ubiquitously expressed in many cells and tissues, and its activation results in a variety of physiological effects that range from vasodilatation and increased vascular permeability to hyperalgesia and natriuresis (1). Recent studies with B 2 R knock-out mice also point to a protective role of the B 2 R in the process of aging and in diabetes (2). After extracellular binding of its endogenous agonists, of the nona-peptide bradykinin (BK), or of kallidin (Lys-BK), the B 2 R undergoes conformational changes that turn it into a guanine nucleotide exchange factor for the G proteins G q/11 and G i , thus leading to the activation of G protein-dependent signaling cascades. Among other events, this results in phosphatidylinositol hydrolysis and activation of MAPK pathways. As reported for many GPCRs, desensitization of the B 2 R comes along with phosphorylation of serine/threonine residues in its C terminus by G protein-coupled receptor kinases (GRKs) or second messenger kinases as well as recruitment of ␤-arrestins and ends with the sequestration of the receptor into intracellular compartments (1). Upon short term stimulation the receptor gets recycled to the plasma membrane, whereas long term stimulation leads to the down-...

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Section: Discussionsupporting

confidence: 73%

mentioning

confidence: 99%

Helix 8 Plays a Crucial Role in Bradykinin B2 Receptor Trafficking and Signaling

Feierler

Wirth

Welte

et al. 2011

Journal of Biological Chemistry

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“…In fact, the H8 domain is critical for G protein coupling as observed for rhodopsin 23 and for other GPCRs (see 24 ). More interestingly, the H8 of the muscarinic M3R was shown to cross-link with the Gαq protein in an agonist-dependent manner 25 suggesting a close proximity between the receptor H8 domain and the G protein.…”

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confidence: 94%

Propagation of conformational changes during μ-opioid receptor activation

Sounier¹,

Mas²,

Steyaert³

et al. 2015

Nature

231

225

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μ-Opioid receptors (μOR) are G protein coupled receptors (GPCRs) that are activated by a structurally diverse spectrum of natural and synthetic agonists including endogenous endorphin peptides, morphine and methadone. The recent structures of the μOR in inactive1 and agonist-induced active states (companion article) provide snapshots of the receptor at the beginning and end of a signaling event, but little is known about the dynamic sequence of events that span these two states. Here we report the use of solution-state NMR to examine the process of μOR activation. We obtained spectra of the μOR in the absence of ligand, and in the presence of the high-affinity agonist BU72 alone, or with BU72 and a G protein mimetic nanobody. Our results show that conformational changes in transmembrane segments (TM) 5 and 6, which are required for the full engagement of a G protein, are almost completely dependent on the presence of both the agonist and the G protein mimetic nanobody revealing a weak allosteric coupling between the agonist binding pocket and the G protein coupling interface (TM5 and TM6) similar to what has been observed for the β2-adrenergic receptor2. Unexpectedly, in the presence of agonist alone, we observe larger spectral changes involving intracellular loop 1 (ICL1) and helix 8 (H8), when compared to changes in TM5 and TM6. These results suggest that one or both of these domains may play a role in the initial interaction with the G protein, and that TM5 and TM6 are only engaged later in the process of complex formation. The initial interactions between the G protein and ICL1 and/or H8 may play a role in G protein coupling specificity as has been suggested for other family A GPCRs.

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“…This helical region has been shown to be involved in the G protein activation, trafficking and function of the receptors [32, 52, 53]. Highly concentrated, positively charged residues in helix 8 have been defined to be essential for the cell surface expression of some GPCRs [2–5].…”

Section: Discussionmentioning

confidence: 99%

A single mutation in helix 8 enhances the angiotensin II type 1a receptor transport and signaling

Zhu

Zhang

Davis

et al. 2015

Cellular Signalling

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The amphipathic helix 8 in the membrane-proximal C-terminus is a structurally conserved feature of class A seven transmembrane-spanning G protein-coupled receptors (GPCRs). Mutations of this helical motif often cause receptor misfolding, defective cell surface transport and dysfunction. Surprisingly, we demonstrated here that a single point mutation at Lys308 in helix 8 markedly enhanced the steady-state surface density of the angiotensin II type 1a receptor (AT1aR). Consistent with the enhanced cell surface expression, Lys308 mutation significantly augmented AT1aR-mediated mitogen-activated protein kinase ERK1/2 activation, inositol phosphate production, and vascular smooth muscle cell migration. This mutation also increased the overall expression of AT1aR without altering receptor degradation. More interestingly, Lys308 mutation abolished AT1aR interaction with β-COP, a component of COPI transport vesicles, and impaired AT1aR responsiveness to the inhibition of Rab6 GTPase involved in the Golgi-to-ER retrograde pathway. Furthermore, these functions of Lys308 were largely dependent on its positively charged property. These data reveal previously unappreciated functions of helix 8 and novel mechanisms governing the cell surface transport and function of AT1aR.

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Helix 8 of the M₁Muscarinic Acetylcholine Receptor: Scanning Mutagenesis Delineates a G Protein Recognition Site

Cited by 29 publications

References 41 publications

Helix 8 Plays a Crucial Role in Bradykinin B2 Receptor Trafficking and Signaling

Helix 8 Plays a Crucial Role in Bradykinin B2 Receptor Trafficking and Signaling

Propagation of conformational changes during μ-opioid receptor activation

A single mutation in helix 8 enhances the angiotensin II type 1a receptor transport and signaling

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Helix 8 of the M1Muscarinic Acetylcholine Receptor: Scanning Mutagenesis Delineates a G Protein Recognition Site

Cited by 29 publications

References 41 publications

Helix 8 Plays a Crucial Role in Bradykinin B2 Receptor Trafficking and Signaling

Helix 8 Plays a Crucial Role in Bradykinin B2 Receptor Trafficking and Signaling

Propagation of conformational changes during μ-opioid receptor activation

A single mutation in helix 8 enhances the angiotensin II type 1a receptor transport and signaling

Contact Info

Product

Resources

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Helix 8 of the M₁Muscarinic Acetylcholine Receptor: Scanning Mutagenesis Delineates a G Protein Recognition Site