A monoclonal antibody raised against a thermo-stabilised β1-adrenoceptor interacts with extracellular loop 2 and acts as a negative allosteric modulator of a sub-set of β1-adrenoceptors expressed in stable cell lines

Soave, Mark; Cseke, G.; Hutchings, C. J.; Brown, Alastair; Woolard, Jeanette; Hill, Stephen J.

doi:10.1016/j.bcp.2017.10.015

Cited by 11 publications

(6 citation statements)

References 53 publications

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“…To accomplish this, we first determined that all three CDR loops of sdAb JN241 contribute to its binding and antagonist func-tion. The mechanistic understanding of JN241 functional activity was further refined by the structural information and shown to involve engagement of APJ ECL2, which is consistent with the observations reported for other GPCRs (16,35). In comparison, we observed that sdAbs binding to APJ N terminus alone were nonfunctional, supporting the notion that antibodies binding the N terminus alone cannot fully antagonize class A GPCRs (19,36).…”

Section: Discussionsupporting

confidence: 88%

Structure-guided discovery of a single-domain antibody agonist against human apelin receptor

Ma¹,

Ding²,

Song³

et al. 2020

Sci. Adv.

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Developing antibody agonists targeting the human apelin receptor (APJ) is a promising therapeutic approach for the treatment of chronic heart failure. Here, we report the structure-guided discovery of a single-domain antibody (sdAb) agonist JN241-9, based on the cocrystal structure of APJ with an sdAb antagonist JN241, the first cocrystal structure of a class A G protein-coupled receptor (GPCR) with a functional antibody. As revealed by the structure, JN241 binds to the extracellular side of APJ, makes critical contacts with the second extracellular loop, and inserts the CDR3 into the ligand-binding pocket. We converted JN241 into a full agonist JN241-9 by inserting a tyrosine into the CDR3. Modeling and molecular dynamics simulation shed light on JN241-9-stimulated receptor activation, providing structural insights for finding agonistic antibodies against class A GPCRs. RESULTS Generation of a potent sdAb antagonist JN241 against human APJWe reconstituted thermally and conformationally stabilized APJ proteins in nanodiscs and proteoliposomes. APJ nanodiscs were used as immunogens to generate immune repertoires in camels. APJ proteoliposomes were used as antigens to isolate APJ-specific sdAbs from the camel immune sdAb library by phage display. This strategy yielded 186 unique sdAbs with the CDR3 lengths ranging from 13 to 23 amino acids and the affinities ranging from single-digit nanomolar

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Section: Discussionsupporting

confidence: 88%

Structure-guided discovery of a single-domain antibody agonist against human apelin receptor

Ma¹,

Ding²,

Song³

et al. 2020

Sci. Adv.

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“…Issues with GPCR‐targeted antibodies can be tackled using several approaches. For example, the use of purified or thermostabilised receptor populations for immunisation, immunisation of the target receptor DNA directly into the host organism, or immunisation with peptide fragments have all yielded antibodies which can specifically bind their target GPCR [ 31 , 32 , 33 ]. These advances have led to the development of novel GPCR antibodies, providing new opportunities to visualise endogenous receptor localisation.…”

Section: Challenges With Studying Endogenous Gpcrsmentioning

confidence: 99%

Detection of genome‐edited and endogenously expressed G protein‐coupled receptors

et al. 2021

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G protein-coupled receptors (GPCRs) are the largest family of membrane receptors and major targets for FDA-approved drugs. The ability to quantify GPCR expression and ligand binding characteristics in different cell types and tissues is therefore important for drug discovery. The advent of genome editing along with developments in fluorescent ligand design offers exciting new possibilities to probe GPCRs in their native environment. This review provides an overview of the recent technical advances employed to study the localisation and ligand binding characteristics of genome-edited and endogenously expressed GPCRs.

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“…β 1 -AA is produced against β 1 -AR-ECII [16]. In 1987, Wallukat et al [17] found this autoantibody in dilated cardiomyopathy for the first time.…”

Section: Discussionmentioning

confidence: 99%

“…This problem was evident in this study, which may be owing to the differences in the binding sites of β 1 -AA and β 1 -AR blockers. Metoprolol recognizes the β 1 -AR ligand-binding pocket [23,41,42], whereas β 1 -AA binds to the second extracellular loop of β 1 -AR [16]. Animal studies have demonstrated that the combination of the β 2 -AR agonist fenoterol and β 1 -AR blocker metoprolol can improve the heart function of rats with HF.…”

Section: Discussionmentioning

confidence: 99%

Biased activation of β2-AR/Gi/GRK2 signal pathway attenuated β1-AR sustained activation induced by β1-adrenergic receptor autoantibody

et al. 2021

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Heart failure is the terminal stage of many cardiac diseases, in which β1-adrenoceptor (β1-AR) autoantibody (β1-AA) has a causative role. By continuously activating β1-AR, β1-AA can induce cytotoxicity, leading to cardiomyocyte apoptosis and heart dysfunction. However, the mechanism underlying the persistent activation of β1-AR by β1-AA is not fully understood. Receptor endocytosis has a critical role in terminating signals over time. β2-adrenoceptor (β2-AR) is involved in the regulation of β1-AR signaling. This research aimed to clarify the mechanism of the β1-AA-induced sustained activation of β1-AR and explore the role of the β2-AR/Gi-signaling pathway in this process. The beating frequency of neonatal rat cardiomyocytes, cyclic adenosine monophosphate content, and intracellular Ca2+ levels were examined to detect the activation of β1-AA. Total internal reflection fluorescence microscopy was used to detect the endocytosis of β1-AR. ICI118551 was used to assess β2-AR/Gi function in β1-AR sustained activation induced by β1-AA in vitro and in vivo. Monoclonal β1-AA derived from a mouse hybridoma could continuously activate β1-AR. β1-AA-restricted β1-AR endocytosis, which was reversed by overexpressing the endocytosis scaffold protein β-arrestin1/2, resulting in the cessation of β1-AR signaling. β2-AR could promote β1-AR endocytosis, as demonstrated by overexpressing/interfering with β2-AR in HL-1 cells, whereas β1-AA inhibited the binding of β2-AR to β1-AR, as determined by surface plasmon resonance. ICI118551 biasedly activated the β2-AR/Gi/G protein-coupled receptor kinase 2 (GRK2) pathway, leading to the arrest of limited endocytosis and continuous activation of β1-AR by β1-AA in vitro. In vivo, ICI118551 treatment attenuated myocardial fiber rupture and left ventricular dysfunction in β1-AA-positive mice. This study showed that β1-AA continuously activated β1-AR by inhibiting receptor endocytosis. Biased activation of the β2-AR/Gi/GRK2 signaling pathway could promote β1-AR endocytosis restricted by β1-AA, terminate signal transduction, and alleviate heart damage.

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A monoclonal antibody raised against a thermo-stabilised β1-adrenoceptor interacts with extracellular loop 2 and acts as a negative allosteric modulator of a sub-set of β1-adrenoceptors expressed in stable cell lines

Abstract: Graphical abstract

Cited by 11 publications

References 53 publications

Structure-guided discovery of a single-domain antibody agonist against human apelin receptor

Structure-guided discovery of a single-domain antibody agonist against human apelin receptor

Detection of genome‐edited and endogenously expressed G protein‐coupled receptors

Biased activation of β2-AR/Gi/GRK2 signal pathway attenuated β1-AR sustained activation induced by β1-adrenergic receptor autoantibody

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