Molecular characterization of feline melanocortin 4 receptor and melanocortin 2 receptor accessory protein 2

Habara, Makoto; Mori, Nobuko; Okada, Yuki; Kawasumi, Koh; Nakao, Nobuhiro; Tanaka, Yoshikazu; Arai, Toshiro; Yamamoto, Ichiro

doi:10.1016/j.ygcen.2018.01.020

Cited by 8 publications

(7 citation statements)

References 37 publications

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“…In addition, MRAP2deficient mice have targeted deletion in Sim1-positive hypothalamic neurons, which express MC4R, develop severe obesity and have several phenotypic similarities with the MC4R-null mice (Novoselova et al 2016). Further studies have also demonstrated that MRAP2 is able to modify the pharmacological properties of MC4R, which is involved in the regulation of energy balance in several vertebrate species, such as chicken (Zhang et al 2017), cats (Habara et al 2018), zebrafish (Josep Agulleiro et al 2013;Sebag et al 2013) and sea lampreys (Zhu et al 2019). All of these findings support the hypothesis that, within the hypothalamus, MC4R and MRAP2 can form a functional complex and play a pivotal role in governing energy balance such as food intake, growth, fatness and obesity in mice and humans, and in other vertebrates (e.g.…”

Section: Introductionmentioning

confidence: 99%

The Asp298Asn polymorphism of melanocortin‐4 receptor (MC4R) in pigs: evidence for its potential effects on MC4R constitutive activity and cell surface expression

Zhang

et al. 2020

Animal Genetics

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Summary In humans and mice, melanocortin receptor 4 (MC4R) and melanocortin receptor accessory protein 2 (MRAP2) can form a complex and control energy balance, thus regulating body weight and obesity. In pigs, a missense variant (p.Asp298Asn) of MC4R has been suggested to be associated with growth and fatness; however, the effect of Asp298Asn substitution on MC4R function is controversial, limiting its application in animal breeding. Here we examined the effect of this polymorphism on MC4R constitutive activity, cell surface expression and signaling, and its interaction with MRAP2 in pigs. We found that: (i) both pig MC4RAsp and MC4RAsn can be activated by its ligands (α‐MSH and ACTH) and stimulate cAMP/PKA signaling pathway, as detected by pGL3–CRE–luciferase reporter assay, indicating that, like pMC4RAsp, pMC4RAsn is coupled to the cAMP/PKA signaling pathway; (ii) compared with pMC4RAsp, pMC4RAsn loses the basal constitutive activity and shows a decreased surface expression, as detected by dual‐luciferase reporter assay and Nano‐HiBiT system; (iii) as in other vertebrates, both pMC4RAsp and pMC4RAsn can interact with pMRAP2, thus decreasing receptor surface expression and enhancing ligand sensitivity, although, in contrast to pMC4RAsp, the basal constitutive activity of pMC4RAsn cannot be affected by pMRAP2; and (iv) RNA‐seq data analysis revealed a co‐expression of MC4R and MRAP2 in pig hypothalamus. Taken together, our data provide convincing evidence that Asp298Asn substitution decreases the constitutive activity and cell surface expression of MC4R or MC4R–MRAP2 complex, which may affect energy balance and be a valuable selection marker for breeding programs in pigs.

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Section: Introductionmentioning

confidence: 99%

The Asp298Asn polymorphism of melanocortin‐4 receptor (MC4R) in pigs: evidence for its potential effects on MC4R constitutive activity and cell surface expression

Zhang

et al. 2020

Animal Genetics

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“…Melanocortin 4 receptor (MC4R) binds one of melanocortins α-melanocyte-stimulating hormone (α-MSH), which is considered is important in obesity. Habara et al isolated melanocortin receptor 4 and its regulator Melanocortin 2 receptor accessory protein 2 (MRAP2) from cats and analyzed the heterodimerization of these proteins [29]. Leory et al characterized several mutants of Janus Kinase 2 in signaling by a transmembrane cytokine receptor, erythropoietin receptor [30].…”

Section: Application Of Nanobit For Analysis Of Protein-protein Intermentioning

confidence: 99%

Protein-Protein Interaction Assays Using Split-NanoLuc

Ohmuro‐Matsuyama¹,

Ueda²

2019

Bioluminescence - Analytical Applications and Basic Biology

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Protein-protein interaction assays are fundamental to basic biology, drug discovery, diagnostics, screening, and immunoassays. Protein-fragment complementation (PCA) is one of such useful protein-protein interaction assays. PCA when performed using luciferase is a reversible approach, whereas when performed using green fluorescent protein analogs is an irreversible approach. The NanoLuc technology developed in 2012 utilizes a small and structurally robust luciferase that is capable of producing very bright luminescence. NanoLuc PCA has been used to detect many protein-protein interactions and for screening purposes. Methods developed from NanoLuc PCA include the HiBiT technology and NanoLuc ternary technology. These novel technologies are promising in various fields and further developments are anticipated.Bioluminescence -Analytical Applications and Basic Biology 2 very bright luminescence. Based on this attractive enzyme, PCA systems were developed [13,14]. This innovation on the NanoLuc PCA improves the luminescent signal, which is markedly better than the conventional PCA signal obtained using other luciferases. Herein, we will focus on the new PCA technology and its application, and further discuss potential improvements in the system. PCA using NanoLucVerhoef et al. constructed a PCA system using NanoLuc [14]. They made several pairs of NanoLuc fragments by cutting at several loop regions, and selected a pair comprised of the N-terminal 52-amino acid (aa) fragment and the C-terminal 119-aa fragment (Figure 2A). These fragments were used to successfully detect the interaction between the transactivation domain fragment of p53 and Mdm2.At almost the same time, Dixon et al. developed another NanoLuc-based PCA system designated NanoLuc Binary Technology (NanoBiT) [13]. This was devised by first identifying a dissection site from 90 candidate sites. An 18-kDa N-terminal fragment and 13-aa C-terminal fragment were selected. The K D value between these fragments was 6 μM. This low affinity was suitable for PCA, but their use was hampered by the very low stability of the N-terminal fragment. The sequence of the N-terminal fragment was optimized from an N-terminal library containing 15,000 variants. The optimization increased the luminescent signal by 300-fold when the two fragments were interacting, which was 37% that of the wild-type NanoLuc. However, the affinity between the N-and C-terminal fragments became too strong for PCA (K D = 900 nM). As a next step, the sequence of the C-terminal peptide was optimized from 350 variants. Finally, two fragments were obtained. They were designated LgBiT (18 kDa) and SmBiT (11 aa). These exhibited significantly low

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“…For GPCR dimerization purposes, NanoBiT ® has been implemented to detect ligand-dependent modulation of D 2L R − D 2L R homodimers [40,112]. The methodology has also been used to perform live-cell monitoring of the dynamics of the interaction between the Melanocortin 4 receptor (MC4R), and the Melanocortin 2 receptor accessory protein 2 (MRAP2), as well as of MC4R homodimerization [113].…”

Section: Luminescence-based Complementation Assaysmentioning

confidence: 99%

“…To follow the dynamics of GPCR interactions in living cells in real time or to monitor the influence of ligand-dependent modulation on the level of dimerization, one can use a real-time PCA-based method for a certain amount of time. For example, to evaluate the effect of the α-melanocyte-stimulating hormone (α-MSH) on the interaction between MC4R and MRAP2, Habara et al [113] (2018) measured this interaction with a NanoBiT ® -based PCA for up to 120 s before and after the addition of α-MSH. By doing so, the authors could demonstrate that the stimulation of MC4R with α-MSH slightly decreased the NanoBiT ® signal, which led to the postulation that the activated structural change of MC4R negatively impacts the interaction with MRAP2.…”

Section: Guidelines To Perform Accurate Pca-based Assaysmentioning

confidence: 99%

Luminescence- and Fluorescence-Based Complementation Assays to Screen for GPCR Oligomerization: Current State of the Art

Wouters

Vasudevan

Crans

et al. 2019

IJMS

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G protein-coupled receptors (GPCRs) have the propensity to form homo- and heterodimers. Dysfunction of these dimers has been associated with multiple diseases, e.g., pre-eclampsia, schizophrenia, and depression, among others. Over the past two decades, considerable efforts have been made towards the development of screening assays for studying these GPCR dimer complexes in living cells. As a first step, a robust in vitro assay in an overexpression system is essential to identify and characterize specific GPCR–GPCR interactions, followed by methodologies to demonstrate association at endogenous levels and eventually in vivo. This review focuses on protein complementation assays (PCAs) which have been utilized to study GPCR oligomerization. These approaches are typically fluorescence- and luminescence-based, making identification and localization of protein–protein interactions feasible. The GPCRs of interest are fused to complementary fluorescent or luminescent fragments that, upon GPCR di- or oligomerization, may reconstitute to a functional reporter, of which the activity can be measured. Various protein complementation assays have the disadvantage that the interaction between the reconstituted split fragments is irreversible, which can lead to false positive read-outs. Reversible systems offer several advantages, as they do not only allow to follow the kinetics of GPCR–GPCR interactions, but also allow evaluation of receptor complex modulation by ligands (either agonists or antagonists). Protein complementation assays may be used for high throughput screenings as well, which is highly relevant given the growing interest and effort to identify small molecule drugs that could potentially target disease-relevant dimers. In addition to providing an overview on how PCAs have allowed to gain better insights into GPCR–GPCR interactions, this review also aims at providing practical guidance on how to perform PCA-based assays.

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Molecular characterization of feline melanocortin 4 receptor and melanocortin 2 receptor accessory protein 2

Cited by 8 publications

References 37 publications

The Asp298Asn polymorphism of melanocortin‐4 receptor (MC4R) in pigs: evidence for its potential effects on MC4R constitutive activity and cell surface expression

The Asp298Asn polymorphism of melanocortin‐4 receptor (MC4R) in pigs: evidence for its potential effects on MC4R constitutive activity and cell surface expression

Protein-Protein Interaction Assays Using Split-NanoLuc

Luminescence- and Fluorescence-Based Complementation Assays to Screen for GPCR Oligomerization: Current State of the Art

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