Development of a genetically encoded melanocortin sensor for high sensitivity in vivo imaging

IF 6.3 2区医学 Q1 ENDOCRINOLOGY & METABOLISM

Molecular Metabolism Pub Date : 2025-11-01 Epub Date: 2025-09-18 DOI:10.1016/j.molmet.2025.102254

Yoon Namkung , Tal Slutzki , Joao Pedroso , Xiaohong Liu , Paul V. Sabatini , Maia V. Kokoeva , Stéphane A. Laporte

{"title":"Development of a genetically encoded melanocortin sensor for high sensitivity in vivo imaging","authors":"Yoon Namkung , Tal Slutzki , Joao Pedroso , Xiaohong Liu , Paul V. Sabatini , Maia V. Kokoeva , Stéphane A. Laporte","doi":"10.1016/j.molmet.2025.102254","DOIUrl":null,"url":null,"abstract":"<div><h3>Objective</h3><div>The central melanocortin system, composed of peptides derived from pro-opiomelanocortin (POMC) such as the melanocyte-stimulating hormones (α-, β-, γ-MSH) and melanocortin 4 receptors (MC4R), along with the agouti-related protein (AgRP), plays a pivotal role in controlling energy balance. To elucidate the dynamic role of α-MSH release in regulating appetite, specific, sensitive, and spatiotemporally resolved genetic sensors are required.</div></div><div><h3>Methods</h3><div>The melanocortin 1 receptor (MC1R) scaffold was leveraged for its robust plasma membrane expression, high affinity for melanocortins and low affinity for AgRP to design a α-MSH selective sensor for <em>in vivo</em> use. This was achieved by integrating circularly permuted green fluorescent protein (cpGFP) into the receptor, which we named <strong>Fl</strong>uorescence <strong>A</strong>mplified <strong>Re</strong>ceptor sensor for Melanocortin (FLARE<sub>MC</sub>).</div></div><div><h3>Results</h3><div>The FLARE<sub>MC</sub> sensor has high potency and selectivity in heterologous and homologous expressing cells for α-MSH and the synthetic melanocortin agonist MTII but not to the inverse agonist AgRP. The sensor exhibited impaired signaling, with reduced G protein activation, no β-arrestin coupling, and failed to internalize upon agonist stimulation. <em>In vitro</em>, FLARE<sub>MC</sub> displayed high photostability and reversible photoactivation. These properties suggest that the FLARE<sub>MC</sub> is suitable for long-term activity recording in the brain without desensitizing or interfering with endogenous melanocortin receptor signaling. When expressed in the paraventricular nucleus (PVN) of the mouse hypothalamus, the primary site of anorexigenic α-MSH signaling, FLARE<sub>MC</sub> demonstrated its effectiveness in detecting changes associated with melanocortin responses <em>in vivo</em>.</div></div><div><h3>Conclusions</h3><div>FLARE<sub>MC</sub> enables the study of melanocortin system in cultured cells and <em>in vivo</em>. This first of its class highly sensitive melanocortin sensor will serve as a valuable tool to advance our understanding of the complex dynamics governing melanocortin-dependent appetite regulation and related processes in the brain.</div></div>","PeriodicalId":18765,"journal":{"name":"Molecular Metabolism","volume":"101 ","pages":"Article 102254"},"PeriodicalIF":6.3000,"publicationDate":"2025-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Molecular Metabolism","FirstCategoryId":"3","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S2212877825001619","RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2025/9/18 0:00:00","PubModel":"Epub","JCR":"Q1","JCRName":"ENDOCRINOLOGY & METABOLISM","Score":null,"Total":0}

引用次数: 0

Abstract

Objective

The central melanocortin system, composed of peptides derived from pro-opiomelanocortin (POMC) such as the melanocyte-stimulating hormones (α-, β-, γ-MSH) and melanocortin 4 receptors (MC4R), along with the agouti-related protein (AgRP), plays a pivotal role in controlling energy balance. To elucidate the dynamic role of α-MSH release in regulating appetite, specific, sensitive, and spatiotemporally resolved genetic sensors are required.

Methods

The melanocortin 1 receptor (MC1R) scaffold was leveraged for its robust plasma membrane expression, high affinity for melanocortins and low affinity for AgRP to design a α-MSH selective sensor for in vivo use. This was achieved by integrating circularly permuted green fluorescent protein (cpGFP) into the receptor, which we named Fluorescence Amplified Receptor sensor for Melanocortin (FLARE_MC).

Results

The FLARE_MC sensor has high potency and selectivity in heterologous and homologous expressing cells for α-MSH and the synthetic melanocortin agonist MTII but not to the inverse agonist AgRP. The sensor exhibited impaired signaling, with reduced G protein activation, no β-arrestin coupling, and failed to internalize upon agonist stimulation. In vitro, FLARE_MC displayed high photostability and reversible photoactivation. These properties suggest that the FLARE_MC is suitable for long-term activity recording in the brain without desensitizing or interfering with endogenous melanocortin receptor signaling. When expressed in the paraventricular nucleus (PVN) of the mouse hypothalamus, the primary site of anorexigenic α-MSH signaling, FLARE_MC demonstrated its effectiveness in detecting changes associated with melanocortin responses in vivo.

Conclusions

FLARE_MC enables the study of melanocortin system in cultured cells and in vivo. This first of its class highly sensitive melanocortin sensor will serve as a valuable tool to advance our understanding of the complex dynamics governing melanocortin-dependent appetite regulation and related processes in the brain.

查看原文本刊更多论文

用于高灵敏度体内成像的遗传编码黑素皮质素传感器的研制。

目的：由促黑素细胞激素（α-、β-、γ-MSH）和黑素皮质素4受体（MC4R）等源自促黑素皮质素前体（POMC）的多肽以及刺痛酶相关蛋白（AgRP）组成的中枢黑素皮质素系统在控制能量平衡中起关键作用。为了阐明α-MSH释放在食欲调节中的动态作用，需要特异性、敏感性和时空分辨的遗传传感器。方法：利用黑素皮质素1受体（melanocortin 1 receptor, MC1R）在质膜上的强表达、对黑素皮质素的高亲和力和对AgRP的低亲和力，设计α-MSH选择性传感器用于体内。这是通过将循环排列的绿色荧光蛋白（cpGFP）整合到受体中实现的，我们将其命名为黑色素皮质素荧光扩增受体传感器（FLAREMC）。结果：FLAREMC传感器在α-MSH和合成黑素皮质素激动剂MTII的异源和同源表达细胞中具有较高的效价和选择性，而对反相激动剂AgRP的效价和选择性不高。传感器表现出信号传导受损，G蛋白活化降低，没有β-阻滞蛋白偶联，并且在激动剂刺激下无法内化。在体外，FLAREMC表现出高的光稳定性和可逆的光激活。这些特性表明，FLAREMC适合长期记录大脑活动，而不会脱敏或干扰内源性黑素皮质素受体信号。当FLAREMC在小鼠下丘脑室旁核（PVN）中表达时（PVN是缺氧α-MSH信号的主要部位），FLAREMC证明了其在体内检测黑素皮质素反应相关变化的有效性。结论：FLAREMC能够在培养细胞和体内研究黑素皮质素系统。这是同类中第一个高灵敏度的黑素皮质素传感器，将作为一个有价值的工具，促进我们对黑素皮质素依赖的食欲调节和大脑中相关过程的复杂动力学的理解。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

求助全文

约1分钟内获得全文求助全文

来源期刊

Molecular Metabolism ENDOCRINOLOGY & METABOLISM-

CiteScore

14.50

自引率

2.50%

发文量

219

审稿时长

43 days

期刊介绍： Molecular Metabolism is a leading journal dedicated to sharing groundbreaking discoveries in the field of energy homeostasis and the underlying factors of metabolic disorders. These disorders include obesity, diabetes, cardiovascular disease, and cancer. Our journal focuses on publishing research driven by hypotheses and conducted to the highest standards, aiming to provide a mechanistic understanding of energy homeostasis-related behavior, physiology, and dysfunction. We promote interdisciplinary science, covering a broad range of approaches from molecules to humans throughout the lifespan. Our goal is to contribute to transformative research in metabolism, which has the potential to revolutionize the field. By enabling progress in the prognosis, prevention, and ultimately the cure of metabolic disorders and their long-term complications, our journal seeks to better the future of health and well-being.