Novel Fluorescently Labeled PACAP and VIP Highlight Differences between Peptide Internalization and Receptor Pharmacology
Novel Fluorescently Labeled PACAP and VIP Highlight Differences between Peptide Internalization and Receptor Pharmacology
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DOI：
10.1021/acsptsci.2c00124
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发表时间：
2022-12-07
影响因子：
--
通讯作者：
Walker, Christopher S.
中科院分区：
文献类型：
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作者：
Alexander, Tyla I.;Tasma, Zoe;Walker, Christopher S.
相关肽垂体腺苷酸环酶激活多肽（PACAP）和血管活性肠肽（VIP）在外周组织和中枢神经系统中具有多种生物学功能。因此，这些肽和它们的三种受体代表了几种疾病的潜在药物靶点，包括神经和疼痛相关疾病。然而，关于这些肽如何通过内化等过程调节其受体，人们知之甚少。因此，我们开发了工具，通过铜介导的Cy5荧光团的1,3-偶极环加成合成PACAP-38、PACAP-27和VIP的荧光标记类似物来研究受体调节。cy5标记肽在其受体上的功能在cAMP积累试验中得到证实。然后在转染细胞的两种不同的PAC(1)受体剪接变体，VPAC(1)和VPAC(2)受体中检测和量化cy5标记肽的内化。所有标记的肽都是功能性的，表现出与未标记的肽相当的cAMP药理学，并以时间依赖性的方式内化。在PAC（1n）、PAC（1s）、VPAC(1)和VPAC(2)受体上，cy5标记肽的内化谱在时间上存在差异。有趣的是，cy5标记肽的活性模式在cAMP积累和内化方面有所不同，这表明这些肽刺激cAMP积累和内化的方式不同，因此表现出偏倚激动作用。这种对pacap反应性受体信号传导和内化的新见解可能为未来的治疗发展提供独特的途径。本文描述的荧光标记PACAP和VIP肽，我们验证了它们是研究受体内化的工具，将在广泛的应用中发挥作用，并为该受体家族提供更深入的了解。
The related peptides pituitary adenylate cyclaseactivating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) have diverse biological functions in peripheral tissues and the central nervous system. Therefore, these peptides and their three receptors represent potential drug targets for several conditions, including neurological and pain-related disorders. However, very little is known about how these peptides regulate their receptors through processes such as internalization. Therefore, we developed tools to study receptor regulation through the synthesis of fluorescently labeled analogues of PACAP-38, PACAP-27, and VIP using copper-mediated 1,3-dipolar cycloaddition of the Cy5 fluorophore. The functionality of Cy5-labeled peptides at their receptors was confirmed in cAMP accumulation assays. Internalization of the Cy5-labeled peptides was then examined and quantified at two distinct PAC(1) receptor splice variants, VPAC(1) and VPAC(2) receptors in transfected cells. All labeled peptides were functional, exhibiting comparable cAMP pharmacology to their unlabeled counterparts and underwent internalization in a time-dependent manlier. Temporal differences in the internalization profiles were observed between Cy5-labeled peptides at the PAC(1n), PAC(1s), VPAC(1), and VPAC(2) receptors. Interestingly, the pattern of Cy5-labeled peptide activity differed for cAMP accumulation and internalization, indicating that these peptides differentially stimulate cAMP accumulation and internalization and therefore display biased agonism. This novel insight into PACAP-responsive receptor signaling and internalization may provide a unique avenue for future therapeutic development. The fluorescently labeled PACAP and VIP peptides described herein, which we validated as tools to study receptor internalization, will have utility across a broad range of applications and provide greater insight into this receptor family.