Alexander Tyla I, Tasma Zoe, Siow Andrew, Rees Tayla A, Brimble Margaret A, Harris Paul W R, Hay Debbie L, Walker Christopher S
Department of Pharmacology and Toxicology, The University of Otago, Dunedin 9054, New Zealand.
Maurice Wilkins Centre for Molecular Biodiscovery, The University of Auckland, Auckland 1010, New Zealand.
ACS Pharmacol Transl Sci. 2022 Dec 8;6(1):52-64. doi: 10.1021/acsptsci.2c00124. eCollection 2023 Jan 13.
The related peptides pituitary adenylate cyclase-activating 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 receptor splice variants, VPAC and VPAC receptors in transfected cells. All labeled peptides were functional, exhibiting comparable cAMP pharmacology to their unlabeled counterparts and underwent internalization in a time-dependent manner. Temporal differences in the internalization profiles were observed between Cy5-labeled peptides at the PAC, PAC, VPAC, and VPAC 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.
相关肽类垂体腺苷酸环化酶激活多肽（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，这些肽及其三种受体代表了多种病症（包括神经和疼痛相关疾病）的潜在药物靶点。然而，对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，通过使用Cy5荧光团的铜介导1,3-偶极环加成反应合成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积累测定中得到了证实。然后在转染细胞中的两种不同PAC受体剪接变体VPAC和VPAC受体上检查并定量Cy5标记肽的内化。所有标记肽均具有功能，在cAMP药理学方面与其未标记的对应物相当，并以时间依赖性方式发生内化。在PAC、PAC、VPAC和VPAC受体上的Cy5标记肽之间观察到内化谱的时间差异。有趣的是，Cy5标记肽活性的模式在cAMP积累和内化方面有所不同，表明这些肽以不同方式刺激cAMP积累和内化，因此表现出偏向性激动作用。对PACAP反应性受体信号传导和内化的这一新见解可能为未来的治疗发展提供一条独特途径。本文所述的荧光标记PACAP和VIP肽，我们已将其验证为研究受体内化的工具，将在广泛的应用中发挥作用，并能更深入地了解这个受体家族。
