Novel Fluorescently Labeled PACAP and VIP Highlight Differences between Peptide Internalization and Receptor Pharmacology

Tyla I Alexander; Zoe Tasma; Andrew Siow; Tayla A Rees; Margaret A Brimble; Paul W R Harris; Debbie L Hay; Christopher S Walker

doi:10.1021/acsptsci.2c00124

Novel Fluorescently Labeled PACAP and VIP Highlight Differences between Peptide Internalization and Receptor Pharmacology

ACS Pharmacol Transl Sci. 2022 Dec 8;6(1):52-64. doi: 10.1021/acsptsci.2c00124. eCollection 2023 Jan 13.

Authors

Tyla I Alexander^{1

2}, Zoe Tasma^{2

3}, Andrew Siow⁴, Tayla A Rees^{2

3}, Margaret A Brimble^{2

4}, Paul W R Harris^{2

4}, Debbie L Hay^{1

2}, Christopher S Walker^{2

3}

Affiliations

¹ Department of Pharmacology and Toxicology, The University of Otago, Dunedin 9054, New Zealand.
² Maurice Wilkins Centre for Molecular Biodiscovery, The University of Auckland, Auckland 1010, New Zealand.
³ School of Biological Sciences, The University of Auckland, Auckland 1010, New Zealand.
⁴ School of Chemical Sciences, The University of Auckland, Auckland 1010, New Zealand.

Abstract

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 PAC₁ receptor splice variants, VPAC₁ and VPAC₂ 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_1n, PAC_1s, VPAC₁, and VPAC₂ 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.