Peptide Modifications Differentially Alter G Protein‐Coupled Receptor Internalization and Signaling Bias

Abstract

Although G protein‐coupled receptors (GPCRs) are targeted by more clinically used drugs than any other type of protein, their ligand development is particularly challenging. Humans have four neuropeptide Y receptors: hY1R and hY5R are orexigenic, while hY2R and hY4R are anorexigenic, and represent important anti‐obesity drug targets. We show for the first time that PEGylation and lipidation, chemical modifications that prolong the plasma half‐lives of peptides, confer additional benefits. Both modifications enhance pancreatic polypeptide preference for hY2R/hY4R over hY1R/hY5R. Lipidation biases the ligand towards arrestin recruitment and internalization, whereas PEGylation confers the opposite bias. These effects were independent of the cell system and modified residue. We thus provide novel insights into the mode of action of peptide modifications and open innovative venues for generating peptide agonists with extended therapeutic potential.

Fluorescently labeled adrenomedullin allows real-time monitoring of adrenomedullin receptor trafficking in living cells

“…For the development of peptide derived drugs such as ADM analogues, it is indispensable not only to elucidate receptor binding and activation properties, but also to investigate the fate of the peptide after the initial signaling event, that is, G protein signaling. Recent discoveries impressively demonstrated the ability of peptide ligands to differentially alter receptor responses in terms of internalization behavior [7]. Elucidating the ADM-induced internalization of the AM 1 receptor by direct observation in living cells is therefore of particular interest for possible therapeutic applications of ADM-derived peptide drugs.…”

“…C-terminal fusion of enhanced yellow fluorescent protein (eYFP) to CLR was achieved by modification of a HA-hY 2 R-eYFP_pVITRO2 construct (hygromycin resistance) [7,12]. Therefore, the CLR sequence was amplified from a CLR_pCDNA3 vector (by courtesy of Dr. Ingo Flamme, Bayer HealthCare AG, Wuppertal), introducing unique EcoRV and SalI restriction sites at the 5' and 3' end, respectively.…”

“…For potential therapeutic applications, recent studies have highlighted the contribution of receptor surface availability, trafficking, and the fate of the ligand for efficient targeting of GPCRs [7,8]. For the AM 1 receptor, it has been shown previously that stimulation with ADM leads to internalization of the receptor complex [9].…”

Development of Potent and Metabolically Stable APJ Ligands with High Therapeutic Potential

“…Interestingly, an exchange of the terminal pGlu for Gln led to an almost complete restoration of function (Table ). Another approach to effectively change the ADME properties of peptidic drugs is the modification by fatty acids . Therefore, we analyzed whether palmitoylated (Palm) apelin analogues show increased stability but retain biological activity.…”

“…Thereby, the peptide is shielded due to formation of micelles, and further protection is achieved by binding to serum proteins . However, this most often leads to decreased aqueous solubility but can be reversed by the introduction of hydrophilic residues such as glutamate . In the case of apelin, the solubility was only weakly affected, as the peptide sequence consists of several charged and polar amino acids, and thus no linker was necessary.…”