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.

C-terminal motif of human neuropeptide Y4 receptor determines internalization and arrestin recruitment

“…The hY 4 R is known to co-internalize with its ligand hPP [18] which raised the question whether distinct internalization behavior of hY 4 R mutants is reflected by hPP uptake. With the help of a hPP-derivative that was N-terminally modified with a TAMRA fluorophore, ligand uptake was estimated by fluorescence microscopy (Fig.…”

“…The hY 4 R was cloned into the pcDNA3 vector to result in a C-terminal fusion protein with the Renilla Luciferase 8 variant ( R Luc8) using AsiS I and Sbf I restrictions sites [18]. Bovine arr-3 was cloned into the mCherry-NE/S vector for live cell imaging or N-terminally fused to Venus and cloned into the pcDNA3 vector for bioluminescence resonance energy transfer (BRET) experiments [19].…”

“…Peptides were synthesized by solid-phase peptide synthesis according to 9-fluorenylmethoxycarbonyl/ tert -butyl (Fmoc/ t Bu) strategy as reported recently [18]. …”

“…Non-stimulated cells were treated with CHX and BFA only. Washing and [ 125 I]-hPP binding experiments were performed as described recently [18]. …”

“…Arr-3 interaction with individual hY 4 R constructs was determined by BRET assay as reported earlier [18]. Cells were stimulated with 10 −9 , 10 −8 and 10 −7 M hPP for 10 min.…”