Acylation of the incretin peptide exendin-4 directly impacts GLP-1 receptor signalling and trafficking
biorxiv(2021)
摘要
The glucagon-like peptide-1 receptor (GLP-1R) is a class B G protein-coupled receptor and mainstay therapeutic target for the treatment of type 2 diabetes and obesity. Recent reports have highlighted how biased agonism at the GLP-1R affects sustained glucose-stimulated insulin secretion through avoidance of desensitisation and downregulation. A number of GLP-1R agonists (GLP-1RAs) feature a fatty acid moiety to promote albumin binding in order to prolong their pharmacokinetics, but the potential for these ligand changes to influence GLP-1R signalling has rarely been investigated beyond potency assessments for cyclic adenosine monophosphate (cAMP). In this work we directly compare the prototypical GLP-1RA exendin-4 with its C-terminally acylated analogue, exendin-4-C16, for their relative propensities to recruit and activate G proteins and β-arrestins, endocytic and post-endocytic trafficking profiles, and interactions with model and cellular membranes. Both ligands had similar cAMP potency but the exendin-4-C16 showed ∼2.5-fold bias towards G protein recruitment and a ∼60% reduction in β-arrestin-2 recruitment efficacy compared to exendin-4, as well as reduced GLP-1R endocytosis and preferential targeting towards recycling pathways. These effects were associated with a reduced ability to promote the movement of the GLP-1R extracellular domain, as determined using a conformational biosensor approach, and a ∼70% increase in insulin secretion. Interactions with plasma membrane lipids were enhanced by the acyl chain. Exendin-4-C16 showed extensive albumin binding and was highly effective for lowering of blood glucose in mice over at least 72 hours. Overall, our study highlights the importance of a broad approach to the evaluation of GLP-1RA pharmacology.
Significance statement Acylation is a common strategy to enhance the pharmacokinetics of peptide-based drugs. Our work shows how acylation can also affect various other pharmacological parameters, including biased agonism, receptor trafficking and interactions with the plasma membrane, which may be therapeutically important.
### Competing Interest Statement
The authors have declared no competing interest.
* BRET
: Bioluminescence resonance energy transfer
BSA
: Bovine serum albumin
CyOFP
: Cyan-excitable orange fluorescent protein
DERET
: Diffusion-enhanced resonance energy transfer
FCS
: Fluorescence correlation spectroscopy
FITC
: Fluorescein isothiocyanate
GFP
: Green fluorescent protein
GLP-1(R)(A)
: Glucagon-like peptide-1 (receptor) (agonist)
GUV
: Giant unilameller vesicle
MβCD
: Methyl-β-cyclodextrin
Nb37
: Nanobody-37
POPC
: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine
T2D
: Type 2 diabetes
TMR
: Tetramethylrhodamine
TR-FRET
: Time-resolved Förster resonance energy transfer
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关键词
incretin peptide
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