Modifying a Hydroxyl Patch in Glucagon-like Peptide 1 Produces Biased Agonists with Unique Signaling Profiles

Area of Science:

• Biochemistry
• Molecular Biology
• Endocrinology

Background:

• Glucagon-like peptide-1 (GLP-1) is known to lower blood glucose by stimulating insulin secretion.
• However, GLP-1 possesses other poorly understood signaling properties.
• Understanding these properties is crucial for developing targeted therapies.

Purpose of the Study:

• To investigate the role of hydroxy amino acids in GLP-1(7-36) in directing cell signaling.
• To determine how specific hydroxyl groups influence receptor affinity and downstream signaling pathways.
• To explore the potential for biased signaling through targeted mutations.

Main Methods:

• Site-directed mutagenesis of hydroxy amino acids (Thr11, Ser14, Ser17, Ser18) in GLP-1(7-36) to alanine.
• Measurement of receptor affinity, cAMP production, β-arrestin-2 recruitment, ERK1/2 phosphorylation, and Ca2+ signaling.
• Assessment of insulin secretion in cell-based assays and in vivo using mice.

Main Results:

• Single mutations of hydroxy amino acids reduced receptor affinity and cAMP production tenfold.
• Specific mutations differentially affected β-arrestin-2 and ERK1/2 signaling.
• Mutating three serines selectively silenced ERK1/2 signaling, demonstrating biased signaling.
• Complete mutation of the hydroxyl patch maintained membrane localization but potently stimulated cAMP and insulin secretion.

Conclusions:

• Hydrogen bonding among hydroxy amino acids in GLP-1 cooperatively controls cell signaling pathways.
• A specific hydroxyl patch in GLP-1 acts as a regulatory element for class B G protein-coupled receptors.
• These findings provide novel insights into hormone-receptor interactions and biased signaling.