Ligand function at constitutively active receptor mutants is affected by two distinct yet interacting mechanisms

Area of Science:

• Biochemistry
• Molecular Biology
• Pharmacology

Background:

• Constitutive activity in G-protein-coupled receptors (GPCRs) is often associated with enhanced partial agonist signaling.
• The cholecystokinin type 2 receptor (CCK-2R) serves as a model to investigate this phenomenon.
• Diverse activating mutations in GPCRs may exhibit varied effects on receptor function.

Purpose of the Study:

• To explore whether constitutive activity in GPCRs universally enhances partial agonist signaling.
• To investigate the functional impact of diverse activating mutations on the CCK-2R.
• To elucidate the mechanisms underlying heterogeneous responses to activating mutations in GPCRs.

Main Methods:

• Utilized the cholecystokinin type 2 receptor (CCK-2R) as a model system.
• Introduced various amino acid substitutions to induce constitutive activity in CCK-2R.
• Assessed the impact of these mutations on partial agonist efficacy and signaling.

Main Results:

• Mutations in the third intracellular loop systematically increased partial agonist activities.
• Activating mutations in transmembrane domains showed variable effects, increasing efficacy for some compounds while decreasing or not changing others.
• Combined mutations revealed signaling changes influenced by both equilibrium shifts and ligand-specific interactions.

Conclusions:

• Functional heterogeneity exists among constitutively active GPCR mutants.
• A subset of activating mutations can alter ligand-receptor interactions, deviating from established criteria for constitutive activity.
• These findings challenge the universality of proposed hallmark criteria for GPCR constitutive activity.