The effect of pH on beta(2) adrenoceptor function. Evidence for protonation-dependent activation

Area of Science:

• Biochemistry
• Molecular Biology
• Pharmacology

Background:

• G protein-coupled receptors (GPCRs) mediate cellular responses to various stimuli.
• Protonation is implicated in the activation mechanisms of rhodopsin and alpha(1B) adrenergic receptors.
• Understanding pH effects on GPCRs is crucial for elucidating their signaling pathways.

Purpose of the Study:

• To investigate the role of protonation in the activation of the beta(2) adrenergic receptor (beta(2)AR).
• To examine the impact of pH on ligand-induced conformational changes and G protein activation of beta(2)AR.
• To determine if Asp(130) is essential for the pH-dependent activation of beta(2)AR.

Main Methods:

• Utilized purified, NBD-labeled beta(2)AR for biophysical analysis of conformational changes.
• Measured agonist-induced fluorescence changes at different pH levels (6.5 and 8.0).
• Performed functional studies to assess beta(2)AR-mediated Galpha(s) activation at varying pH.

Main Results:

• The rate of agonist-induced conformational change in beta(2)AR was 2-fold higher at pH 6.5 compared to pH 8.0.
• Agonist affinity for beta(2)AR was reduced at lower pH (6.5).
• Basal activation of Galpha(s) by beta(2)AR was significantly greater at pH 6.5 than at pH 8.0.
• Mutation of Asp(130) did not abolish the pH sensitivity of beta(2)AR activation.

Conclusions:

• Protonation enhances beta(2)AR basal activity, likely by destabilizing the inactive receptor conformation.
• The findings suggest a role for protonation in GPCR activation beyond conserved acidic residues.
• Beta(2)AR activation exhibits pH sensitivity, independent of the homologous residue in rhodopsin and alpha(1B) adrenergic receptors.