G proteins allosterically regulate agonist, inverse agonist AND antagonist binding to GPCRs

Seminar by Roger Sunahara, Department of Pharmacology, University of Michigan Medical School.

Abstract

The notion that agonists tightly regulate the activation of G proteins by G protein-coupled receptors (GPCRs) serves as the cornerstone for the mechanism of hormone action.  Receptor activation results in the promotion of GDP release from the G protein and the subsequent binding of GTP. These data would imply that G proteins, in turn, can influence the binding of the hormones themselves.  Here we provide evidence that G proteins can allosterically modulate the binding of not just agonists, but they are also capable of modulating the binding of inverse agonists and "neutral antagonists".  We utilize High Density Lipoprotein (HDL) particles as a platform to reconstitute purified preparations of three Class A GPCRs: β2-adrenergic receptor, bovine rhodopsin and the μ-opioid receptor. The nanometer-scale particles are capable of containing a limited, but defined number of receptors in a controlled phospholipid bilayer environment.  We have isolated monomeric forms of all three receptors, demonstrated efficient activation of G proteins and observed clear and direct allosteric modulation of agonist binding by G proteins. The tight G protein coupling and degree of homogeneity of these detergent-free preparations have also allowed for functional characterization employing biophysical approaches.  Using fluorescence spectroscopy approaches we have been able to recapitulate the observed allosteric role of G proteins on agonist, and even inverse agonist binding.  More importantly we demonstrate that the nucleotide-free form of the G protein-receptor complex represents the high affinity site for agonists.  Somewhat unexpected is our observation that what are considered "neutral" antagonists cannot bind to this nucleotide-free form of the G protein-receptor complex.  These data have tremendous implications on our interpretation of radioligand binding assays using antagonist probes, the gold standard method for pharmacological profiling of GPCRs.