Agonist-selective recruitment of engineered protein probes and of GRK2 by opioid receptors in living cells

Summary:

The manuscript is well written and the work well executed, with many controls reported. Only one minor comment was raised during the discussion.

The only further and useful addition would be a consideration of the proposed structural changes induced by the different agonists in the Discussion section. Since crystal structures are known, some speculation about the differently activated states would add a mechanistic/structural component to the manuscript.

We are pleased that the reviewers appreciate the findings and consider them important for understanding GPCR signal transduction.

The reviewers raise an important point and we have included a new paragraph in the revised Discussion section that focuses on the possible structural / biophysical basis for the observed selectivity of protein recruitment to opioid receptors promoted by different agonists (please see the fourth paragraph of the Discussion section):

First, we discuss that distinct allosteric complexes may reflect unique receptor conformations. More specifically, available structures of MOR in complex with either nanobody or heterotrimeric Gi show differences in the conformation of intracellular loop 3 (ICL3). Therefore, differential recruitment of Nb and mGsi may reflect agonist-selective stabilization of distinct active receptor conformations, possibly involving distinct ICL3 arrangements.

Second, we outline the possibility that agonists may promote receptors to adopt similar conformations but that distinct sensor characteristics (kinetics of sensor binding, sensor concentration, and/or sensor affinity) contribute to the differential recruitment.

We think it is likely that both mechanisms contribute to the agonist-selective allosteric effects that we observe in intact cells and may similarly be used by endogenous GPCR interaction partners.