G-protein coupled receptors (GPCR) serve as relays for recognizing signals outside the cell, which are transmitted through the membrane to initiate cellular signaling cascades. Their diverse physiological responses in living cells established GPCRs as important drug targets. Binding of extracellular modulators either induce, inhibit, or alter the activation of GPCRs by stimulating different signaling pathways. Despite increasing structural information of GPCRs, complemented by intensive computational studies, the signaling mechanism in GPCRs remains elusive. Here, we analyzed how different extracellular modulators can stimulate different signaling pathways in the β2 adrenoreceptor (β2AR) and the μ-opioid receptor (MOR) by using rigidity analysis. To this end, we address a mechanism of signaling that emerges from altered GPCR dynamics. Binding of a modulator partially rigidifies the biomolecule. The rigidification then percolates through the network of interactions within the biomolecule and can be felt at a distant site. (Changes in) network rigidity were analyzed with the CNA (Constraint Network Analysis) software, which characterizes biomolecular flexibility and rigidity for understanding the relationship between biomolecular structure, (thermo-) stability, and function. We used an in silico perturbation approach to identify networks of residues for mediating signaling in β2AR and MOR. CNA was applied on conformational ensembles derived from MD trajectories of β2AR and MOR in complex with sets of different modulators. Remarkably, based on altered stability characteristics, we can discriminate between agonist, antagonist, and inverse agonist binding. Furthermore, our results show different pathways of connected residues in both, β2AR and MOR, in dependence of the type of extracellular modulator. Understanding how different signaling pathways in GPCRs can be stimulated by extracellular modulators provides an exciting opportunity to gain structure-based insights into the diverse signaling mechanisms of GPCRs, which is a prerequisite for the rational design of functionally selective modulators.

Signaling pathways in transmembrane receptors deduced from network rigidity

255th National Meeting (2018)

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