Unusually persistent Gαi-signaling of the neuropeptide Y2 receptor depletes cellular Gi/o pools and leads to a Gi-refractory state

Background: A sensitive balance between receptor activation and desensitization is crucial for cellular homeostasis. Like many other GPCR, the human neuropeptide Y-2 receptor (hY(2)R) undergoes ligand dependent activation and internalization into intracellular compartments, followed by recycling to the plasma membrane. This receptor is involved in the pathophysiology of distinct diseases e.g. epilepsy and cancer progression and conveys anorexigenic signals which makes it an interesting and promising anti-obesity target. However, Y2R desensitization was observed after daily treatment with a selective PYY13-36 analog in vivo by a yet unknown mechanism. Materials: We studied the desensitization and activatability of recycled Y2R in transiently transfected HEK293 cells as well as in endogenously Y2R expressing SH-SY5Y and SMS-KAN cells. Results were evaluated by one-way ANOVA and Tukey post test. Results: We observed strong desensitization of the Y2R in a second round of stimulation despite its reappearance at the membrane. Already the first activation of the Y2R leads to depletion of the functional cellular G alpha(i/o) protein pool and consequently desensitizes the linked signal transduction pathways, independent of receptor internalization. This desensitization also extends to other G alpha(i/o)-coupled GPCR and can be detected in transfected HEK293 as well as in SH-SY5Y and SMS-KAN cell lines, both expressing the Y2R endogenously. By overexpression of chimeric G alpha(qi) proteins in a model system, activation has been rescued, which identifies a critical role of the G protein status for cellular signaling. Furthermore, Y2R displays strong allosteric coupling to inhibitory G proteins in radioligand binding assays, and loses 10-fold affinity in the G protein-depleted state observed after activation, which can be largely abrogated by overexpression of the G alpha(i)-subunit. Conclusion: The unusually persistent G alpha(i)-signaling of the Y2R leads to a state of cellular desensitization of the inhibitory G alpha(i)-pathway. The strong allosteric effects of the Y2R-G alpha(i)-interaction might be a mechanism that contributes to the burst of G alpha(i)-signaling, but also serves as a mechanism to limit the Y-2-mediated signaling after recycling. Thus, the cell is left in a refractory state, preventing further G alpha(i)-signaling of the Y2R itself but also other G alpha(i/o)-coupled receptors by simply controlling the repertoire of downstream effectors.