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

Abstract

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, Y 2 R desensitization was observed after daily treatment with a selective PYY 13-36 analog in vivo by a yet unknown mechanism. Materials: We studied the desensitization and activatability of recycled Y 2 R in transiently transfected HEK293 cells as well as in endogenously Y 2 R 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 Y 2 R in a second round of stimulation despite its reappearance at the membrane. Already the first activation of the Y 2 R leads to depletion of the functional cellular Gα i/o protein pool and consequently desensitizes the linked signal transduction pathways, independent of receptor internalization. This desensitization also extends to other Gα 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 Y 2 R endogenously. By overexpression of chimeric Gα qi proteins in a model system, activation has been rescued, which identifies a critical role of the G protein status for cellular signaling. Furthermore, Y 2 R 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 Gα i-subunit. Conclusion: The unusually persistent Gα i-signaling of the Y 2 R leads to a state of cellular desensitization of the inhibitory Gα i-pathway. The strong allosteric effects of the Y 2 R-Gα i-interaction might be a mechanism that contributes to the burst of Gα 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 Gα i-signaling of the Y 2 R itself but also other Gα i/o-coupled receptors by simply controlling the repertoire of downstream effectors.

Shuttling of Peptide‐Drug Conjugates by G Protein‐Coupled Receptors Is Significantly Improved by Pulsed Application

“…Despite the constant reduction of internalized peptide after certain cycles of stimulation, still a high amount of TAMRA‐NPY was taken up by the cells, which also can be seen in the accumulation experiment (Figure 8B). This confirms the Y 1 R receptor to be a powerful and suitable shuttling system, contrary to the recently found impaired activation and internalization of the related Y 2 R [35] . These findings underline the importance of comprehensive knowledge about receptor signaling and trafficking.…”

“…Furthermore, quantification of internalized peptide after simultaneous application of recycling inhibitor NH 4 Cl as well as expression and transport inhibitor CHX /BFA verified the high contribution of recycled receptors after single (Figure 2) and multiple cycles (Figures 2, 3, and 7) as the proportion of ingested NPY was dramatically reduced when recycling was blocked. Obviously, this is an endogenous function of the receptor as the closely related Y 2 R is not able to recover and re‐internalize [35] . Receptors mediate signals through a cyclic process of receptor activation, desensitization (termination and inactivation of downstream signals), and resensitization (reactivation for next wave of stimulation), which independently regulate GPCR function.…”

Mechanisms of Nausea and Vomiting: Current Knowledge and Recent Advances in Intracellular Emetic Signaling Systems

“…The neuropeptide Y hormone receptor family are activated by the three endogenous ligands: neuropeptide Y, peptide YY (PYY), and the pancreatic polypeptide processes [ 93 , 101 , 102 ]. Neuropeptide Y receptors consists of five G-protein coupled receptor subtypes (Y 1 , Y 2 , Y 4 , Y 5 , and Y 6 ), which mediate a large variety of physiological and pathophysiological processes [ 93 , 101 , 102 ]. Following secretion by intestinal neuroendocrine L cells, peptide YY is hydrolyzed rapidly to PYY (3–36) [ 103 ].…”

NPY and Gene Therapy for Epilepsy: How, When,... and Y

“…NPY receptors are predominantly expressed at the cell surface and sequence motifs essential for endoplasmic reticulum export and delivery to the membrane have been identified, particularly in the C-terminal portion of the protein (Walther et al, 2011(Walther et al, , 2012. Y2Rs display desensitization (Ziffert et al, 2020a) but can undergo arrestin beta3-dependent and independent internalization only when exposed to high concentrations of agonist (Lundell et al, 2011;Walther et al, 2011). The low rate of Y2R internalization may depend on the presence of a N-terminal extracellular domain rich in acidic/anionic residues (Parker et al, 2001;Gicquiaux et al, 2002).…”