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

G protein-coupled receptors (GPCRs) can be used to shuttle peptide-drug conjugates into cells. But, for efficient therapy, a high concentration of cargo needs to be delivered. To explore this, we studied the pharmacologically interesting neuropeptide Y1 receptor (Y1 R) in one recombinant and three oncogenic cell systems that endogenously express the receptor. We demonstrate that recycled receptors behave identically to newly synthesized receptors with respect to ligand binding and internalization pathways. Depending on the cell system, biosynthesis, recycling efficiency, and peptide uptake differ partially, but shuttling was efficient in all systems. However, by comparing continuous application of the ligand for four hours to four cycles of internalization and recycling in between, a significantly higher amount of peptide uptake was achieved in the pulsed application (150-250 % to 300-400 %). Accordingly, in this well-suited drug shuttle system pulsed application is superior under all investigated conditions and should be considered for innovative, targeted drug delivery in general.

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 Y2 receptor (hY2R) 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 Gαi/o protein pool and consequently desensitizes the linked signal transduction pathways, independent of receptor internalization. This desensitization also extends to other Gαi/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 Gαqi 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 Gαi-subunit. CONCLUSION: The unusually persistent Gαi-signaling of the Y2R leads to a state of cellular desensitization of the inhibitory Gαi-pathway. The strong allosteric effects of the Y2R-Gαi-interaction might be a mechanism that contributes to the burst of Gαi-signaling, but also serves as a mechanism to limit the Y2-mediated signaling after recycling. Thus, the cell is left in a refractory state, preventing further Gαi-signaling of the Y2R itself but also other Gαi/o-coupled receptors by simply controlling the repertoire of downstream effectors. Video abstract.

Biased agonists at the human Y1 receptor lead to prolonged membrane residency and extended receptor G protein interaction.

Functionally selective ligands to address specific cellular responses downstream of G protein-coupled receptors (GPCR) open up new possibilities for therapeutics. We designed and characterized novel subtype- and pathway-selective ligands. Substitution of position Q34 of neuropeptide Y to glycine (G34-NPY) results in unprecedented selectivity over all other YR subtypes. Moreover, this ligand displays a significant bias towards activation of the Gi/o pathway over recruitment of arrestin-3. Notably, no bias is observed for an established Y1R versus Y2R selective ligand carrying a proline at position 34 (F7,P34-NPY). Next, we investigated the spatio-temporal signaling at the Y1R and demonstrated that G protein-biased ligands promote a prolonged localization at the cell membrane, which leads to enhanced G protein signaling, while endosomal receptors do not contribute to cAMP signaling. Thus, spatial components are critical for the signaling of the Y1R that can be modulated by tailored ligands and represent a novel mode for biased pathways.