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 (Y1R) 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. The sensitive equilibrium of receptor activation and desensitization is complex, and detailed knowledge is required for applications that use GPCRs as a shuttle system for intracellular drug delivery. As Y1R is overexpressed in various cancer subtypes, its shuttling capacity was quantitatively examined in diverse cell systems and by different methods to exploit its natural ligand‐induced endocytosis for targeted therapy.