The к-Opioid receptor (OPRK) belongs to the family of G-protein coupled receptors (GPCRs), is mainly localized at axon terminals in different brain regions, and is involved in e.g., nociception, consciousness, or mood. Like all GPCRs, OPRK mediates downstream signaling via e.g. heterotrimeric G proteins, consisting of Gα-, Gβ-, and Gγ-subunits, or β-arrestins. OPRK is reported to primarily signal via the inhibitory Gαi/o family and also exhibits interactions with Gα12, however, how OPRK mediates this preference has not yet been elucidated.In this project we therefore aim to explore the molecular determinants underlying OPRK Gα subunit selectivity employing information driven mutagenesis.For this purpose, computational analyses were used to identify important amino acid residues (aa) responsible for OPRK Gα preference. Following OPRK mutagenesis, we employed the Bioluminiscence Resonance Energy Transfer based TRUPATH assay to analyse OPRK wildtype and mutants coupling selectivity to different Gαβγ subunit combinations.In addition to the already reported OPRK Gα12 interaction, we were able to show that OPRK wildtype can also signal via the Gα15- subunit. Furthermore, the characterization of mutations allowed us to identify several critical aa responsible for the observed OPRK signal transduction mediated by Gα12 and Gα15​.While it remains unclear if the acquired knowledge can be extrapolated to other GPCRs, our results significantly deepen our insights into OPRK-signaling.