Trafficking of luteinizing hormone receptor directs the differential signal activation between luteinizing hormone and chorionic gonadotropin.

Luteinizing hormone (LH) and human choriogonadotropin (hCG) support distinct reproductive events via differential activation of the luteinizing hormone receptor (LHCGR). LH-mediated LHCGR trafficking is known to be key in activating and regulating its signal responses, yet whether LH and hCG differentially direct LHCGR trafficking is unknown. Bioluminescence resonance energy transfer (BRET) trafficking biosensors and high-resolution TIRF imaging demonstrated that LH induces rapid internalization and recycling via an APPL1-linked very early endosomal pathway, while hCG-mediated receptor trafficking and recycling is slower, and preferentially involves β-arrestins and accumulation in endocytic compartments positive for early and late endosomal markers. Receptor internalization was differentially required for Gq, Gi and Gs protein-mediated signals, revealing distinct LH- vs hCG-trafficking signatures that may be fundamental to preserving unique hormone signalling patterns and their impact at the genomic level. This study supports different LH vs hCG modes of action on the LHCGR through differential post-endocytic sorting of the receptor, providing a potential 'location bias' mechanism underlying the distinct physiological roles of these two gonadotropins. Short title: LH vs hCG receptor internalization.