Adhesion Organogenesis, neurodevelopment, reproduction, and other functions rely on G-protein-coupled receptors (aGPCRs) The Stachel sequence is a conserved internal (tethered) agonist sequence that activates several aGPCRs. GPR133 and GPR114, two aGPCRs in complex with Gs, were studied using cryogenic electron microscopy (cryo-EM). Both receptors' Stachel sequences adopt a helical-bulge sheet structure and insert into a binding site generated by the transmembrane domain, according to the structures (TMD). The majority of intramolecular interactions with the TMD are mediated by a hydrophobic interaction motif (HIM) within the Stachel sequence. Biochemical study of the HIM motif, when combined with cryo-EM structures, gives insight into the cross-reactivity and selectivity of the Stachel sequences.