Towards a molecular understanding of the role of helix 8 in GPCR trafficking.

Helix 8 in G protein-coupled receptors (GPCRs) has recently been linked to receptor internalization (Schmidt et al.,2025, Sci. Adv. 11, eadv1499), but the molecular basis of this relationship remains unclear. Here, we examined a proposed mechanism in which helix 8 functions as a surface-active amphipathic element that promotes membrane binding, senses membrane curvature, and thereby facilitates GPCR trafficking. We analyzed six amphipathic peptides, including helix 8 segments from five GPCRs spanning a broad range of internalization rates, using molecular dynamics simulations and circular dichroism spectroscopy. Secondary structure in a membrane environment showed the strongest relationship with internalization, indicating that helix-forming propensity is a key determinant of trafficking behavior. Membrane binding showed a weaker and non-significant association with internalization. Taken together with the attachment to the helix 7 and putative lipid anchors at palmitoylation sites of common helix 8s, membrane binding was assessed not to be a dominant factor for internalization. Curvature sensing, as modeled here, did not correlate positively with internalization, although all constructs favored positive curvature and curvature-related hydrophobic phase width matched the internalization trend. Together, the results suggest that, while membrane binding and curvature-related properties likely contribute to the process, the ability of helix 8 to form amphipathic helices is the principal factor underlying differential GPCR internalization. These findings support a broader model in which surface-active amphipathic motifs tune membrane protein trafficking by coupling dynamic structural propensities to membrane interactions.