Abstract
Endophilin plays key roles during endocytosis of cellular receptors, including generating membrane curvature to drive internalization. Electrostatic interactions between endophilin’s BIN/Amphiphysin/Rvs domain and anionic membrane lipids have been considered the major driving force in curvature generation. However, the SH3 domain of endophilin also interacts with the proline-rich third intracellular loop (TIL) of various G-protein-coupled receptors (GPCRs), and it is unclear whether this interaction has a direct role in generating membrane curvature during endocytosis. To examine this, we designed model membranes with a membrane density of 1400 receptors per μm2 represented by a covalently conjugated TIL region from the β1-adrenergic receptor. We observed that TIL recruits endophilin to membranes composed of 95 mol% of zwitterionic lipids via the SH3 domain. More importantly, endophilin recruited via TIL tubulates vesicles and gets sorted onto highly curved membrane tubules. These observations indicate that the cellular membrane bending and curvature sensing activities of endophilin can be facilitated through detection of the TIL of activated GPCRs in addition to binding to anionic lipids. Furthermore, we show that TIL electrostatically interacts with membranes composed of anionic lipids. Therefore, anionic lipids can modulate TIL/SH3 domain binding. Overall, our findings imply that an interplay between TIL, charged membrane lipids, BAR domain, and SH3 domain could exist in the biological system and that these components may act in coordination to regulate the internalization of cellular receptors.
Highlights
Endocytosis is an important regulatory pathway for G-protein-coupled receptor (GPCR)-mediated cell signaling processes [1, 2]
Electrostatic interactions between BAR domains and anionic lipid headgroups are considered essential for recruitment of BAR proteins to the membrane, but they are critical for curvature sensing and generation of these proteins [20, 52,53,54,55]
The Src homology 3 (SH3) domain is known to mediate membrane recruitment of endophilin via protein–protein interactions, to what extent this recruitment pathway contributes to the curvature generation activity has so far remained unexplored
Summary
Endocytosis is an important regulatory pathway for G-protein-coupled receptor (GPCR)-mediated cell signaling processes [1, 2]. It has been suggested that molecular interactions occur between the endophilin SH3 domain and the third intracellular loop (TIL) of several GPCR family members during their internalization through the FEME pathway [10, 16, 17]. Still, it has remained unclear how membrane remodeling by endophilin is functionally coupled with protein–protein interactions mediated by the SH3 domain. Our results reveal that endophilin retains its curvature sensing and generation properties when recruited via its SH3 domain, which has implications for our understanding of how endophilin’s BAR domains interact with the membrane and are involved in receptor internalization processes
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