GPCR cargo modifies lipid order in clathrin-coated pits

Abstract

<b>Abstract ID 15819</b> <b>Poster Board 128</b> Endocytosis of G protein-coupled receptors (GPCRs) <i>via</i> clathrin-coated pits (CCPs) is crucial for receptor desensitization and signaling. The core protein machinery that mediate CCP initiation, maturation, and scission have been heavily studied. Although this core protein machinery is broadly distributed and shared between all CCPs, these structures are initiated at spatially non-random sites on the membrane and are restricted to specialized regions in some cell types. Whether the physical properties of the membrane itself contribute to this specialization is not well understood. Here, we use high resolution multichannel total internal reflection fluorescence microscopy (TIR-FM) to measure lipid order in the plasma membrane using a dual channel ratiometric fluorescence readout from a solvatochromic pyrene-based probe. Lipid order imaging in CCP clusters showed that CCPs represent membrane domains of a lower order relative to the rest of the plasma membrane. The lipid order in endocytic clusters exhibited time-resolved changes during CCP maturation. The prototypical GPCR, β₂-adrenergic receptor (β2AR) clusters into a subset of CCPs in response to activation by its agonist isoproterenol. Interestingly, β2AR-containing CCPs exhibited higher lipid order relative to CCPs that did not contain the receptor. However, such a difference was not observed in case of another class A GPCR, the delta opioid receptor (DOR), and the constitutively internalizing cargo, transferrin. β2AR, unlike DOR, has been shown to extend CCP lifetimes and receptor residence times on the membrane via a C-terminal PDZ ligand that links the receptor to the actin cytoskeleton. We show here that the difference in lipid order between β2AR-containing CCPs and CCPs that do not contain β2AR was abrogated upon disruption of the β2AR C-terminal PDZ ligand. Overall, our results show that CCPs represent specialized membrane domains with a distinct lipid order, and that lipid order is an important determinant of cargo-specificity in GPCR endocytosis.