Biophysical Society Conference | Estes Park 2023

Membrane Budding and Fusion

Poster Abstracts

38-POS Board 13 GPCR CARGO MODIFIES LIPID ORDER IN CLATHRIN-COATED PITS

G Aditya Kumar 1 ; Manojkumar A Puthenveedu 1 ; 1 University of Michigan Medical School, Department of Pharmacology, Ann Arbor, MI, USA

Endocytosis of G protein-coupled receptors (GPCRs) via 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 these core protein components are 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. In addition, lipid order correlates with CCP dynamics such that cells that with lower lipid order exhibit a larger fraction of CCPs with shorter lifetimes, and an overall reduction in the number of productive CCPs relative to more ordered cells The prototypical GPCR, beta-2-adrenergic receptor (B2AR) clusters into a subset of CCPs in response to activation by its agonist isoproterenol. Interestingly, B2AR-containing CCPs exhibit higher lipid order relative to CCPs that did not contain the receptor. B2AR extends CCP lifetimes and receptor residence times on the membrane via a C-terminal PDZ ligand that links the receptor to the actin cytoskeleton. Our data show that the difference in lipid order between B2AR-containing CCPs and CCPs that do not contain B2AR was abrogated upon disruption of the B2AR C-terminal PDZ ligand. Overall, our results show that CCPs represent specialized membrane domains with a distinct lipid order, and specialized GPCR cargo modify local membrane lipid order to regulate CCP dynamics.

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