Entropic Control of Receptor Recycling Using Engineered Ligands

Abstract

Receptor internalization by endocytosis regulates diverse cellular processes, from the rate of nutrient uptake to the timescale of essential signaling events. The established view is that internalization is tightly controlled by specific protein-binding interactions. However, recent work suggests that physical aspects of receptors influence the process in ways that cannot be explained by biochemistry alone. Specifically, work from several groups suggests that increasing the steric bulk of receptors may inhibit their uptake by multiple types of trafficking vesicles. How do biochemical and biophysical factors work together to control internalization? Here, we show that receptor uptake is well described by a thermodynamic trade-off between receptor-vesicle binding energy and the entropic cost of confining receptors within endocytic vesicles. Specifically, using large ligands to acutely increase the size of engineered variants of the transferrin receptor, we demonstrate that an increase in the steric bulk of a receptor dramatically decreases its probability of uptake by clathrin-coated structures. Further, in agreement with a simple thermodynamic analysis, all data collapse onto a single trend relating fractional occupancy of the endocytic structure to fractional occupancy of the surrounding plasma membrane, independent of receptor size. This fundamental scaling law provides a simple tool for predicting the impact of receptor expression level, steric bulk, and the size of endocytic structures on receptor uptake. More broadly, this work suggests that bulky ligands could be used to drive the accumulation of specific receptors at the plasma membrane surface, providing a biophysical tool for targeted modulation of signaling and metabolism from outside the cell.

Synergy between intrinsically disordered domains and structured proteins amplifies membrane curvature sensing

“…All of these chimeric proteins contained the intracellular and transmembrane domains of transferrin receptor (TfR-Δecto, human, amino acids 1-88; AAA61153) and GFP (EGFP; AAB02574). For this present work, a A206K mutation was added to GFP to prevent dimerization of GFP-containing proteins 53 . The mutation was performed using site-directed mutagenesis (primers in Supplementary Table 4 ).…”

“…GFP and PEG40K-GFP were prepared as previously described 53 . Briefly, non-dimerizable hexa-his-tagged EGFP A206K (his-GFP; AAB02574) was made suitable for maleimide-PEG conjugation by mutating an exposed glycine residue in the N-terminal linker region to a cysteine.…”

Molecular mechanisms of force production in clathrin‐mediated endocytosis

“…However, extracellular proteins in the cavity of the developing CCP also exert steric forces, countering those produced by cytoplasmic proteins. Above a certain point, bulky cargoes are excluded from the nascent pit . The extreme case of maximal crowding at equal density on both faces of the membrane could double or triple the total energetic cost of forming a vesicle .…”

The Ascending GLP-1 Road From Clinical Safety to Reduction of Cardiovascular Complications

“…Although the current exenatide-based drugs have not yet been shown to clearly reduce MACE events in CVOTs (18,19), there is no evidence that these agents are less effective GLP-1R agonists, relative to human GLP-1R agonists, when tested in GLP-1R signaling studies. Intriguingly, recent experimental evidence from studies of the transferrin receptor indicates the critical importance of ligand size for determining the extent of receptor recycling and, ultimately, expression and signaling at the plasma membrane (31). Whether ligand size constraints impact signaling via the GLP-1R has not been extensively examined.…”