The uptake of receptors by clathrin-mediated endocytosis underlies signaling, nutrient import, and recycling of transmembrane proteins and lipids. In the complex, crowded environment of the plasma membrane, receptors are internalized when they bind to components of the clathrin coat, such as the major adaptor protein, AP2. Receptors with higher affinity for AP2 are known to be more strongly internalized compared to receptors with lower affinity. However, it remains unclear how receptors with different affinities compete for space within crowded endocytic structures. To address this question, we constructed receptors with varying affinities for AP2 and allowed them to compete against one another during internalization. As expected, the internalization of a receptor with high affinity for AP2 was reduced when it was coexpressed with a competing receptor of similar affinity. However, receptors of low affinity for AP2 were surprisingly difficult to displace from endocytic structures, even when expressed alongside receptors with much higher affinity. To understand how these low-affinity receptors are protected from competition, we looked at AP2 heterogeneity across clathrin-coated structures. When we examined structures with lower-than-average AP2 content, we found that they were relatively enriched in cargo of low affinity for AP2 and depleted of cargo with high affinity. These findings suggest that the heterogeneity of adaptor protein content across the population of endocytic structures enables the internalization of diverse receptors. Given the critical role that internalization plays in signaling, this effect may help to prevent strongly internalized receptors from interfering with the cell’s ability to process signals from weakly internalized receptors.

Clathrin-mediated endocytosis is essential for the removal of transmembrane proteins from the plasma membrane in all eukaryotic cells. Many transmembrane proteins are glycosylated. These proteins collectively comprise the glycocalyx, a sugar-rich layer at the cell surface, which is responsible for intercellular adhesion and recognition. Previous work has suggested that glycosylation of transmembrane proteins reduces their removal from the plasma membrane by endocytosis. However, the mechanism responsible for this effect remains unknown. To study the impact of glycosylation on endocytosis, we replaced the ectodomain of the transferrin receptor, a well-studied transmembrane protein that undergoes clathrin-mediated endocytosis, with the ectodomain of MUC1, which is highly glycosylated. When we expressed this transmembrane fusion protein in mammalian epithelial cells, we found that its recruitment to endocytic structures was substantially reduced in comparison to a version of the protein that lacked the MUC1 ectodomain. This reduction could not be explained by a loss of mobility on the cell surface or changes in endocytic dynamics. Instead, we found that the bulky MUC1 ectodomain presented a steric barrier to endocytosis. Specifically, the peptide backbone of the ectodomain and its glycosylation each made steric contributions, which drove comparable reductions in endocytosis. These results suggest that glycosylation constitutes a biophysical signal for retention of transmembrane proteins at the plasma membrane. This mechanism could be modulated in multiple disease states that exploit the glycocalyx, from cancer to atherosclerosis.

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Endocytic uptake of receptors is a fundamental process that underlies multiple cellular events from signal transduction and nutrient import to recycling transmembrane proteins and lipids. Since receptor recycling plays a role in most signaling pathways, it is important to understand how receptors are selected for internalization. In order to be internalized, receptors must bind to protein components of the endocytic coat, such as the major adaptor protein, AP2. Receptors with greater affinities for AP2 are more strongly internalized compared to receptors with lower affinity. How do receptors with different affinities compete for space within crowded endocytic structures? To address this question, we constructed receptors with varying affinities for AP2 and allowed them to compete against each other during internalization. Internalization of a receptor with high affinity for AP2 was reduced when co-expressed with a competing receptor of high affinity. Based on these results, we expected that the weaker the affinity of a receptor, the easier it would be for a strongly internalized receptor to compete it out of coated pits. Instead, we found that the fractional change in internalization of our model receptors upon competition with a strongly internalized receptor decreased with decreasing affinity for AP2. These observations suggest that weakly internalized receptors are protected from competition with more strongly internalized competitors. This protection could be due to a small fraction of the coated vesicle capacity being occupied by weakly internalized receptors. Therefore, to drive them out, a strongly internalized competitor has to saturate nearly the entire coated vesicle. Given the critical role that internalization plays in receptor signaling, this effect may serve as a protection mechanism that prevents overexpression of strongly internalized receptors from interfering with the cell's ability to process signals from weakly internalized receptors.

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