Weakly Internalized Receptors Use Coated Vesicle Heterogeneity to Evade Competition during Endocytosis

Abstract

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.

Endocytosis blocks the vesicular secretion of exosome marker proteins

“…In the case of proteins that carry YxxΦ-type endocytosis signals, ligand-mediated inhibition of AP-2/mu2-mediated endocytosis involves binding competition, which reflects the limiting amount of AP-2 in the cell, and protein crowding effects, which reflect the competition of ligands for packaging into clathrin-coated pits and vesicles. Thus, our discovery that highlevel expression of CD63 inhibited protein endocytosis (3.0-fold) and drove the plasma membrane accumulation of itself, Lamp1, and Lamp2, is broadly consistent with our modern understanding of protein endocytosis (32,(42)(43)(44)(45)(46).…”

“…The model that emerges from these data is that endocytosis inhibits CD63's vesicular secretion and that inhibitors of endocytosis, including syntenin, induce CD63's budding from the cell. Because high-level expression of other endocytosis signal-containing protein is known to inhibit AP-2, through a combination of competition for limited amounts of mu2/AP-2 and protein crowding effects (32,(42)(43)(44)(45)(46), we next tested whether high-level expression of CD63 might do the same, resulting in the inhibition of endocytosis and the induction of CD63's vesicualr secretion from the cell. To investigate this issue, we first created a Tet-on version of CD63 −/− 293F cells (47) and then made derivatives of it that carry dox-inducible transgenes designed to express wild-type (WT) CD63, which has the endocytosis signal YEVM, CD63-YQRF [YQRF has a particularly high affinity for mu2 (32)], CD63-YQTI, or CD63-AEMV, which lacks the tyrosine at position −4 that is critical for binding mu2.…”

“…Given that CD63 expression inhibits endocytosis (32,(42)(43)(44)(45)(46), we next examined the effect of the specific sequence of CD63's endocytosis signal, and the level of CD63 expression, on the plasma membrane display of CD63 and two other mu2-binding, endocytosis signal-containing, lysosomal proteins, Lamp1 and Lamp2. To do so, we grew all four of the CD63 transgenic cell lines ± dox, chilled the cells to 4°C, stained them with fluorescently labeled antibodies specific for CD63, Lamp1, Lamp2, CD81, and CD9, and then examined them by flow cytometry.…”

“…5G). Thus, while other YxxΦ motif-containing proteins inhibit AP-2 by a combination of competitive inhibition and protein crowding effects (32,(42)(43)(44)(45)(46), high-level expression of CD63 also inhibits mu2 by driving its vesicular secretion and cellular depletion.…”

“…The endocytosis field has known for >20 years ago that high-level expression of endocytosis signal-containing proteins inhibits protein endocytosis (32,(42)(43)(44)(45)(46). In the case of proteins that carry YxxΦ-type endocytosis signals, ligand-mediated inhibition of AP-2/mu2-mediated endocytosis involves binding competition, which reflects the limiting amount of AP-2 in the cell, and protein crowding effects, which reflect the competition of ligands for packaging into clathrin-coated pits and vesicles.…”