Characterizing biochemical and biophysical mechanisms that impact uptake of membrane proteins by clathrin-mediated endocytosis
Creators: name: Gollapudi, Sadhana
Publisher: name: The University of Texas at Austin
Year: 2023
Subjects: Clathrin-mediated endocytosis
Plasma membrane receptors
Receptor uptake
Glycosylation
Glycans
MUC1
Galectins
Galectin-3

description: The uptake of membrane receptors by endocytosis is a crucial process that plays a significant role in regulating diverse cellular processes, including signal transduction, nutrient uptake, and cell-cell interactions. Clathrin-mediated endocytosis (CME) is the most well-studied type of endocytosis. The selection of receptors for endocytosis is a complex process influenced by numerous biochemical factors. For example, the biochemical internalization motifs on the cytoplasmic tails of receptors heavily influence their recruitment into endocytic structures. Notably, in the past decade, it has become evident that biochemical factors alone cannot predict receptor selection for endocytosis. Recent studies have also shown that biophysical characteristics, such as receptor size, are crucial in determining the degree to which a receptor is recruited into endocytic structures. Furthermore, post-translational modifications such as glycosylation and ubiquitination can also influence the endocytosis of transmembrane proteins. Interestingly, receptor clustering is also known to influence the recruitment of receptors into endocytic structures and the dynamics of CME. All these factors together create a delicate balance that determines the strength and degree to which receptors undergo endocytosis. Therefore, investigating these factors and their interplay is vital for elucidating the cellular processes dependent on the uptake of membrane proteins. In my thesis, I first characterized the impact of the affinity of biochemical internalization motifs on transmembrane receptors for CME adaptor proteins and their ability to compete for space within endocytic structures. We found that fusion receptors with a weaker affinity for AP2 are protected from being competed out of endocytic structures by receptors with higher affinities. These findings explain how weakly internalized receptors can evade competition and undergo endocytosis, thus regulating their associated downstream effects. I then investigated the contribution of glycosylation to receptor size and its impact on uptake by CME. These studies revealed that glycosylation contributes to as much as 50% of the steric bulk of membrane protein ectodomains. Furthermore, we showed that glycosylated membrane proteins were more weakly recruited into endocytic structures than analogous glycosylated membrane proteins. Currently, I am working on further characterizing the influence of glycans appended to membrane protein on their uptake by CME. My work investigates how galectins, such as galectin-3, that bind and cluster glycosylated membrane proteins can impact membrane protein endocytosis. Collectively, these results provide a framework for understanding the interplay between biochemical and biophysical characteristics that determine the selection of receptors for uptake by endocytosis.