Novel insights into arrestin mediated receptor trafficking
Abstract
G protein-coupled receptors (GPCRs) represent the largest and most ubiquitous family of cell surface receptors. These receptors respond to a variety of extracellular stimuli and regulate numerous biological processes including cell growth, differentiation, development, neurotransmission, sight, and smell. Upon prolonged stimulation, GPCRs become refractory to further agonist stimulation, a process referred to as desensitization. This process is initiated by G protein-coupled receptor kinase (GRK) mediated GPCR phosphorylation followed by the recruitment and high affinity binding of arrestins. Arrestin binding physically blocks the interaction between the receptor and G protein, leading to signal termination. Although first discovered for its role in desensitization, arrestin has since been shown to regulate multiple processes, including internalization, intracellular trafficking, G protein independent signaling, and transcription. Here I set out to study the mechanisms that regulate the interaction between arrestin2 and clathrin and analyze the role of arrestin in intracellular trafficking. While the interaction of the non-visual arrestins with clathrin is an important step in mediating the proper internalization of GPCRs, little is known about how this interaction is prevented in the basal state. Thus, I set out to analyze the binding of arrestin to a GST fusion protein containing residues 1-363 of the clathrin terminal domain (TD). Interestingly, clathrin binding was enhanced following truncation of the arrestin2 C-terminal tail or addition of an N-terminal tag. Using site-directed mutagenesis I identified three acidic residues within the C-tail (Glu-404, Glu-405, and Glu-406) and four basic resides in the N-terminus (Lys-4, Arg-7, Lys-10, Lys-11), which are involved in regulating clathrin binding. Also, charge reversal at both Glu-389 and Asp-390, which appear to be positioned between the N- and C-terminus in the arrestin2 crystal structure, leads to enhanced clathrin binding. These results suggest that arrestin2 contains a series of intramolecular interactions that regulate its interaction with clathrin, highlighting this interaction as a critical step in regulating receptor trafficking. Arrestins have also been shown to regulate the internalization and intracellular trafficking of a number of cell surface receptors and proteins outside of the GPCR family. I thus set out to analyze the role of arrestin in regulating the trafficking of the cationindependent mannose 6-phosphate receptor (CI-MPR). Interestingly, upon knockdown of either arrestin2 or arrestin3 I observed decreased CI-MPR levels at the cell surface and TGN, and increased CI-MPR degradation. I also found that processing of the lysosomal hydrolase cathepsin D was differentially affected by arrestin2 and arrestin3. Arrestin3, but not arrestin2, binds directly to residues 2352-2367 within the CI-MPR C-tail. Immunofluorescence studies show that endogenous arrestin3 colocalizes with CI-MPR while cells expressing arrestin3-GFP show arrestin colocalization with CI-MPR at the endosomes and TGN. These results suggest that the non-visual arrestins regulate the retrograde trafficking of CI-MPR, thus indirectly effecting lysosomal hydrolase processing. Taken together, these studies reveal a novel mechanism regulating the basal arrestin/clathrin interaction and a novel function of arrestin in regulating the retrograde trafficking of CI-MPR. I hypothesize that regulation of clathrin binding serves an important role in controlling internalization and the subsequent trafficking of multiple classes of receptors. In addition I’ve identified CI-MPR as a novel arrestin binding partner and uncovered a role of arrestin in a new intracellular trafficking pathway.
Subject Area
Molecular biology|Biochemistry
Recommended Citation
Kern, Ronald C, "Novel insights into arrestin mediated receptor trafficking" (2009). ProQuest ETD Collection - Thomas Jefferson University. AAI3413167.
https://jdc.jefferson.edu/dissertations/AAI3413167
