Abstract/Details

Novel insights into arrestin mediated receptor trafficking


2009 2009

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Abstract (summary)

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.

Indexing (details)


Subject
Molecular biology;
Biochemistry
Classification
0307: Molecular biology
0487: Biochemistry
Identifier / keyword
Pure sciences; Biological sciences; Arrestin; G-protein coupled receptors; Receptor trafficking
Title
Novel insights into arrestin mediated receptor trafficking
Author
Kern, Ronald C.
Number of pages
216
Publication year
2009
Degree date
2009
School code
0272
Source
DAI-B 71/09, Dissertation Abstracts International
Place of publication
Ann Arbor
Country of publication
United States
ISBN
9781124134901
Advisor
Benovic, Jeffrey L.
Committee member
Benovic, Jeffrey L.; Hoek, Jan B.; Keen, James H.; Wedegaertner, Philip B.; Williams, John C.
University/institution
Thomas Jefferson University
Department
Biochemistry and Molecular Biology
University location
United States -- Pennsylvania
Degree
Ph.D.
Source type
Dissertations & Theses
Language
English
Document type
Dissertation/Thesis
Dissertation/thesis number
3413167
ProQuest document ID
746780773
Copyright
Database copyright ProQuest LLC; ProQuest does not claim copyright in the individual underlying works.
Document URL
http://search.proquest.com/docview/746780773
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