Abstract/Details

Novel insights into arrestin mediated receptor trafficking


2009 2009

Other formats: Order a copy

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
Access the complete full text

You can get the full text of this document if it is part of your institution's ProQuest subscription.

Try one of the following:

  • Connect to ProQuest through your library network and search for the document from there.
  • Request the document from your library.
  • Go to the ProQuest login page and enter a ProQuest or My Research username / password.