Molecular mechanisms of regulation of G protein-coupled receptor signaling by arrestins

1999 1999

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Abstract (summary)

G protein-mediated signal transduction by seven transmembrane G protein-coupled receptors (GPRs) is one of the most prevalent signaling systems in the body. Rapid agonist-induced desensitization of GPRs involves phosphorylation by G protein-coupled receptor kinases (GRKs) and binding of arrestins. The goal of this research thesis was to uncover the molecular mechanisms of regulation of GPR signaling by arrestins.

To investigate which GPR cytoplasmic domains are involved in interaction with arrestins, synthetic peptides from rhodopsin were assessed for their ability to modulate arrestin binding. The third, and to a lesser extent the first, cytoplasmic loop peptides of rhodopsin specifically modulated arrestin interaction, and the binding site(s) for these peptides was localized predominantly to the N-terminal half of arrestin. To investigate the proposed binding competition between arrestins and G proteins for the GPR, arrestin and the G protein transducin were assessed for their interaction with nonphosphorylated and phosphorylated rhodopsin. Phosphorylation of rhodopsin by GRKs enhanced arrestin interaction and decreased transducin interaction thus allowing arrestin to effectively compete with transducin for binding to phosphorylated light-activated rhodopsin.

It was recently demonstrated that the nonvisual arrestins, β-arrestin and arrestin3, promote internalization of GPRs. To shed light on a possible mechanism we investigated the ability of arrestins to interact with clathrin. β-arrestin and arrestin3 interacted specifically with clathrin in a high-affinity and stoichiometric fashion. Moreover, the GPR and clathrin binding functions were both critical for nonvisual arrestin-promoted β2AR internalization. Importantly, immunofluorescence in intact cells demonstrated colocalization of the β2AR, β-arrestin and clathrin in the presence of agonist. To localize the clathrin binding domain in nonvisual arrestins, truncation mutants, glutathione S-transferase fusion proteins, and alanine scanning and site-directed mutants of arrestin3 were assessed for clathrin interaction. The clathrin binding domain in arrestin3 was localized to a small discrete region in its far C-terminus, consisting of critical hydrophobic and charged residues. Moreover, an arrestin3 mutant containing three critical hydrophobic residues mutated to alanine was significantly impaired in promoting β 2AR internalization.

To uncover the molecular mechanism(s) underlying the sequestration defect in recently characterized nonvisual arrestin mutants containing a Val to Asp mutation in their far N-terminus, we assessed the ability of β-arrestin V53D to interact with both GPRs and clathrin. While β-arrestin V53D bound well to clathrin, it was defective in interaction with phosphorylated agonist-activated GPRs. However, β-arrestin V53D was a poor dominant-negative construct in sequestration studies. Importantly, a construct expressing the C-terminal 100 amino acids of β-arrestin, β-arrestin (319–418), bound to clathrin, was completely devoid of GPR binding, and was an effective dominant-negative construct in sequestration studies.

The studies described here have greatly improved our understanding of the molecular mechanisms underlying regulation of G protein-coupled receptor signaling by arrestins. Since internalization of GPRs is likely to be a mechanism for resensitization, these studies shed light on the involvement of arrestins in both the desensitization and resensitization of GPRs.

Indexing (details)

Molecular biology;
Cellular biology
0307: Molecular biology
0379: Cellular biology
Identifier / keyword
Biological sciences; Arrestins; G protein-coupled receptor; Signaling
Molecular mechanisms of regulation of G protein-coupled receptor signaling by arrestins
Krupnick, Jason Garrett
Number of pages
Publication year
Degree date
School code
DAI-B 60/02, Dissertation Abstracts International
Place of publication
Ann Arbor
Country of publication
United States
0599202890, 9780599202894
Benovic, Jeffrey L.
Thomas Jefferson University
University location
United States -- Pennsylvania
Source type
Dissertations & Theses
Document type
Dissertation/thesis number
ProQuest document ID
Database copyright ProQuest LLC; ProQuest does not claim copyright in the individual underlying works.
Document URL
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