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Title page for ETD etd-08042011-074210


Type of Document Dissertation
Author Coffa, Sergio
Author's Email Address sergio.coffa@vanderbilt.edu
URN etd-08042011-074210
Title Non-visual arrestins bind mitogen activated protein kinases and regulate their signaling
Degree PhD
Department Pharmacology
Advisory Committee
Advisor Name Title
Benjamin Spiller Committee Chair
Brian E. Wadzinski Committee Member
Charles Sanders Committee Member
H. Alex Brown Committee Member
Vsevolod V. Gurevich Committee Member
Keywords
  • arrestin
Date of Defense 2011-07-27
Availability unrestricted
Abstract
Arrestins are multifunctional signaling proteins, important for the regulation of signal transduction and the trafficking of G protein-coupled receptors (GPCRs). Recently, GPCR-arrestin interactions have been proposed to be necessary for activation of G-protein-independent signaling pathways, one of which is the activation of mitogen activated protein kinases (MAPKs). To investigate potential arrestin-MAPK interactions, we have used a variety of molecular tools including the co-expression of the individual domains of arrestin with single components of the c-Raf1-MEK1-ERK2 signaling cascade. We found that non-visual arrestins bind all three kinases, assembling c-Raf1, MEK1, and ERK2 along their short axis, with each kinase directly interacting with both domains.

To further investigate the interactions between arrestins and MAPK, we used alanine-scanning mutagenesis of residues on the non-receptor-binding surface of arrestin that are conserved between arrestin-2 and arrestin-3. We found that the substitution of arginine 307 with an alanine significantly reduced arrestin-2 binding to c-Raf1, whereas the interactions of this mutant with active phosphorylated receptors and the downstream kinases MEK1 and ERK2 were not affected. In contrast to wild type arrestin-2, Arg307Ala mutant failed to rescue arrestin-dependent ERK1/2 activation in arrestin-2/3 knockout MEFs. Interestingly, alanine substitution of the homologous arrestin-3 residue (lysine 308) did not significantly affect c-Raf1 binding or its ability to promote ERK1/2 activation. Together, these findings suggest that the two non-visual arrestins perform the same function via distinct molecular mechanisms. To further elucidate arrestin-MAPK interactions, we performed in vitro binding assays using pure proteins, and demonstrated that ERK2 directly binds free arrestin-2 and arrestin-3, as well as receptor-associated arrestin-1, arrestin-2, and arrestin-3. We have also shown that the arrestin-2 and arrestin-3 association with beta2-adrenergic receptors (β2ARs) significantly enhances ERK2 binding, yet has virtually no effect upon arrestins interactions with the upstream kinases c-Raf1 and MEK1.

Arrestins exist in three conformational states: free, receptor-bound, and microtubule (MT)-bound. Using conformationally biased arrestin mutants, we found that ERK2 prefers two conformations: MT-bound, mimicked by “constitutively inactive” arrestin-Δ7, and receptor-bound, mimicked by “pre-activated” arrestin-3A. Both mutants were able to rescue arrestin-mediated ERK1/2 activation in arrestin-2/3 double knockout fibroblasts. Lastly, we found that the arrestin-2 interaction with c-Raf1 is enhanced by receptor binding, whereas the interaction between arrestin-3 and c-Raf1 is not, thus suggesting that the two non-visual arrestins execute similar functions via diverse mechanisms.

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