[Abstract Title]. - Society for Neuroscience

Title: Role of dimerization for PICK1 function
Authors: *I. AMMENDRUP-JOHNSEN 1 , K. L. MADSEN 1 , D. S. HAN 2 , M. B. JENSEN 1 , V.
K. BHATIA 1 , H. A. WEINSTEIN 2 , U. GETHER 1 ;
1 Univ. of Copenhagen, Copenhagen, Denmark; 2 Dept. of Physiol. and Biophysics, Weill Cornell
Med. Col., Cornell Univ., New York, NY
Abstract: PICK1 (Protein Interacting with C-Kinase 1) is a scaffolding protein that is widely
expressed in the CNS and interacts via its N-terminal PDZ domain with several receptors,
transporters and kinases including e.g. the AMPA receptor GluR2/3 subunits, the metabotropic
glutamate receptor mGluR7, the dopamine transporter (DAT) and protein kinase Cα. PICK1 is
believed to fulfil its biological role either by regulating trafficking of its binding partners, or by
recruiting PKCα to facilitate their phosphorylation. PICK1 contains in its C-terminal half a BAR
domain that can dimerize to yield a membrane curvature sensing unit with a key role in
mediating these functions. Recently, we showed that in the absence of PDZ ligand the activity of
the BAR domain is inhibited by the PDZ domain, and that unmasking of BAR domain activity is
not just a consequence of ligand binding to the PDZ domain but requires as well recruitment of
PICK1 to a specific membrane compartment. Here we hypothesize that activation of the BAR
domain involves transitions between monomeric and dimeric/multimeric states of PICK1. To test
this hypothesis we have employed FRET (Fluorescence Resonance Energy Transfer) based
techniques. For studies in living cells, we have tagged full length PICK1 as well as two different
truncation mutants with eCFP- and eYFP. Application of the three-filter FRET technique showed
no FRET signal for cytoplasmic full-length PICK1; however, a significant FRET signal was seen
for PICK1 co-expressed with a transmembrane ligand that localized the protein to recycling
endosomes. A clear FRET signal was also observed for PICK1 with truncated PDZ domain
(PICK1 Γ101) that constitutively localized to endosomes. Importantly, the FRET signal was
reduced in a mutation of PICK1 Γ101 predicted based on homology modelling to disrupt BAR
domain dimerization. Additional cellular and biochemical experiments should further clarify the
significance of these observations and their functional implications.
Disclosures: I. Ammendrup-Johnsen , None; K.L. Madsen, None; M.B. Jensen, None; V.K.
Bhatia, None; H.A. Weinstein, None; U. Gether, None; D.S. Han, None.
Poster
238. LTD: Hippocampus and Cortex
Time: Sunday, November 16, 2008, 1:00 pm - 5:00 pm
Program#/Poster#: 238.9/D54
Topic: B.08.f. Long-term depression ( LTD )