Fog, Jacob2; Vægter, Christian Bjerggaard4; Gether, Ulrik2
1 Department of Medical Biochemistry, Faculty of Health Sciences, Aarhus University, Aarhus University2 University of Copenhagen3 Department of Biomedicine - Forskning og uddannelse, Vest, Department of Biomedicine, Health, Aarhus University4 Department of Biomedicine - Forskning og uddannelse, Vest, Department of Biomedicine, Health, Aarhus University
PICK1 has been shown to interact with the distal dopamine transporter (DAT) C-terminus via its PDZ domain. Although we recently have shown that ER export and targeting of the DAT to the cell surface is critically dependent on discrete epitopes in the distal C-terminus, these events do not require canonical PDZ domain interactions with proteins such as PICK1. To clarify the actual role of PDZ domain interactions for DAT function we have expressed the wild type DAT and a number of C-terminal mutants either alone or together with PICK1 in HEK293, N2A neuroblastoma and PC12 cells. Data obtained from analysis of these cell lines strongly suggest that the DAT interaction with PICK1 might play a role in recruiting PICK1 to the membrane rather than a role of PICK1 in recruiting DAT to the surface. In HEK293 cells we have also data indicating that the strength of the DAT-PICK1 complex is much higher than what would be expected from the affinity of C-terminal DAT peptides to recombinant PICK1. A DAT mutant incapable of binding PICK1, +Ala, does not display the same characteristics. The tight interaction between DAT and PICK1 is further supported by the finding that PICK1 is co-internalized with DAT upon stimulation with phorbol esters. Currently we are performing experiments aimed at further addressing the functional consequences of the tight interaction between PICK1 and DAT. In parallel to these studies we are currently exploring the function of several putative DAT interacting proteins identified either by yeast two-hybrid screens or proteomics based approaches.
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Society for Neuroscience 34th annual meeting, 2004