Received for publication December 1, 2005.
Revised April 5, 2006.
Accepted for publication April 5, 2006.

Agonist-induced Cell Surface Trafficking of an Intracellularly Sequestered D1 Dopamine Receptor Homo- oligomer

Abstract

The role of oligomerization in D1 dopamine receptor trafficking to the cell surface was examined using conformationally distinct variants of this receptor. Substitution of the highly conserved aspartic acid (Asp¹⁰³) in transmembrane domain 3 resulted in a constitutively active receptor, D103A, that did not bind agonists or antagonists, but trafficked to the cell surface as oligomers. Co-expression of D103A with the wild-type D1 receptor in HEK293t cells resulted in inhibition of cell surface expression of the D1 receptor, due to receptor oligomerization, causing intracellular retention of both proteins. Rescue of the intracellularly retained oligomer could be achieved only by membrane permeable full and partial agonists, which resulted in cell surface expression of the D1 receptor, whereas cell permeable antagonists and cell impermeable agonists had no effect. Cell surface fluorescence resonance energy transfer studies of cells co-expressing D103A and D1 revealed no signal before agonist treatment but a robust signal after agonist treatment, indicating that the intact D1/D103A oligomer reached the cell surface only after agonist treatment but not under basal conditions. This suggests that rescue of the retained D1/D103A oligomer to the cell surface was a result of an agonist-induced change in the conformation of D1, permitting cell surface trafficking of the D1/D103A receptor oligomeric complex from the endoplasmic reticulum.

Key words: Dopamine, cAMP, Receptor synthesis/trafficking, Fluorescence techniques, Mutagenesis/Chimeric approaches, Receptor binding studies