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Received for publication February 8, 2007.
Revised August 14, 2007.
Accepted for publication August 15, 2007.
There is accumulating evidence that G protein-coupled receptor signaling is regulated by localization in lipid raft microdomains. In this report, we determined that the D1 dopamine receptor (D1R) is localized in caveolae, a subset of lipid rafts, by sucrose gradient fractionation and confocal microscopy. Through co-immunoprecipitation and bioluminescence resonance energy transfer assays, we demonstrated that this localization was mediated by an interaction between caveolin-1 and D1R in COS7 cells and an isoform selective interaction between D1R and caveolin-1
in rat brain. We determined that the D1R interaction with caveolin-1 required a putative caveolin binding motif identified in transmembrane domain 7. Agonist stimulation of D1R caused translocation of D1R into caveolin-1 enriched sucrose fractions which was determined to be a result of D1R endocytosis through caveolae. This was found to be PKA-independent and a kinetically slower process than clathrin mediated endocytosis. Site directed mutagenesis of the caveolin binding motif at amino acids F313 and W318 significantly attenuated caveolar endocytosis of D1R. We also found that these caveolin binding mutants had a diminished capacity to stimulate cAMP production which was determined to be due to constitutive desensitization of these receptors. In contrast, we found that D1Rs had an enhanced ability to maximally generate cAMP in chemically induced caveolae disrupted cells. Taken together, these data suggest that caveolae has an important role in regulating D1R turnover and signaling in brain.
Key words:
Dopamine, Gs family, cAMP, Lipid rafts/microdomains, Sequestration/Internalization, Fluorescence techniques, Mutagenesis/Chimeric approaches, Receptor binding studies
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