Regulation of D1 Dopamine Receptor Trafficking and Signaling by Caveolin-1

Abstract

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 coimmunoprecipitation and bioluminescence resonance energy transfer assays, we demonstrated that this localization was mediated by an interaction between caveolin-1 and D1R in COS-7 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 protein kinase A-independent and a kinetically slower process than clathrin-mediated endocytosis. Site-directed mutagenesis of the caveolin binding motif at amino acids Phe313 and Trp318 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.

Caveolin proteins and estrogen signaling in the brain

“…Additionally, caveolae can rapidly modulate signaling through an endocytotic mechanism by which the caveolae internalize receptors or other signaling components so that they are non-functional. These endocytosed structures can remain near the membrane surface so that they can be made available for signaling by reintroduction to the membrane surface facilitated by the caveolae [46,118,119].…”

“…Surface receptors in which trafficking to the membrane has been reported to be dependent on caveolin function include, but are not limited to, the D1 dopamine receptor [46], M1 muscarinic receptor [47], angiotensin II type 1 receptor [48], and glucagon-like peptide 1 receptor [49]. Notably, caveolins also play a role in receptor endocytosis [47,[50][51][52][53][54] providing an additional regulatory mechanism to modulate cell signaling.…”

β-Arrestin Recruitment and G Protein Signaling by the Atypical Human Chemokine Decoy Receptor CCX-CKR

“…The occurrence of parallel internalization pathways for one receptor is not unprecedented. For instance, the dopamine D 1 receptor internalizes through both caveolae and clathrin-coated pits (34,35). Interestingly, inhibition of clathrin-coated pit-dependent endocytosis by various methods did .…”

“…not affect D 1 receptor internalization, suggesting that ␤-arrestin and clathrin-coated pits might not be the dominant pathway for this receptor (34).…”

Psychostimulants, Brain Membrane Lipids and Dopamine Transmission

“…D1Rs are primarily localized in lipid raft microdomains in HEK293 and COS-7 cells and can physically interact with caveolae, a raft-associated protein [49,50]. D1Rs and caveolae can form a functional complex in lipid rafts and regulate D1R-mediated G protein signaling.…”