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-1α 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.
Footnotes
-
This work was supported by the National Institute on Drug Abuse and the Canadian Institutes of Health Research. S.R.G is a Canada Research Chair in Molecular Neuroscience.
-
Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
-
doi:10.1124/mol.107.034769.
-
ABBREVIATIONS: GPCR, G protein-coupled receptor; D1R, D1 dopamine receptor; H89, N-[2-(4-bromocinnamylamino)ethyl]-5-isoquinoline; TM, transmembrane; SCH23390, R-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine; GTPγS, guanosine 5′-O-(3-thio)triphosphate; GFP, green fluorescent protein; mRFP, monomeric red fluorescent protein; eGFP, enhanced green fluorescent protein; Rluc, Renilla reniformis luciferase; GRK, G protein receptor kinase; HEK, human embryonic kidney; PAGE, polyacrylamide gel electrophoresis; ARF, ADP ribosylation factor; HA, hemagglutinin; HRP, horseradish peroxidase; PBS, phosphate-buffered saline; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate; O/N, overnight; RT, room temperature; SKF 81297, (±)-6-chloro-7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrobromide; BRET, bioluminescence resonance energy transfer; mβCD, methyl-β-cyclodextrin; PKA, protein kinase A; CBM, caveolin binding motif.
- Received February 4, 2007.
- Accepted August 15, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
MolPharm articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|