D1 dopamine receptor signaling involves caveolin-2 in HEK-293 cells.
BACKGROUND
Dopamine receptors in the kidney, especially those belonging to the D1-like receptor family, are important in the regulation of renal function and blood pressure. Because of increasing evidence that G protein-coupled receptors (GPCRs) are associated with caveolae and lipid rafts, we tested the hypothesis that the D1 dopamine receptor (D1R) and signaling molecules are regulated by caveolin in caveolae or lipid rafts.


METHODS
Six experimental approaches were used: (1) construction of tagged human D1Rs (hD1Rs) and transfectants; (2) cell culture [human embryonic kidney (HEK)-293 and immortalized rat renal proximal tubule cells] and biotinylation; (3) cell fractionation by sucrose gradient centrifugation; (4) immunoprecipitation and immunoblotting; (5) immunofluorescence and confocal microscopy; and (6) adenylyl cyclase assays.


RESULTS
hD1Rs, heterologously expressed in HEK-293 cells, formed protein species with molecular mass ranging from 50 to 250 kD, and were localized in lipid rafts and nonraft plasma membranes. The hD1Rs cofractionated with caveolin-2, G protein subunits, and several signaling molecules. Both exogenously expressed hD1Rs and endogenously expressed rat D1Rs colocalized and coimmunoprecipitated with caveolin-2. A D1R agonist (fenoldopam) increased the amount of caveolin-2beta associated with hD1Rs and activated adenylyl cyclase to a greater extent in lipid rafts than in nonraft plasma membranes. Reduction in the expression of caveolin-2 with antisense oligonucleotides attenuated the stimulatory effect of fenoldopam on cyclic adenosine monophosphate (cAMP) accumulation.


CONCLUSIONS
The majority of hD1Rs are distributed in lipid rafts. Heterologously and endogenously expressed D1Rs in renal cells are associated with and regulated by caveolin-2.
Yu
Peiying
P
Department of Pediatrics, Georgetown University School of Medicine, Washington, D.C., USA. yup@georgetown.edu
Yang
Zhiwei
Z
Jones
John E
JE
Wang
Zheng
Z
Owens
Shaun A
SA
Mueller
Susette C
SC
Felder
Robin A
RA
Jose
Pedro A
PA
D000262:Adenylyl Cyclases / Q000378:metabolism; D051243:Caveolin 2; D022461:Caveolins / Q000378:metabolism*; D002458:Cell Fractionation; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000502:physiology*; D021962:Membrane Microdomains / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D017447:Receptors, Dopamine D1 / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*; D013395:Sucrose; D014162:Transfection
D051243:Caveolin 2; D022461:Caveolins; D008565:Membrane Proteins; D016376:Oligonucleotides, Antisense; D017447:Receptors, Dopamine D1; C106354:flotillins; D013395:Sucrose; D000242:Cyclic AMP; D000262:Adenylyl Cyclases