Hormones – Cytokines – Signalling
Kidney International (2004) 66, 2167–2180; doi:10.1111/j.1523-1755.2004.66007.x
D1 dopamine receptor signaling involves caveolin-2 in HEK-293 cells
PEIYING YU, ZHIWEI YANG, JOHN E JONES, ZHENG WANG, SHAUN A OWENS, SUSETTE C MUELLER, ROBIN A FELDER and PEDRO A JOSE
Department of Pediatrics, Georgetown University School of Medicine, Washington, D.C.; Department of Physiology and Biophysics, Georgetown University School of Medicine, Washington, D.C.; Department of Oncology, Lombardi, Comprehensive Cancer Center, Georgetown University, Washington, D.C.; and Department of Pathology, The University of Virginia, Charlottesville, Virginia
Correspondence: Peiying Yu, M.D., Georgetown University Medical Center, Department of Pediatrics, 3800 Reservoir Road, NW, Washington, D.C. 20007. E-mail:yup@georgetown.edu
Received 29 December 2003; Revised 30 April 2004; Accepted 22 June 2004.
Abstract
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-2
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.
Conclusion
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.
Keywords:
D1 dopamine receptor, caveolin-2, flotillin, renal proximal tubule, cAMP
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