The vitamin D receptor is present in caveolae-enriched plasma membranes and binds 1 alpha,25(OH)2-vitamin D3 in vivo and in vitro.
The steroid hormone 1 alpha,25(OH)(2)-vitamin D(3) (1,25D) regulates gene transcription through a nuclear receptor [vitamin D receptor (VDR)] and initiation of rapid cellular responses through a putative plasma membrane-associated receptor (VDR(mem)). This study characterized the VDR(mem) present in a caveolae-enriched membrane fraction (CMF), a site of accumulation of signal transduction agents. Saturable and specific [(3)H]-1,25D binding in vitro was found in CMF of chick, rat, and mouse intestine; mouse lung and kidney; and human NB4 leukemia and rat ROS 17/2.8 osteoblast-like cells; in all cases the 1,25D K(D) binding dissociation constant = 1-3 nM. Our data collectively support the classical VDR being the VDR(mem) in caveolae: 1) VDR antibody immunoreactivity was detected in CMF of all tissues tested; 2) competitive binding of [(3)H]-1,25D by eight analogs of 1,25D was significantly correlated between nuclei and CMF (r(2) = 0.95) but not between vitamin D binding protein (has a different ligand binding specificity) and CMF; 3) confocal immunofluorescence microscopy of ROS 17/2.8 cells showed VDR in close association with the caveolae marker protein, caveolin-1, in the plasma membrane region; 4) in vivo 1,25D pretreatment reduced in vitro [(3)H]-1,25D binding by 30% in chick and rat intestinal CMF demonstrating in vivo occupancy of the CMF receptor by 1,25D; and 5) comparison of [(3)H]-1,25D binding in VDR KO and WT mouse kidney tissue showed 85% reduction in VDR KO CMF and 95% reduction in VDR KO nuclear fraction. This study supports the presence of VDR as the 1,25D-binding protein associated with plasma membrane caveolae.
Department of Biochemistry, University of California, Riverside, California 92521, USA.
Johanna A
JA
Huhtakangas
Christopher J
CJ
Olivera
June E
JE
Bishop
Laura P
LP
Zanello
Anthony W
AW
Norman
D000818:Animals; D001667:Binding, Competitive; D002117:Calcitriol / Q000032:analysis / Q000378:metabolism*; D021941:Caveolae / Q000737:chemistry* / Q000378:metabolism; D051242:Caveolin 1; D022461:Caveolins / Q000032:analysis; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D002467:Cell Nucleus / Q000737:chemistry / Q000378:metabolism; D002645:Chickens; D006801:Humans; D051379:Mice; D051381:Rats; D018167:Receptors, Calcitriol / Q000032:analysis* / Q000378:metabolism*; D014018:Tissue Distribution
C495540:CAV1 protein, human; C495541:Cav1 protein, mouse; C495537:Cav1 protein, rat; D051242:Caveolin 1; D022461:Caveolins; D018167:Receptors, Calcitriol; D002117:Calcitriol