Ribozyme knockdown functionally links a 1,25(OH)2D3 membrane binding protein (1,25D3-MARRS) and phosphate uptake in intestinal cells.
We used a ribozyme loss-of-function approach to demonstrate that the protein product of a cDNA encoding a multifunctional membrane-associated protein binds the seco-steroid 1,25(OH)(2)D(3) and transduces its stimulatory effects on phosphate uptake. These results are paralleled by studies in which the ability of the hormone to stimulate phosphate uptake in isolated chick intestinal epithelial cells is abolished by preincubation with Ab099 directed against the amino terminus of the protein. We now report the complete sequence of the cloned chicken cDNA for the 1,25D(3)-MARRS (membrane-associated, rapid-response steroid-binding) protein and reveal it to be identical to the multifunctional protein ERp57. Functional studies showed that active ribozyme, but not a scrambled control, decreased specific membrane-associated 1,25(OH)(2)D(3) binding, but did not affect binding to the nuclear receptor for 1,25(OH)(2)D(3). Seco-steroid-dependent stimulation of protein kinase C activity was diminished as 1,25D(3)-MARRS protein levels were reduced in the presence of the ribozyme, as judged by Western blot analyses. Phosphate uptake in isolated cells is an index of intestinal phosphate transport that occurs during growth and maturation. Whereas cells and perfused duodena robustly responded to 1,25(OH)(2)D(3) in preparations from young birds, older animals no longer responded with stimulated phosphate uptake or transport. The age-related decline was accompanied by a decrease in 1,25D(3)-MARRS mRNA that was apparent up to 1 year of age. Together, these studies functionally link phosphate transport in the chick duodenum with the 1,25D(3)-MARRS protein and point to a previously uncharacterized role for this multifunctional protein class.
Nemere
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I
Department of Nutrition and Food Sciences, and Center for Integrated BioSystems, Utah State University, Logan, UT 84322-8700, USA. nemere@cc.usu.edu
Farach-Carson
M C
MC
Rohe
B
B
Sterling
T M
TM
Norman
A W
AW
Boyan
B D
BD
Safford
S E
SE
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002117:Calcitriol / Q000378:metabolism*; D002645:Chickens; D017931:DNA Primers; D007413:Intestinal Mucosa / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D010710:Phosphates / Q000378:metabolism; D016337:RNA, Catalytic / Q000378:metabolism*
D017931:DNA Primers; D008565:Membrane Proteins; D010710:Phosphates; D016337:RNA, Catalytic; D002117:Calcitriol