Vitamin D receptor phosphorylation in transfected ROS 17/2.8 cells is localized to the N-terminal region of the hormone-binding domain

Barbara B. Jones, Peter Jurutka, Carol A. Haussler, Mark R. Haussler, G. Kerr Whitfield

Research output: Contribution to journalArticle

28 Citations (Scopus)

Abstract

The human 1,25-dihydroxyvitamin D3 receptor (hVDR) has been recently shown to be phosphorylated in vitro by casein kinase-II. Most of the residues phosphorylated by this enzyme were shown to reside between Asn160 and Asp232, a region near the N-terminal boundary of the hormone-binding domain. We report here that the hVDR is also phosphorylated in vivo after transfection into ROS 17/2.8 cells. In addition to testing full-length hVDR, we analyzed several internally deleted hVDR mutants. The expression and phosphorylation of full-length and mutated hVDRs were monitored in transfected cells by metabolic labeling with either [35S]methionine or [32P]orthophosphate, followed by immunopurification using monoclonal anti-VDR antibody linked to agarose beads. Transfected hVDR is distinguishable from the endogenous rat VDR when the immunoprecipitated proteins are resolved on sodium dodecyl sulfate-polyacrylamide gels. Significant phosphorylation of transfected full-length hVDR was observed in ROS 17/2.8 cells, and it was less dependent on the presence of 1,25-dihydroxyvitamin D3 than that of the endogenous rat receptor. Most importantly, the region of in vivo phosphorylation, as defined by internal deletion mutants, resides between Met197 and Val234. Therefore, we have localized the major site of phosphorylation of hVDR to residues in the N-terminal region of the hormonebinding domain. The boundaries of this region fall within the amino acid segment defined for phosphorylation of hVDR by casein kinase-II in vitro, suggesting that VDR is an in vivo substrate for cas-ein kinase-II or a related protein kinase.

Original languageEnglish (US)
Pages (from-to)1137-1146
Number of pages10
JournalMolecular Endocrinology
Volume5
Issue number8
StatePublished - 1991
Externally publishedYes

Fingerprint

Calcitriol Receptors
Phosphorylation
Hormones
Casein Kinase II
Calcitriol
Sodium Dodecyl Sulfate
Methionine
Sepharose
Protein Kinases
Transfection
Anti-Idiotypic Antibodies
Phosphotransferases
Phosphates
Monoclonal Antibodies
Amino Acids
Enzymes

ASJC Scopus subject areas

  • Molecular Biology
  • Endocrinology, Diabetes and Metabolism

Cite this

Vitamin D receptor phosphorylation in transfected ROS 17/2.8 cells is localized to the N-terminal region of the hormone-binding domain. / Jones, Barbara B.; Jurutka, Peter; Haussler, Carol A.; Haussler, Mark R.; Whitfield, G. Kerr.

In: Molecular Endocrinology, Vol. 5, No. 8, 1991, p. 1137-1146.

Research output: Contribution to journalArticle

Jones, Barbara B. ; Jurutka, Peter ; Haussler, Carol A. ; Haussler, Mark R. ; Whitfield, G. Kerr. / Vitamin D receptor phosphorylation in transfected ROS 17/2.8 cells is localized to the N-terminal region of the hormone-binding domain. In: Molecular Endocrinology. 1991 ; Vol. 5, No. 8. pp. 1137-1146.
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