Serum and monoclonal antibodies against the chick intestinal receptor for 1,25-dihydroxyvitamin D3. Generation by a preparation enriched in a 64,000-dalton protein.

Pike, J. Wesley; Marion, Samuel L.; Donaldson, Carol A.; Haussler, Mark R.

doi:10.1016/s0021-9258(18)33191-0

Cited by 112 publications

(5 citation statements)

References 61 publications

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“…The three monoclonal primary antibodies are likely to react principally with differing epitopes on the VDR, and each may cross-react with other proteins. 9A7, the principal anti-VDR antibody that we used, is a monoclonal IgG recognizing one VDR epitope common to birds and mammals (45,59). We do not presently know if the 9A7 antibody shares a portion of the epitope recognized by the VD2F12 antibody (11-20% overlap between 9A7 and VD2F12 antibodies in an assay based on competition of antibodies for partially purified VDRs) (Barsony, J., and R. E Schurnan, unpublished data [Antibody Resources Inc., Rockville, MD]).…”

Section: Discussionmentioning

confidence: 99%

“…Normal dermal fibroblasts were from three subjects (6). Dermal fibroblasts from seven patients with hereditary resistance to calcitriol (mutant fibroblasts) had VDR functional defects characterized previously (6,19,39,40,59,74).…”

Section: Cell Sourcesmentioning

confidence: 99%

“…3 and No. 7) with decreased VDR binding to DNA, the distribution of VDRs in unstimu- * The VDR functional categories were established in prior reports (6,19,39,40,59,74). Patients and their cells are assigned the same identification symbols as in those reports.…”

Section: Rapid Effects Of Calcitriol In L~broblasts From Patients With Defective Vitamin D Receptor Functionmentioning

confidence: 99%

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Immunocytology with microwave-fixed fibroblasts shows 1 alpha,25-dihydroxyvitamin D3-dependent rapid and estrogen-dependent slow reorganization of vitamin D receptors.

Bársony

Pike

DeLuca

et al. 1990

The Journal of Cell Biology

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Abstract. Prior studies have given no evidence for regulation of vitamin D receptor (VDR) compartmentalization or subcellular organization. Microwave fixation (9-15 s) and an indirect immunodetection system of avidin-biotin enhancement and phycoerythrin fluorophore resulted in sufficient spatial and temporal resolution to allow analysis of these processes. We studied cultured fibroblasts from normals or from patients with four different types of hereditary defect compromising VDR function (mutant cells).Compartmentalization of VDRs in the absence of 1,25-dihydroxyvitamin D3 (calcitriol) was regulated by serum or estrogen. VDRs were mainly cytoplasmic in cells cultured without serum and phenol red, but VDRs were mainly intranuclear after addition of serum or an estrogen to cells for at least 18 h (slow regulation).Calcitriol initiated a rapid and multistep process (rapid regulation) of reorganization in a portion of VDRs: clumping within 15-45 s, alignment of clumps along fibrils within 30-45 s, perinuclear accumulation of clumps within 45-90 s, and intranuclear accumulation of clumps within 1-3 roan. We found similar rapid effects of calcitriol on VDRs in various other types of cultured cells. These sequential VDR pattern changes showed calcitriol dose dependency and calcitriol analogue specificity characteristic for the VDR. In mutant fibroblasts VDR pattern changes after calcitriol were absent or severely disturbed at selected steps. Treatment of normal cells with wheat germ agglutinin, which blocks protein transport through nuclear pores, also blocked calcitriol-dependent translocation of VDRs. We conclude that immunocytology after microwave fixation provides evidence for regulation of VDR organization and localization.H ORMONE binding to the vitamin D receptor (VDR) ~ and other steroid receptor-related proteins initiates a process of receptor activation that allows the receptor to become competent to regulate transcription (7, 27). Steroid-related receptors may also mediate certain rapid (socalled nongenomic) effects of their hormonal agonists (4,17,34,68,70). Initiation of these genomic and nongenomic effects may be similar among steroid-related receptors, through biochemical and physical changes in the receptors, such as changes in phosphorylation (46, 52) and/or changes in binding to other cell components (i.e., identical proteins or other proteins [8,22,36]).Despite many studies, there is controversy concerning receptor compartmentalization and possible subcellular changes in receptor distribution patterns after binding between a steroid hormone and its receptor (44,73). Studies of steroid-related receptors in the 1960s and 1970s, prin- cipally with radioactive hormones, suggested that unstimulated receptors were mainly in the cytoplasm. Parallel studies suggested that steroid hormone binding initiated receptor translocation into the nucleus within minutes (76) or, in most studies, later than 0.5-4 h after hormone exposure (73). Subsequently, immunocytology and cell fractionation suggested that even nona...

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Section: Discussionmentioning

confidence: 99%

Section: Cell Sourcesmentioning

confidence: 99%

Section: Rapid Effects Of Calcitriol In L~broblasts From Patients With Defective Vitamin D Receptor Functionmentioning

confidence: 99%

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Immunocytology with microwave-fixed fibroblasts shows 1 alpha,25-dihydroxyvitamin D3-dependent rapid and estrogen-dependent slow reorganization of vitamin D receptors.

Bársony

Pike

DeLuca

et al. 1990

The Journal of Cell Biology

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“…Attempts to isolate the quantity of receptor at the necessary purity for physical or biochemical studies have met with limited success due to the low abundance of this receptor in tissue extracts (Pike & Haussler, 1979;Simpson & DeLuca, 1982;Simpson et al, 1983;Pike et al, 1983;Dame et al, 1985).…”

mentioning

confidence: 99%

Monoclonal antibodies to the porcine intestinal receptor for 1,25-dihydroxyvitamin D3: interaction with distinct receptor domains

Dame¹,

Pierce²,

Prahl³

et al. 1986

Biochemistry

119

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Monoclonal antibodies to different domains of the porcine intestinal 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] receptor have been produced. A nuclear extract enriched in the 1,25-(OH)2D3 receptor was prepared from small intestinal mucosa of young pigs. The receptor was purified an additional 6600-fold by chromatography on DNA-cellulose, ammonium sulfate precipitation, gel filtration high-performance liquid chromatography, and DEAE-Sepharose chromatography, with an overall yield of 23% and an average purity of 24%. A BALB/c mouse immunized with this material developed serum polyclonal antibodies to the 1,25-(OH)2D3 receptor, as demonstrated by a change in sedimentation of the porcine receptor on sucrose gradients. Spleen cells from this animal were fused with mouse myeloma cells (P3-NSI/1-Ag4-1, SP2/0-Ag14), and 24 hybridomas secreting antibodies to the 1,25-(OH)2D3 receptor were identified by both a radiometric immunosorbent assay and an immunoprecipitation assay. Twenty-one hybridoma lines were cloned by limiting dilution and further characterized as subclass IgG1 antibodies with the exception of one which is an IgA. All but two of the antibodies cross-react with the 1,25-(OH)2D3 receptor from both mammalian (human, monkey, and rat) and avian (chicken) intestine; two antibodies recognize only porcine intestinal receptor. All antibodies are unreactive to the vitamin D serum transport protein. Eight of the antibodies bind denatured receptor on an immunoblot. A solid-phase competition assay was used to identify four groups of antibodies that bind to distinct epitopes on the 1,25-(OH)2D3 receptor. One antibody from each of the four groups was used to examine the effect of antibody binding on the DNA-binding activity of the receptor-hormone complex. One antibody completely inhibited the binding of the 1,25-(OH)2D3 receptor complex to DNA-cellulose, suggesting that the epitope for this antibody may be located in the polynucleotide binding domain of the protein. Antibodies from two additional groups only slightly perturbed DNA binding, while one had no effect, suggesting that these antibodies bind to receptor epitopes distant from the region of the polypeptide directly involved in polynucleotide binding. These antibodies that are directed to several different binding sites on the 1,25-(OH)2D3 receptor provide important new tools to probe the biochemistry and topology of the 1,25-(OH)2D3 receptor and to investigate its role in mediating target tissue response to hormone.

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“…Immunoprecipitation and Gel Electrophoresis. Cell lysates were immunoprecipitated with anti-VDR 4A5y monoclonal antibody (mAb) linked to Sepharose beads (Pike et al, 1983).…”

Section: Methodsmentioning

confidence: 99%

The 1,25-dihydroxyvitamin D3 receptor is phosphorylated in response to 1,25-dihydroxyvitamin D3 and 22-oxacalcitriol in rat osteoblasts, and by casein kinase II, in vitro

Jurutka¹,

Terpening²,

Haussler³

1993

Biochemistry

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We analyzed the endogenous nuclear 1,25-dihydroxy-vitamin D3 (1,25(OH)2D3) receptor (VDR) in rat osteosarcoma (ROS 17/2.8) cells and present biochemical evidence that it is a phosphoprotein. When ROS 17/2.8 cells are labeled metabolically with [35S]methionine, treatment with 10(-8) M 1,25(OH)2D3 elicits a decrease in the electrophoretic mobility of immunoprecipitated VDR in denaturing polyacrylamide gels, a property characteristic of phosphorylated proteins. Similar labeling of cells with [32P]orthophosphate results in a rapid (< or = 30 min), 1,25(OH)2D3-dependent incorporation of 32P into a 54-kDa VDR species that comigrates with the slower migrating receptor species extracted from [35S]methionine-labeled ROS 17/2.8 cells that have been exposed to 1,25(OH)2D3. Alkaline phosphatase treatment of immunoprecipitated VDR from 1,25(OH)2D3-treated cells converts the form of the VDR with reduced mobility to the faster migrating species present in 1,25(OH)2D3-deficient cells. Incubation of ROS 17/2.8 cells with the non-hypercalcemic 1,25(OH)2D3 analog, 22-oxacalcitriol (OCT), produces a level of VDR phosphorylation similar to that elicited by 1,25(OH)2D3 treatment. Transient transfection of osteosarcoma cells with a reporter vector containing a vitamin D responsive element derived from the rat osteocalcin gene yields equivalent transcriptional activation in the presence of either 1,25(OH)2D3 or OCT. Further experiments performed at various 1,25(OH)2D3 concentrations to assess the relationship between receptor phosphorylation and transcriptional activity in intact cells showed a positive correlation between these two parameters, indicating that the 1,25(OH)2D3 hormone stimulates VDR phosphorylation and transcriptional activation in parallel. Finally, highly purified casein kinase II (CK-II) phosphorylates the VDR in a 1,25(OH)2D3-independent, in vitro reaction.(ABSTRACT TRUNCATED AT 250 WORDS)

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Serum and monoclonal antibodies against the chick intestinal receptor for 1,25-dihydroxyvitamin D3. Generation by a preparation enriched in a 64,000-dalton protein.

Cited by 112 publications

References 61 publications

Immunocytology with microwave-fixed fibroblasts shows 1 alpha,25-dihydroxyvitamin D3-dependent rapid and estrogen-dependent slow reorganization of vitamin D receptors.

Immunocytology with microwave-fixed fibroblasts shows 1 alpha,25-dihydroxyvitamin D3-dependent rapid and estrogen-dependent slow reorganization of vitamin D receptors.

Monoclonal antibodies to the porcine intestinal receptor for 1,25-dihydroxyvitamin D3: interaction with distinct receptor domains

The 1,25-dihydroxyvitamin D3 receptor is phosphorylated in response to 1,25-dihydroxyvitamin D3 and 22-oxacalcitriol in rat osteoblasts, and by casein kinase II, in vitro

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