Biomedical subjects

M R Haussler

Publications and source records attributed to M R Haussler.

At least 37 records · Page 2Linked to original sources

PubMed1996

Examination of the potential functional role of conserved cysteine residues in the hormone binding domain of the human 1,25-dihydroxyvitamin D3 receptor.

The significance of conserved cysteines at positions 288, 337, and 369 in the hormone binding domain of the human vitamin D receptor was evaluated by individual site-directed mutagenesis to glycine. Neither nuclear localization nor heterodimerization with retinoid X receptors in binding to the vitamin D-responsive element was appreciably affected by altering these cysteines, but vitamin D hormone (1,25-(OH)2D3) activated transcription was compromised significantly in the C288G and C337G mutants. Only the C288G mutant displayed depressed (3-fold) 1,25-(OH)2D3 ligand binding affinity at 4 degrees C, in vitro, although at elevated temperatures (23-37 degrees C), ligand binding was attenuated severely in C288G, moderately in C337G and very mildly in C369G. The degree of impairment of ligand binding at physiologic temperatures correlated with the requirement for increased concentrations of 1,25-(OH)2D3 ligand to maximally stimulate transcriptional activity in co-transfected COS-7 cells. Thus cysteine 288 and, to a lesser extent, cysteine 337 are important for high affinity hormone binding to the vitamin D receptor, which ultimately leads to ligand-dependent transcriptional activation.

Amino Acid Sequence↗

PubMed1996

Affinity labeling of the 1 alpha,25-dihydroxyvitamin D3 receptor.

Genomic actions of the calciotropic hormone 1 alpha, 25-dihydroxyvitamin D3 (1,25(OH)2D3) involves a multistep process that is triggered by the highly specific binding of 1,25(OH)2D3 to 1 alpha, 25-dihydroxyvitamin D3 receptor, VDR. In order to study this key step in the cascade, we synthesized 1 alpha,25-dihydroxy[26(27)-3H]vitamin D3-3-deoxy-3 beta-bromoacetate (1,25(OH)2[3H]D3-BE) and 1 alpha,25-dihydroxyvitaminD3-3 beta-[1-14C]bromoacetate(1,25(OH)2D3-[14C]BE) binding-site directed analogs of 1,25(OH)2D3, and affinity-labeled baculovirus-expressed recombinant human VDR (with 1,25(OH)2[3H]D3-BE), and naturally occurring VDRs in cytosols from calf thymus homogenate and rat osteosarcoma (ROS 17/2.8) cells (with 1,25(OH)2D3-[14C]BE). In each case, specificity of labeling was demonstrated by the drastic reduction in labeling when the incubation was carried out in the presence of an excess of nonradioactive 1 alpha,25(OH)2D3. These results strongly suggested that 1,25(OH)2[3H]D3-BE and 1,25(OH)2D3-[14C]BE covalently modified the 1,25(OH)2D3-binding sites in baculovirus-expressed recombinant human VDR and naturally occurring calf thymus VDR and rat osteosarcoma VDR, respectively.

Affinity Labels↗

PubMed1996

Inhibition of ligand induced promoter occupancy in vivo by a dominant negative RXR.

BACKGROUND: Retinoid X receptors (RXRs) heterodimerize with other nuclear hormone receptors and control ligand mediated transcription. To address how RXRs function as heterodimers, we investigated activities of truncated RXR alpha and RXR beta that lack approximately 20 conserved C-terminal amino acids. RESULTS: The truncated RXRs formed heterodimers and bound to respective DNA elements in vitro. By transient reporter assays we found that these RXRs act as dominant negative receptors and inhibit ligand dependent transcription by the retinoic acid receptor (RAR) and vitamin D receptor. P19 embryonal carcinoma cells stably expressing the truncated RXR beta (termed delta C2) were deficient in activating the endogenous RAR beta gene and an RA responsive reporter. To study the dominant negative activity of delta C2 further, genomic footprinting analysis was performed for the RAR beta2 promoter. In control P19 clones, the RA responsive element (RARE) and other elements in the promoter were protected after RA treatment. However, in delta C2 clones RA-induced protection was markedly inhibited at all elements. CONCLUSIONS: These results indicate that the C-terminal region of RXR is required for full RARE occupancy in vivo, a RA dependent process that leads to the recruitment of other factors to the promoter and the subsequent transcriptional activation. Thus, RXRs play an integral role in ligand dependent transcription.

Animals↗

PubMed1996

A composite element binding the vitamin D receptor, retinoid X receptor alpha, and a member of the CTF/NF-1 family of transcription factors mediates the vitamin D responsiveness of the c-fos promoter.

The hormonal form of vitamin D, 1 alpha,25-dihydroxyvitamin D3 [1,25- (OH)2D3], transiently stimulates the transcription of the c-fos proto-oncogene in osteoblastic cells. We have identified and characterized a vitamin D response element (VDRE) in the promoter of c-fos. The 1,25-(OH)2D3-responsive region was delineated between residues -178 and -144 upstream of the c-fos transcription start site. A mutation that inhibited binding to the sequence concomitantly abolished 1,25-(OH)2D3-induced transcriptional responsiveness; similarly, cloning to the site upstream of a heterologous promoter conferred copy-number-dependent vitamin D responsiveness to a reporter gene, demonstrating that we have identified a functional response element. The structure of the c-fos VDRE was found to be unusual. Mutational analysis revealed that the c-fos VDRE does not conform to the direct repeat configuration in which hexameric core-binding sites are spaced by a few nucleotide residues. In contrast, the entire 36-bp sequence was essential for binding. We identified the vitamin D receptor and the retinoid X receptor alpha as components of the complex that bound the c-fos VDRE. However, our results also show that a putative CCAAT-binding transcription factor/nuclear factor 1 (CTF/NF-1) family member bound the response element in conjunction with the nuclear hormone receptors. The expression of this CTF/NF-1 family member appeared restricted to bone cells. These data hint at new molecular mechanisms of action for vitamin D.

Base Sequence↗

PubMed1996

Vitamin D receptors from patients with resistance to 1,25-dihydroxyvitamin D3: point mutations confer reduced transactivation in response to ligand and impaired interaction with the retinoid X receptor heterodimeric partner.

Hereditary hypocalcemic vitamin D-resistant rickets is attributable to defects in the nuclear receptor for 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. Two novel point mutations (I314S and R391C) identified in the hormone-binding domain of the human vitamin D receptor (VDR) from patients with hereditary hypocalcemic vitamin D-resistant rickets confer the receptor with sharply reduced 1,25-(OH)2D3-dependent transactivation. These natural mutations, especially R391C, also lead to a second specific consequence, namely impaired heterodimeric interaction with retinoid X receptor (RXR). While the transactivation ability of the I314S mutant can be largely restored by providing excess 1,25-(OH)2D3, R391C activity is more effectively restored with exogenous RXR. These observations are reflected also in the clinical course of each patient: the patient bearing the I314S mutation showed a nearly complete cure with pharmacological doses of a vitamin D derivative, whereas the patient bearing R391C responded only partially to such therapy. Further tests with patient fibroblasts and transfected cells show that the activity of the I314S VDR mutant is augmented somewhat by added RXR, while transactivation by the R391C mutant is best corrected by RXR in the presence of excess hormone. Thus, the effects of hormone vs. RXR in bolstering these mutant VDRs, such that they mediate efficient transactivation, are not entirely separable. The unique properties of these genetically altered receptors establish a new subclass of natural human VDR mutants that illustrate, in vivo, the importance of both 1,25-(OH)2D3 binding and heterodimerization with RXR in VDR action.

Adolescent↗

PubMed1995

The T-box near the zinc fingers of the human vitamin D receptor is required for heterodimeric DNA binding and transactivation.

The T-box mediates binding of retinoid X receptor (RXR) homodimers to DNA while the P- and D-box in the zinc fingers of steroid hormone receptors play roles in DNA-binding specificity and homodimerization, respectively. We investigated the function of these elements in the human vitamin D receptor (hVDR) by mutating a Lys-Glu pair of amino acids in the T-box, and by altering the P- and D-boxes to the corresponding residues of the glucocorticoid receptor (GR). The T-box mutant hVDR displayed attenuated vitamin D responsive element (VDRE) binding in the presence of RXR and was severely compromised in transcriptional activation. In contrast, GR P/D-box mutant hVDRs bound to the rat osteocalcin VDRE and elicited near normal transcriptional activation. The T-box mutant uniquely exhibited dominant negative properties, highlighting the significance of this region of hVDR for heterodimeric transcriptional activation.

Amino Acid Sequence↗

PubMed1995

Transcription factor TFIIB and the vitamin D receptor cooperatively activate ligand-dependent transcription.

The active metabolite of vitamin D, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], regulates gene transcription through binding to the vitamin D receptor (VDR), a member of the nuclear hormone receptor superfamily. Sequence-specific transcription factors, including nuclear hormone receptors, are thought to interact with the basal transcription complex to regulate transcription. In glutathione S-transferase fusion-based protein-protein binding assays we found that VDR specifically binds to TFIIB, a component of the basal complex, and that the interaction requires select domains of each protein. To assess the functional significance of this interaction, transfection assays were performed with a 1,25(OH)2D3-responsive reporter construct. In P19 embryonal carcinoma cells cotransfection of VDR and TFIIB cooperatively activated reporter transcription, while each factor alone gave very low to no activation. This activation was dependent on 1,25(OH)2D3 and the dose of TFIIB and VDR transfected, demonstrating that a nuclear hormone receptor functionally interacts with TFIIB in vivo. In contrast, transfection of NIH 3T3 cells generated strong reporter activation by 1,25(OH)2D3 in the presence of VDR alone, and cotransfection of TFIIB led to specific dose-dependent repression of reporter activity. Taken together, these results indicate that TFIIB-nuclear hormone receptor interaction plays a critical role in ligand-dependent transcription, which is apparently modulated by a cell-type-specific accessory factor.

Adult↗

PubMed1995

1,25(OH)2D3-dependent regulation of calbindin-D28k mRNA requires ongoing protein synthesis in chick duodenal organ culture.

Organ culture of 19-day-old chick embryo duodena was utilized to evaluate the mechanism of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3)-dependent calbindin-D28k (CaBP) expression. Duodenal CaBP and 1,25(OH)2D3 receptor (VDR) expression were assessed by Western blot analysis, while CaBP and VDR mRNA levels were determined by Northern blot analysis. In untreated duodena, both VDR protein and mRNA were present, while CaBP protein and mRNA were undetectable. Treatment of cultured duodena with 25 nM 1,25(OH)2D3 resulted in detectable CaBP mRNA after 4 h which continued to increase during a 24 h time period. Under these conditions, localization of [3H-1 beta]1 alpha,25(OH)2D3 in duodenal chromatin is rapid (< or = 30 min). Thus, the delayed accumulation of detectable CaBP mRNA cannot be explained by slow nuclear binding of 1,25(OH)2D3. The inclusion of 1.6 microM actinomycin D in the organ culture partially inhibited the 1,25(OH)2D3-regulated increase in CaBP mRNA, which implies that there is a transcriptional component involved in the increased CaBP mRNA levels. Similarly, quantitative polymerase chain reaction studies allowed the detection of CaBP pre-mRNA and mRNA sequences 1 h after hormone treatment, suggesting that CaBP gene transcription is initiated rapidly. Treatment of cultures with 36 microM cycloheximide 1 h prior to 1,25(OH)2D3 addition resulted in superinduction of VDR mRNA levels but sharply reduced CaBP steady-state mRNA levels. This dramatic reduction in CaBP mRNA reveals that 1,25(OH)2D3-mediated CaBP expression is dependent on ongoing protein synthesis. Thus, we propose that a labile auxiliary protein or other cofactor, which may or may not be 1,25(OH)2D3-dependent, is necessary for 1,25(OH)2D3-mediated CaBP gene transcription in chick duodena.

Animals↗

PubMed1995

Receptor mediated genomic action of the 1,25(OH)2D3 hormone: expression of the human vitamin D receptor in E. coli.

The nuclear vitamin D receptor (VDR) binds the 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) hormone with high affinity and elicits its actions to stimulate gene expression in target cells by binding to the vitamin D-responsive element (VDRE). VDREs in such positively controlled genes as osteocalcin, osteopontin, beta 3 integrin and vitamin D-24-OHase are direct hexanucleotide repeats with a spacer of three nucleotides. The present studies of VDR/VDRE interaction utilized full-length human vitamin D receptor (hVDR) that was overexpressed in E. coli, purified to near homogeneity (> 95%), and its authenticity confirmed by demonstrating high affinity hormone binding and reactivity to monoclonal antibody 9A7 gamma. The expressed hVDR displays strict dependence on the family of retinoid X receptors (RXRs) for binding to the vitamin D-responsive element (VDRE) in the rat osteocalcin gene. Similar overexpression in E. coli of the DNA binding domain (delta 134), containing only residues 4-133 of hVDR, generated a receptor species that possesses intrinsic DNA binding activity. Both full-length and delta 134 hVDRs retain similar DNA binding specificities when tested with several natural hormone responsive elements, indicating that the N-terminal zinc finger region determines hVDR-DNA sequence selectivity. The C-terminal region of the molecule is required for hormone binding and confers the receptor with the property of very high affinity DNA binding, via heterodimerization between hVDR and RXR. A natural ligand for the RXR co-receptor, 9-cis retinoic acid, suppresses both VDR-RXR binding to the VDRE and 1,25(OH)2D3 stimulated transcription, indicating that 9-cis retinoic acid recruits RXR away from VDR to instead form RXR homodimers.

Base Sequence↗

PubMed1995

New understanding of the molecular mechanism of receptor-mediated genomic actions of the vitamin D hormone.

The nuclear vitamin D receptor (VDR) binds the 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]hormone with high affinity and elicits its actions to regulate gene expression in target cells by binding to vitamin D-responsive elements (VDREs). VDREs in positively controlled genes such as osteocalcin, osteopontin, beta 3-integrin, and vitamin D-24-OHase are direct hexanucleotide repeats with a spacer of three nucleotides. The VDR associates with these VDREs with the greatest affinity as a heterodimer with one of the family of retinoid X receptors (RXRs). VDR consists of an N-terminal zinc finger domain that determines DNA binding, a "hinge" segment and a C-terminal hormone binding domain which also contains two conserved regions that engage in heterodimerization with an RXR on the VDRE. The role of the 1,25(OH)2D3 ligand in transcriptional activation by the VDR-RXR heterodimer is to alter the conformation of the hormone-binding domain of VDR to facilitate strong dimerization with RXR, which results in ligand-enhanced association with the VDRE. Thus RXR is recruited into a heterocomplex by liganded VDR. The natural ligand for the RXR coreceptor, 9-cis retinoic acid, suppresses both VDR-RXR binding to the VDRE and 1,25(OH)2D3-stimulated transcription, indicating that 9-cis retinoic acid diverts RXR away from being the silent partner of VDR to instead form RXR homodimers. Recent data reveal that after binding RXR, a subsequent target for VDR in the vitamin D signal transduction cascade is basal transcription factor IIB (TFIIB).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

Genomic actions of 1,25-dihydroxyvitamin D3.

Recent studies have identified a heterodimer of the vitamin D receptor (VDR) and the retinoid X receptor (RXR) as the active complex for mediating positive transcriptional effects of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], the active hormonal form of vitamin D. The VDR-RXR heterodimer has been shown to bind to direct repeat vitamin D-responsive elements (VDREs) upstream of positively controlled genes in the target tissues for vitamin D, including bone (osteocalcin, osteopontin, and beta 3 integrin), kidney (24-hydroxylase) and intestine (calbindin). Residues that participate in heterodimer formation have been identified in the C-terminal hormone-binding domain by analysis of VDR mutants. The role of the 1,25(OH)2D3 ligand in transcriptional activation by the VDR-RXR heterodimer is not entirely clear, but studies of two natural VDR mutants suggest that the binding of both hormone and RXR are required to induce a receptor conformation that is competent to activate transcription. A final level of complexity is added by recent observations that VDR is modified by phosphorylation. Thus, the VDR-mediated action of 1,25(OH)2D3 is now known to involve multiple factors that may provide a conceptual basis for future understanding of the tissue-specific genomic effects of 1,25(OH)2D3.

Animals↗

PubMed1995

A highly conserved region in the hormone-binding domain of the human vitamin D receptor contains residues vital for heterodimerization with retinoid X receptor and for transcriptional activation.

Residues located between amino acids 244 and 263 in the human vitamin D receptor (hVDR) show extensive homology with other members of the steroid/thyroid/retinoid hormone receptor superfamily. The corresponding region of the glucocorticoid receptor has been shown to interact with the 90-kilodalton heat shock protein (hsp90), yet hVDR does not appear to bind to hsp90. Herein we report a study of hVDR in which the functional role of five conserved residues was tested by replacing Phe-244, Lys-246, Leu-254, Gln-259, and Leu-262 with glycines by site-directed mutagenesis. Initial screening of these mutants indicated that all were significantly impaired in their ability to activate transcription from a vitamin D-responsive reporter construct when expressed in transfected VDR-deficient COS-7 cells. Further characterization revealed two classes of mutants: the predominant class binds the 1,25-dihydroxyvitamin D3 ligand normally but is defective in its ability to form a heterodimeric complex with the retinoid X receptor (RXR) on a vitamin D responsive element (VDRE). A second unique class, represented by a single mutant at Lys-246, is normal both with respect to ligand binding and complex formation but still very impaired in transactivation ability. The distinction between these two classes was confirmed by the demonstration that a member of the first class, with a mutation at Gln-259, could be restored to near wild type transactivation ability by supplying excess RXR, while the Lys-246 mutant could not be so rescued. We therefore conclude that the primary function of this conserved domain in hVDR is the mediation of heterodimerization with RXR, leading to VDRE binding and transactivation. The possibility also exists that the Lys-246 mutant may be impaired in a step of transactivation that is distal to complex formation with RXR on the VDRE, perhaps in interactions with the transcriptional machinery itself.

Amino Acid Sequence↗

PubMed1994

The C-terminal region of the vitamin D receptor is essential to form a complex with a receptor auxiliary factor required for high affinity binding to the vitamin D-responsive element.

The human vitamin D receptor (hVDR) requires another nuclear protein(s), designated receptor auxiliary factor (RAF), for optimal binding to the vitamin D-responsive element (VDRE). To determine the region in hVDR required to form a heterodimer with RAF on the VDRE, mutant hVDR cDNAs were constructed by site-directed mutagenesis and transfected into COS-7 cells. A truncated hVDR, lacking 25 C-terminal amino acids (delta 403-427), showed complex production in combination with endogenous RAF in COS-7 cells. Complex development was markedly enhanced by adding a rat liver nuclear fraction, which contains RAF activity, or either the alpha or beta form of the retinoid-X receptor, which has been reported to be closely related or identical to RAF. In contrast, either a C-terminal truncation of 46 amino acids (delta 382-427) or single point mutations at lysine-382, methionine-383, glutamine-385, or leucine-390 dramatically reduced the ability of hVDR to heterodimerize with RAF. Binding of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] hormone was undetectable in delta 382-427 truncated hVDR, whereas the delta 403-427 mutant hVDR exhibited significant 1,25-(OH)2D3 ligand binding, although the dissociation constant was approximately 10-fold higher than that of the wild-type receptor. Surprisingly, the delta 403-427 mutant hVDR did not mediate measurable transcriptional activation in cotransfection experiments with a VDRE-GH reporter gene construct. These results indicate that hVDR residues between cysteine-403 and serine-427 are required for very high affinity 1,25-(OH)2D3 ligand binding and transcriptional activation, but are not involved in heterodimerization. The region of hVDR between lysine-382 and arginine-402, probably the domain containing heptad 9, plays an essential role in the heterodimerization of hVDR with RAF. However, based upon additional point mutagenesis experiments, it is likely that other regions of the hormone-binding domain, such as that including heptad 4 (leucine-325 to leucine-332), also contribute to the protein-protein interactions required for the high affinity, specific binding of hVDR to the VDRE.

Amino Acid Sequence↗

PubMed1994

New insight into the structure and functions of the vitamin D receptor.

The past 5 years has witnessed marked progress toward a higher understanding of the vitamin D endocrine system. Presently, we have a more complete picture of the VDR itself, the DNA sequences to which it binds, and the other coreceptors with which it interacts. This last feature is perhaps the most startling recent development in the vitamin D endocrine system. It clearly suggests a potential role for retinoids and their vitamin A-derived ligands in the mechanism of vitamin D action. These two fat-soluble vitamins not only have quite discrete functionalities, but they may also play requisite roles in the pathway of each other. This is particularly intriguing because the actions of vitamin D were attributed originally to vitamin A. Only later were these two vitamins functionally dissected. Now, approximately 75 years later, the emerging data suggest that selected actions of these two fat-soluble vitamins may indeed be linked.

Amino Acid Sequence↗

PubMed1993

Purified human vitamin D receptor overexpressed in E. coli and baculovirus systems does not bind 1,25-dihydroxyvitamin D3 hormone efficiently unless supplemented with a rat liver nuclear extract.

We report here that highly purified human vitamin D receptor (hVDR) derived from E. coli or baculovirus expression systems does not exhibit saturable, high affinity 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) ligand binding when these preparations alone are analyzed. Inclusion of rat liver nuclear extract, which does not itself contain detectable 1,25(OH)2D3 binding activity, is required to endow hVDR isolated from bacterial or insect cells with the property of high affinity hormone binding (Kd = 0.13-0.22 nM). This observation should facilitate the valid assay of 1,25(OH)2D3 binding activity and kinetics in samples of overexpressed hVDR. Moreover, since rat liver nuclear extract contains retinoid X receptors and possibly other auxiliary factors capable of forming heterodimers with hVDR that in turn associate with vitamin D responsive elements, we hypothesize that like DNA binding, 1,25(OH)2D3 binding to hVDR requires the cooperation of a co-receptor or some uncharacterized receptor activating/stabilizing factor.

Animals↗

PubMed1993

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

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)

Alkaline Phosphatase↗

PubMed1993

Phosphorylation of the human vitamin D receptor by protein kinase C. Biochemical and functional evaluation of the serine 51 recognition site.

We have reported previously that the human vitamin D receptor (hVDR) is selectively phosphorylated by protein kinase C-beta (PKC-beta), in vitro, on a serine residue in the sequence RRS51MKRK, which is located between the two zinc fingers of hVDR and is potentially important to its transacting function (Hsieh, J.-C., Jurutka, P.W., Galligan, M.A., Terpening, C.M., Haussler, C.A., Samuels, D.S., Shimizu, Y., Shimizu, N., and Haussler, M.R. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 9315-9319). In the present experiments we evaluated this phosphorylation event using a series of hVDR mutants in which serine 51 or its flanking residues were modified. Alteration of serine 51 to a non-phosphorylatable residue resulted in an approximately 60% reduction in basal hVDR phosphorylation in intact cells but did not diminish 1,25-dihydroxyvitamin D3-stimulated phosphorylation. Such mutations also abolished subsequent phosphorylation of immunoprecipitated hVDR by purified PKC-beta, in vitro, as did replacement of basic residues on either side of serine 51. Mutation of serine 51 to glycine (S51G) or to aspartic acid (S51D), as well as altering the basic residues flanking serine 51, abolished the interaction of hVDR with the vitamin D-responsive element (VDRE) as monitored by gel mobility shift analysis. Thus, we conclude that unmodified serine 51 and its surrounding basic residues are crucial not only for PKC-beta substrate recognition but also for the optimal VDRE binding of native hVDR. In transactivation assays, S51G and S51D possessed only 35 and 10% of wild-type hVDR activity, respectively. Mutation of serine 51 to threonine (S51T) restored phosphorylation by PKC-beta, in vitro, to about 40% of wild-type and transactivation to 45% of that of wild-type hVDR. Alteration of serine 51 to alanine, which is the residue in the corresponding position of the glucocorticoid, progesterone, mineral-ocorticoid, and androgen receptors, eliminated PKC-beta phosphorylation but completely preserved the specific DNA binding activity and transactivation capacity of hVDR. Thus, phosphorylation of hVDR at serine 51 is not required for either VDRE binding or transactivation. Finally, incubation of Escherichia coli-expressed hVDR with PKC-beta elicits marked phosphorylation of the receptor and significantly inhibits its ability to complex with the VDRE. We therefore speculate that posttranslational modification of hVDR at serine 51 may constitute a negative regulatory loop which could be operative when target cells are subject to PKC activation events.

Amino Acid Sequence↗

PubMed1993

Phosphorylation of the human 1,25-dihydroxyvitamin D3 receptor by cAMP-dependent protein kinase, in vitro, and in transfected COS-7 cells.

We report that the human 1,25-dihydroxyvitamin D3 receptor is an efficient substrate for cAMP-dependent protein kinase, in vitro. This phosphorylation reaction is rapid and neither dependent upon nor significantly affected by the presence of the 1,25-dihydroxyvitamin D3 ligand. Preliminary mapping experiments utilizing C-terminal truncation mutants reveal that the primary site(s) of phosphorylation, in vitro, is localized between amino acids 133 and 201. Cotransfection of the catalytic subunit of murine cAMP-dependent protein kinase and the human 1,25-dihydroxyvitamin D3 receptor into monkey kidney (COS-7) cells not only results in a dramatic kinase-dependent increase in receptor phosphorylation but also elicits an attenuation in 1,25-dihydroxyvitamin D3-dependent transcriptional activation of a reporter gene. These observations suggest a potential role for cAMP-dependent protein kinase in the modulation of 1,25-dihydroxyvitamin D3 receptor-mediated gene regulation.

Animals↗