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G G Kuiper

Publications and source records attributed to G G Kuiper.

At least 37 records · Page 2Linked to original sources
PubMed1994

Changes in the abundance of androgen receptor isotypes: effects of ligand treatment, glutamine-stretch variation, and mutation of putative phosphorylation sites.

The SDS-polyacrylamide gel electrophoresis (SDS-PAGE) migration pattern of wild-type and mutated human androgen receptors (ARs) expressed in COS-1 cells was analyzed. In the absence of hormone, the wild-type AR migrated as a closely spaced 110-112 kDa doublet. Alkaline phosphatase treatment resulted in a single 110 kDa band showing that the 112 kDa upshift reflects receptors phosphorylation. Deletion of the N-terminal amino acids 46-101 or 100-142 resulted in mutant ARs migrating as single protein bands. Three consensus phosphorylation sites in this region were substituted, and the resulting mutated proteins were analyzed. Two Ser-Pro-directed kinase consensus sites at positions Ser-80 and Ser-93 were both necessary for the AR 112 kDa upshift. Substitution of the putative casein kinase II Ser-118 site had no effect on the AR migration pattern. Surprisingly, deletion of the glutamine repeat, located directly N-terminal of the Ser-Pro sites, resulted also in an AR single form. Lengthening of the glutamine repeat caused an increase in the spacing between the two isotypes of the doublet, showing that the number of glutamine residues determines the extent of the upshift. Hormone treatment induced an extra isotype with an apparent molecular mass of 114 kDa, resulting in a 110-112-114 kDa AR triplet. The hormone-induced upshift was dependent on the Ser-80 consensus phosphorylation site. Mutations in the DNA binding domain caused a different distribution of receptor protein over the three AR isotypes.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

Steroid hormone receptor phosphorylation: is there a physiological role?

All members of the steroid hormone receptor family are phosphoproteins. Additional phosphorylation occurs in the presence of hormone. This hormone-induced phosphorylation, which is 2- to 7-fold more than the basal phosphorylation, is a rapid process. All steroid receptors are phosphorylated at more than one single site. Most phosphorylation sites are located in the N-terminal domain, and phosphorylation occurs mainly on serine residues. Phosphorylation on threonine residues occurs in only a few cases. Phosphorylation on tyrosine residues has been found only for the estrogen receptor. Six different protein kinases are possibly involved in steroid receptor phosphorylation (estrogen receptor kinase; protein kinase A; protein kinase C; casein kinase II; DNA-dependent kinase; Ser-Pro kinases). Steroid receptor phosphorylation has been directly implicated in: activation of hormone binding, nuclear import of steroid receptors, modulation of binding to hormone response elements, and consequently in transcription activation.

Animals↗
PubMed1993

In vitro translation of androgen receptor cRNA results in an activated androgen receptor protein.

Translation of androgen receptor (AR) cRNA in a reticulocyte lysate and subsequent analysis of the translation products by SDS/PAGE showed a protein with an apparent molecular mass of 108 kDa. Scatchard-plot analysis revealed a single binding component with high affinity for R1881 (Kd = 0.3 nM). All AR molecules synthesized specifically bound steroid. No evidence for AR phosphorylation during in vitro synthesis was found. When AR was labelled with [3H]R1881 and analysed on sucrose-density gradients, a complex of approx. 6 S was observed. The complex was shifted to a higher sedimentation coefficient after incubation with a monoclonal AR antibody directed against an epitope in the DNA-binding domain. In the presence as well as the absence of hormone, AR molecules were able to bind to DNA-cellulose without an activation step. Gel retardation assays revealed that the AR forms complexes with a DNA element containing glucocorticoid-responsive element/androgen-responsive element sequences. Receptor-DNA interactions were stabilized by different polyclonal antibodies directed against either the N- or C-terminal part of the AR and were abolished by an antibody directed against the DNA-binding domain of the receptor. In conclusion, translation of AR cRNA in vitro yields an activated AR protein which binds steroid with high affinity. It is proposed that AR antibodies enhance AR-DNA binding by stabilizing AR dimers when bound to DNA.

Adenosine Triphosphate↗
PubMed1993

Localization and hormonal stimulation of phosphorylation sites in the LNCaP-cell androgen receptor.

Phosphorylation of the androgen receptor in human prostate tumour cells (LNCaP) is increased by addition of androgens to intact cells. Double-label studies, using [35S]methionine incorporation into receptor protein, and [32P]P(i) to label metabolically receptor phosphorylation sites, have enabled us to determine the phosphate content, relative to receptor protein, of both nontransformed and transformed and androgen receptors generated in intact LNCaP cells. No net change in the phosphorylation of the intact 110 kDa steroid-binding component of the androgen-receptor complex was found upon transformation to the tight nuclear binding form in the intact cell. Partial proteolysis of androgen receptor protein metabolically labelled with [32P]P(i) and photolabelled with [3H]R1881 (methyltrienolone) revealed that phosphorylation occurs mainly in the N-terminal trans-activation domain, whereas no phosphorylation was detected in the steroid- and DNA-binding domains. The location of most (> 90%) of the hormonally regulated phosphorylation sites in the N-terminal trans-activation domain suggests a role of phosphorylation of the androgen receptor in transcription regulation.

Androgens↗
PubMed1992

The human androgen receptor: structure/function relationship in normal and pathological situations.

Discrete functions have been attributed to precise regions of the human androgen receptor (hAR) by expression of deletion mutants in COS and HeLa cells. A large C-terminal domain constitutes the hormone-binding region and a central basis, cysteine-rich domain is responsible for DNA binding. In addition, separate domains responsible for transactivation and nuclear translocation have been identified. In LNCaP cells (a prostate tumor cell line) the hAR is a heterogeneous protein which is synthesized as a single 110 kDa protein, but becomes rapidly phosphorylated to a 112 kDa protein. Metabolic labeling experiments using radioactive orthophosphate also indicated that the hAR is a phosphoprotein. Structural analysis of the AR gene in LNCaP cells and in 46, XY-individuals displaying androgen insensitivity (AIS) has revealed several different point mutations. In LNCaP cells the mutation affects both binding specificity and transactivation by different steroids. In a person with complete AIS a point mutation was identified in the splice donor site of intron 4, which prevents normal splicing and activates a cryptic splice donor site in exon 4. The consequence is a functionally inactive AR protein due to an in-frame deletion in the steroid-binding domain. In two unrelated individuals with complete AIS, two different single nucleotide alterations in codon 686 (Asp) were found. Both mutations resulted in functionally inactive ARs due to rapidly dissociating hormone-AR complexes. It is concluded that the hAR is a heterogeneous phosphoprotein in which functional errors have a dramatic impact on phenotype and fertility of 46, XY-individuals.

Amino Acid Sequence↗
PubMed1992

The androgen receptor in LNCaP cells contains a mutation in the ligand binding domain which affects steroid binding characteristics and response to antiandrogens.

The human prostate tumor cell line LNCaP contains an abnormal androgen receptor system with broad steroid binding specificity. Progestagens, estradiol and several antiandrogens compete with androgens for binding to the androgen receptor in the cells to a higher extent than in other androgen sensitive systems. Optimal growth of LNCaP cells is observed after addition of the synthetic androgen R1881 (0.1 nM). In addition, estrogens, progestagens and several antiandrogens do not inhibit androgen responsive growth, but have striking growth stimulatory effects and increase EGF receptor level and acid phosphatase secretion. We have found that the androgen receptor in the LNCaP cells contains a single point mutation changing the sense of codon 868 (Thr to Ala) in the ligand binding domain. Expression vectors containing the normal or mutated androgen receptor sequence were transfected into COS or HeLa cells. Androgens, progestagens, estrogens and several antiandrogens bind the mutated androgen receptor protein and activate the expression of an androgen-regulated reporter gene (GRE-tk-CAT), indicating that the mutation directly affects both binding specificity and the induction of gene expression. Interestingly, the antiandrogen casodex showed antiandrogenic properties in growth studies of LNCaP cells and did not induce reporter gene activity in Hela cells transfected with the mutant receptor. The mutated androgen receptor of LNCaP cells is therefore a useful tool in the elucidation of different levels of action of steroids and antisteroids.

Binding Sites↗
PubMed1992

Androgen receptor heterogeneity in LNCaP cells is caused by a hormone independent phosphorylation step.

Androgen receptor synthesis and modification were studied in the human LNCaP cell line. Immunoblotting showed that the androgen receptor migrated as a closely spaced 110-112 kDa doublet on SDS-PAGE gels. Most of the receptor protein is present in the higher molecular mass form. Labelling experiments with [35S]methionine showed that the androgen receptor is synthesized as a single 110 kDa protein which is rapidly converted to a 112 kDa protein. Upon alkaline phosphatase treatment a gradual elimination of the 112 kDa isoform with a concomitant increase of the 110 kDa isoform was seen, indicating that the observed 110 to 112 kDa upshift reflects androgen receptor phosphorylation. Furthermore, it is shown that both isoforms can bind hormone and undergo a hormone dependent transformation to a tight nuclear binding form, indicating that the 110 to 112 kDa conversion is not an obligatory step for hormone binding or receptor transformation.

Autoradiography↗
PubMed1991

Synthesis and post-translational modification of the androgen receptor in LNCaP cells.

Androgen receptor synthesis and modification were studied in the human LNCaP cell line. Immunoblotting with a specific polyclonal antibody showed that the androgen receptor migrated as a closely spaced 110-112 kDa doublet on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels. Most of the receptor protein is present in the higher molecular mass form. Pulse labelling experiments with [35S]methionine showed that the androgen receptor is synthesized as a single 110 kDa protein which is rapidly converted to a 112 kDa protein. Alkaline phosphatase treatment of cytosols from [35S]methionine pulse labelled cells caused a gradual elimination of the 112 kDa isoform with a concomitant increase of the 110 kDa isoform. This indicates that the observed 110 to 112 kDa upshift of the newly synthesized androgen receptor reflects receptor phosphorylation. Both isoforms can bind hormone and can undergo a hormone dependent transformation to a tight nuclear binding form, indicating that the 110 to 112 kDa conversion is not an obligatory step for hormone binding or receptor transformation.

Humans↗
PubMed1991

Androgen receptor abnormalities.

The human androgen receptor is a member of the superfamily of steroid hormone receptors. Proper functioning of this protein is a prerequisite for normal male sexual differentiation and development. The cloning of the human androgen receptor cDNA and the elucidation of the genomic organization of the corresponding gene has enabled us to study androgen receptors in subjects with the clinical manifestation of androgen insensitivity and in a human prostate carcinoma cell line (LNCaP). Using PCR amplification, subcloning and sequencing of exons 2-8, we identified a G----T mutation in the androgen receptor gene of a subject with the complete form of androgen insensitivity, which inactivates the splice donor site at the exon 4/intron 4 boundary. This mutation causes the activation of a cryptic splice donor site in exon 4, which results in the deletion of 41 amino acids from the steroid binding domain. In two other independently arising cases we identified two different nucleotide alterations in codon 686 (GAC; aspartic acid) located in exon 4. One mutation (G----C) results in an aspartic acid----histidine substitution (with negligible androgen binding), whereas the other mutation (G----A) leads to an aspartic acid----asparagine substitution (normal androgen binding, but a rapidly dissociating androgen receptor complex). Sequence analysis of the androgen receptor in human LNCaP-cells (lymph node carcinoma of the prostate) revealed a point mutation (A----G) in codon 868 in exon 8 resulting in the substitution of threonine by alanine. This mutation is the cause of the altered steroid binding specificity of the LNCaP-cell androgen receptor. The functional consequences of the observed mutations with respect to protein expression, specific ligand binding and transcriptional activation, were established after transient expression of the mutant receptors in COS and HeLa cells. These findings illustrate that functional errors in the human androgen receptor have an enormous impact on phenotype and fertility.

Amino Acid Sequence↗
PubMed1991

Substitution of aspartic acid-686 by histidine or asparagine in the human androgen receptor leads to a functionally inactive protein with altered hormone-binding characteristics.

We have identified two different single nucleotide alterations in codon 686 (GAC; aspartic acid) in exon 4 of the human androgen receptor gene in three unrelated families with the complete form of androgen insensitivity. One mutation (G----C) results in an aspartic acid----histidine substitution (with 15-20% of wild-type androgen-binding capacity), whereas the other mutation (G----A) leads to an aspartic acid----asparagine substitution (with normal androgen-binding capacity, but a rapidly dissociating ligand-receptor complex). The mutations eliminate a Hinfl restriction site. Screening for the loss of the Hinfl site in both families with the Asp----Asn mutation resulted in the recognition of heterozygous carriers in successive generations of each. Both mutant androgen receptors were generated in vitro and transiently expressed in COS and HeLa cells. The receptor proteins produced had the same altered binding characteristics as those measured in fibroblasts from the affected subjects. R1881-activated transcription of a GRE-tk-CAT reporter gene construct was strongly diminished by both mutant receptors and was only partially restored using a 100-fold higher concentration of ligand compared with wild-type receptor. Thus, aspartic acid-686 appears essential for normal androgen receptor function. Substitution of this amino acid residue, by either histidine or asparagine, results in androgen insensitivity and lack of androgen-dependent male sexual differentiation.

Adult↗
PubMed1990

The androgen receptor: functional structure and expression in transplanted human prostate tumors and prostate tumor cell lines.

The growth of the majority of prostate tumors is androgen-dependent, for which the presence of a functional androgen receptor is a prerequisite. Tumor growth can be inhibited by blockade of androgen receptor action. However, this inhibition is transient. To study the role of the androgen receptor in androgen-dependent and androgen-independent prostate tumor cell growth, androgen receptor mRNA expression was monitored in six different human prostate tumor cell lines and tumors, which were grown either in vitro or by transplantation on (male) nude mice. Androgen receptor mRNA was clearly detectable in three androgen-dependent (sensitive) tumors and absent or low in three androgen-independent tumors. Growth of the LNCaP prostate tumor cell line can be stimulated both by androgens and by fetal calf serum. In the former situation androgen receptor mRNA expression is downregulated, whereas in the latter no effect on androgen receptor mRNA levels can be demonstrated. Sequence analysis showed that the androgen receptor gene from LNCaP cells contains a point mutation in the region encoding the steroid-binding domain, which confers an ACT codon encoding a threonine residue to GCT, encoding alanine.

Amino Acid Sequence↗
PubMed1990

A mutation in the ligand binding domain of the androgen receptor of human LNCaP cells affects steroid binding characteristics and response to anti-androgens.

LNCaP prostate tumor cells contain an abnormal androgen receptor system. Progestagens, estradiol and anti-androgens can compete with androgens for binding to the androgen receptor and can stimulate both cell growth and excretion of prostate specific acid phosphatase. We have discovered in the LNCaP androgen receptor a single point mutation changing the sense of codon 868 (Thr to Ala) in the ligand binding domain. Expression vectors containing the normal or mutated androgen receptor sequence were transfected into COS or Hela cells. Androgens, progestagens, estrogens and anti-androgens bind the mutated androgen receptor protein and activate the expression of an androgen-regulated reporter gene construct (GRE-tk-CAT). The mutation therefore influences both binding and the induction of gene expression by different steroids and antisteroids.

Binding Sites↗
PubMed1990

Aberrant splicing of androgen receptor mRNA results in synthesis of a nonfunctional receptor protein in a patient with androgen insensitivity.

Androgen insensitivity is a disorder in which the correct androgen response in an androgen target cell is impaired. The clinical symptoms of this X chromosome-linked syndrome are presumed to be caused by mutations in the androgen receptor gene. We report a G----T mutation in the splice donor site of intron 4 of the androgen receptor gene of a 46,XY subject lacking detectable androgen binding to the receptor and with the complete form of androgen insensitivity. This point mutation completely abolishes normal RNA splicing at the exon 4/intron 4 boundary and results in the activation of a cryptic splice donor site in exon 4, which leads to the deletion of 123 nucleotides from the mRNA. Translation of the mutant mRNA results in an androgen receptor protein approximately 5 kDa smaller than the wild type. This mutated androgen receptor protein was unable to bind androgens and unable to activate transcription of an androgen-regulated reporter gene construct. This mutation in the human androgen receptor gene demonstrates the importance of an intact steroid-binding domain for proper androgen receptor functioning in vivo.

Animals↗
PubMed1989

Structure and function of the androgen receptor.

The androgen receptor in several species (human, rat, calf) is a monomeric protein with a molecular mass of 100-110 kDa. The steroid binding domain is confined to a region of 30 kDa, while the DNA-binding domain has the size of approx. 10 kDa. A 40 kDa fragment containing both the DNA and steroid binding domain displayed a higher DNA binding activity than did the intact 100 kDa molecule. cDNA encoding the major part of the human androgen receptor was isolated. The cDNA contains an open reading frame of 2,277 bp but still lacks part of the 5'-coding sequence. Homology with the progesterone and glucocorticoid receptor was about 80% in the DNA binding domain and 50% in the steroid binding domain. The present data provide evidence that the androgen receptor belongs to the superfamily of ligand responsive transcriptional regulators and consists of three distinct domains each with a specialized function.

Amino Acid Sequence↗
PubMed1989

The human androgen receptor: domain structure, genomic organization and regulation of expression.

The domain structure and the genomic organization of the human androgen receptor (hAR) has been studied after molecular cloning and characterization of cDNA and genomic DNA encoding the hAR. The cDNA sequence reveals an open reading frame of 2751 nucleotides encoding a protein of 917 amino acids with a calculated molecular mass of 98,845 D. The N-terminal region of the hAR is characterized by a high content of acidic amino acid residues and by several homopolymeric amino acid stretches. The DNA-binding domain showed a high homology with the DNA-binding domain of the human glucocorticoid receptor (hGR) and the human progesterone receptor (hPR). The predominantly hydrophobic steroid binding domain of the hAR is 50-55% homologous with the ligand binding domains of the hGR and hPR. Transient expression of recombinant AR cDNA in COS-cells resulted in the production of a 110 kDa protein with the expected binding specificity of androgen receptors. Co-transfection with a reporter-gene construct [CAT(chloramphenicol acetyl transferase) under direction of the androgen regulated MMTV-promoter] showed that the protein is functionally active with respect to transcription regulation. In the LNCaP prostate carcinoma cell line two major (11 and 8 kb) and one minor (4.7 kb) mRNA species can be found which can be down-regulated by androgens. The hAR protein coding region was shown to be divided over eight exons with an organization similar to that of the progesterone and oestrogen receptor. The sequence encoding the N-terminal domain was found in one large exon. The two DNA-binding fingers were encoded by two small exons; the information for the androgen-binding domain was found to be distributed over five exons. Southern blot analysis of genomic DNA revealed that the hAR is encoded by one single gene, which is situated on the X-chromosome.

Animals↗
PubMed1989

Mechanism of androgen action: recent observations on the domain structure of androgen receptors and the induction of EGF-receptors by androgens in prostate tumor cells.

In this paper two different aspects of androgen action are reviewed. Polyacrylamide gel electrophoresis of androgen receptors, photoaffinity labeled with R1881 showed that receptors isolated from both human prostate cells and calf uterine cytosol cells are proteins with a molecular mass of approx 110 kD. Purification to homogeneity of this form of the receptor from calf uterus also yielded a 110 kD protein. A molecular model for the DNA-binding form of the receptor is presented in which one polypeptide comprises three active domains: one for ligand binding, one for interaction with nuclear acceptor sites, and a third domain which modulates nuclear interaction. Mild digestion with chymotrypsin or a protease from rat prostates removes the modulating domain and leaves the ligand binding and nuclear interaction domain intact. Trypsin treatment yields a fragment of lower molecular mass containing the ligand binding domain with some affinity for RNA, but not DNA. In vitro studies with a human prostate tumor cell line (LNCaP), suggest that androgens not only directly effect cell growth, but also act indirectly. Both epidermal growth factor (EGF) and androgens stimulate cell growth. In addition androgens stimulate synthesis of receptors for EGF. Thus androgens effect tumor cell growth by autocrine or paracrine mechanisms by making the cells more sensitive for growth factor mediated stimuli.

Androgens↗
PubMed1989

The N-terminal domain of the human androgen receptor is encoded by one, large exon.

Using specific cDNA hybridization probes, the first coding exon of the human androgen receptor gene was isolated from a genomic library. The exon contained an open reading frame of 1586 bp, encoding an androgen receptor amino-terminal region of 529 amino acids. The deduced amino acid sequence was characterized by the presence of several poly-amino acid stretches of which the long poly-glycine stretch (16 residues) and the poly-glutamine stretch (20 residues) were most prominent. Androgen receptor cDNAs from different sources contained information for poly-glycine stretches of variable size (23 and 27 residues, respectively). The androgen receptor amino-terminal domain was found to be hydrophilic and have a net negative charge. Combined with the previously described, partially overlapping cDNA clone 7A2M27 (Trapman et al. (1988) Biochem. Biophys. Res. Commun. 153, 241-248), the complete human androgen receptor was deduced to have a size of 910 amino acids.

Amino Acid Sequence↗