Biomedical subjects

G G Kuiper

Publications and source records attributed to G G Kuiper.

43 records · Page 3Linked to original sources

PubMed1989

Structural organization of the human androgen receptor gene.

The complete coding region of the human androgen receptor gene has been isolated from a genomic library. The information for the androgen receptor was found to be divided over eight exons and the total length of the gene exceeded 90 kb. The sequence encoding the N-terminal region is present in one large exon. The two putative DNA-binding fingers are encoded separately by two small exons. The information for the hormone-binding domain is split over five exons. Positions of introns are identical to those reported for the chicken progesterone receptor and the human oestrogen receptor genes. Southern blot analysis of genomic DNA with various specific probes reveal that the human androgen receptor is encoded by a single-copy gene.

Amino Acid Sequence↗

PubMed1988

Cloning, structure and expression of a cDNA encoding the human androgen receptor.

A cDNA clone has been isolated from a library prepared of mRNA of human breast cancer T47D cells with an oligonucleotide probe homologous to part of the region encoding the DNA-binding domain of steroid receptors. The clone has a size of 1505 bp and sequence analysis revealed an open reading frame of 1356 bp. The deduced amino acid sequence displays two highly conserved regions identified as the putative DNA-binding and hormone binding domains respectively of steroid receptors. Expression of this cDNA clone in COS cells produces a nuclear protein with all the binding characteristics of the human androgen receptor (hAR). The gene encoding the cDNA is assigned to the human X-chromosome. High levels of three hybridizing mRNA species of 11, 8.5 and 4.7 kb respectively are found in the human prostate cancer cell line (LNCaP), which contains elevated levels of hAR. The present data provide evidence that we have isolated a cDNA that encodes a major part of the human androgen receptor.

Amino Acid Sequence↗

PubMed1988

In situ photolabelling of the human androgen receptor.

In situ photoaffinity labelling of the human androgen receptor has been performed in the LNCaP (Lymph Node Carcinoma of the Prostate) cell line. The covalently labelled receptors were identified by SDS-PAGE. Intact LNCaP cells, incubated with [3H]-R1881 and subsequently irradiated with u.v. light and directly solubilized in SDS-buffer, revealed two photolabelled protein bands at 110 and 50 kDa. Irradiation of intact cells and subsequent isolation of nuclei followed by extraction with 0.5 M NaCl resulted in one major photolabelled protein band at 110 kDa. The labelling of this band could be completely suppressed by a 100-fold molar excess of non-radioactive R1881. Photolabelling of androgen receptors in a cytosolic preparation of LNCaP cells after anion exchange chromatography resulted in a much lower labelling efficiency compared with the in situ labelling procedure, although the androgen receptor was purified 100-fold. The steroid binding domain of the human androgen receptor has been partially mapped with chymotrypsin and S. aureus V8 protease digestion. Proteolytic digestion with chymotrypsin of purified photoaffinity-labelled 110 kDa human androgen receptor resulted in the generation of a 15 kDa peptide which still contains the covalently linked hormone. It is concluded that the in situ photoaffinity labelling technique can be applied successfully for characterization of the steroid binding domain of androgen receptors in prostate cancer cells and in other androgen target cells. Furthermore, it was demonstrated that the human androgen receptor is a monomer with a molecular mass of 110 kDa, of which the steroid binding site is confined to a 15 kDa domain.

Affinity Labels↗

PubMed1987

Characterization of androgen receptors in a transplantable human prostatic adenocarcinoma (PC-82).

The transplantable human prostatic adenocarcinoma, PC-82, has been shown to be a suitable model for the study of several aspects of androgen-regulated tumor growth. This tumor contains an androgen receptor, and the purpose of the present investigation was to characterize this androgen receptor with respect to hormone specificity, sedimentation coefficient, dissociation constant, Stokes radius, ionic properties, and molecular mass. Cytosol was prepared from tumor tissues grown in athymic nude mice, which were castrated 10 days before harvesting the tumor. Scatchard plot analysis revealed a binding protein with a Kd of 0.1 nM for R1881 (methyltrienolone) and binding capacity of 120 fmol/mg protein. The receptor showed a high affinity for R1881, testosterone, and 5 alpha-dihydrotestosterone, respectively, whereas no or little affinity was found for progesterone and estradiol. In the presence of 10 mM molybdate the androgen receptor in PC-82 cytosol eluted from an FPLC anion exchange column (Mono Q) at 0.32 M NaCl, which is identical to what has been found for androgen receptors from rat prostate and calf uterine cytosol. Photoaffinity labeling of the [3H]R1881-androgen receptor complex and subsequent analysis on SDS-polyacrylamide gels resulted in a covalently labeled protein with a molecular mass of approximately 50 kD. The androgen receptor of the PC-82 tumor had a sedimentation coefficient of 4S and a Stokes radius of 3.3 nm at high ionic strength (0.4 M NaCl). It is concluded that the PC-82 tumor contains a binding protein with the properties described for androgen receptors present in prostate tissue.

Adenocarcinoma↗

PubMed1987

Analysis of steroid- and DNA-binding domains of the calf uterine androgen receptor by limited proteolysis.

The DNA-binding form of the calf uterine androgen receptor (AR) was subjected to limited protease digestion using chymotrypsin, trypsin and a rat prostate cytosol protease. The properties of the generated polypeptide fragments were identified and compared with those of the intact AR. Physicochemical characterization was achieved through sedimentation analysis, gel filtration chromatography and DEAE anion exchange chromatography. Intactness of functional binding domains was evaluated by measuring the retention of steroid- and DNA-binding capacity. Under non-denaturing conditions the intact AR is a highly asymmetrical molecule with a Stokes radius (RS) of 45A, a sedimentation coefficient of 4.3S and a relative molecular mass of 80,000 daltons. This form of AR has an intrinsic binding affinity for DNA and was eluted from DNA-cellulose with 9 mM MgCl2. Chymotrypsin produced a more globular polypeptide (RS: 31A; 3.1S; 41,000 daltons) with a decreased net negative charge. This fragment also displayed DNA-binding affinity but required a higher concentration of MgCl2 (14 mM) for DNA-cellulose elution, indicating an increased affinity for DNA. The observed reduction in molecular size upon chymotrypsin treatment was confirmed when analysed by SDS-polyacrylamide gel electrophoresis after covalently labelling of the AR with [3H]R1881. Rat prostate cytosol contains a protease which is very active in generating an AR polypeptide with an increased affinity for DNA, without changing the AR net negative charge (RS: 33A; 3.7S; 51,000 daltons). The specificity of this protease remained unknown since none of a large number of inhibitors was able to inactivate this enzyme. The fragment generated is different from that obtained with chymotrypsin since significant differences in size as well as in charge were measured. Trypsin treatment generated a much smaller polypeptide (RS: 25A; 2.9S; 30,000 daltons) which had lost its DNA-binding capacity, but not its steroid binding site. This form probably represents the so-called meroreceptor. When intact AR was treated sequentially with prostate cytosol and trypsin, a polypeptide fragment with identical properties was obtained, indicating the spatial separation of two of the proteolytic cleavage sites. These studies provide evidence for the distinct nature of the molecular domains for androgen and DNA interaction on the calf uterine AR.

Animals↗

PubMed1986

Characterization of androgen receptors after photoaffinity labelling with [3H]methyltrienolone (R1881).

The synthetic androgen 17 beta-hydroxy-17 alpha-[3H]methyl-4,9,11-estratrien-3-one (R1881) has been used as photoaffinity label to characterize androgen receptors in rat prostate, in a human transplantable prostatic adenocarcinoma (PC-82) and in calf uterus. Androgen receptors preparations were partially purified either via differential chromatography on 2',5'-ADP-Sepharose (rat prostate), via anion exchange fast protein liquid chromatography (rat prostate and PC-82) or via DNA-cellulose chromatography (calf uterus). Purification factors obtained with the three different methods were: 245, 75 and 40 respectively. Photolabelling of receptor preparations was performed via irradiation with a high pressure mercury lamp either before or after partial purification. Polyacrylamide gel electrophoresis under denaturing conditions showed that the DNA-binding form of the androgen receptor in calf uterus cytosol is a protein with a molecular mass of approx 95 kD. The covalent attachment of [3H]R1881 to the 95 kD protein could be completely suppressed by a 200-fold molar excess of dihydrotestosterone. In rat prostate cytosol an androgen receptor with a molecular mass of approx 50 kD could be photoaffinity labelled with R1881. A similar size was found for the androgen receptor in the human prostatic adenocarcinoma. Our results show that photoaffinity labelling of androgen receptors with [3H]R1881 as ligand can be applied for characterization of partial purified androgen receptor preparations.

Affinity Labels↗

PubMed1985

Photoaffinity labelling of androgen receptors with 17 beta-hydroxy-17 alpha-[3H]methyl-4,9,11-estratrien-3-one.

The synthetic androgen 17 beta-hydroxy-17 alpha-[3H]methyl-4,9,11-estratrien-3-one (R1881) has been used as photoaffinity label to characterize androgen receptors in calf uterus and rat prostate. Polyacrylamide gel electrophoresis under denaturing conditions showed that the DNA-binding form of the androgen receptor in calf uterus cytosol is a protein with a molecular mass of 98 kD. In rat prostate cytosol an androgen receptor with a molecular mass of 46 kD could be photoaffinity labelled with R1881. The photoaffinity labelling procedure described here provides a method for studying the hormone binding domain of androgen receptors in partial purified preparations.

Affinity Labels↗