NEW TOOLS FOR THE STUDY OF HUMAN ANDROGEN RESISTANCE

Project: Research project

Description

The purpose of these proposed studies is the development of new tools for
the analysis of the structure and function of the human androgen receptor
protein. The specific goals of the proposal are: 1) the development of
high affinity monoclonal antibodies capable of recognizing the human
androgen receptor, 2) the application of photoaffinity labeling techniques
to produce a radiolabeled steroid-receptor complex covalently linked at the
steroid binding site, and 3) the further elucidation of the process by
which androgen-receptor complexes acquire DNA-binding capacity. These
tools will be applied to the study of the clinical syndromes of androgen
resistance, a diverse clinical spectrum of disorders ranging from complete
failure of male phenotypic development to the occurrence of unexplained
oligospermia in otherwise normal men. The availability of the tools and
techniques described will open new avenues for the elucidation of the
molecular defects responsible for androgen resistance. The production of monoclonal antibodies that recognize the human androgen
receptor will be approached by two routes. A conventional approach
involving partial purification of the androgen receptor protein by affinity
chromatography coupled with DNA-cellulose chromatography will be used to
provide androgen receptor for immunization and production of monoclonal
antibodies. In addition, anti-idiotypic antibodies against a purified high
affinity monoclonal anti-dihydro-testosterone antibody will be produced and
tested for their ability to recognize the androgen binding site of the
authentic receptor. The production of these antibodies will be pursued
using both conventional immunizations as well as newer in vitro techniques
of immunization. In a separate series of experiments covalent linkage of
photoreactive ligands to partially purified androgen receptors will be
attempted; the photolabeled products will be analyzed by polyacrylamide gel
electrophoresis under denaturing conditions. Finally, the role of partial
proteolysis in the generation of DNA-binding androgen receptor complexes,
and in the lability of some androgen receptor complexes under transforming
conditions will be evaluated using a DNA-cellulose binding assay coupled
with the use of assays for cytosolic proteases using synthetic fluorogenic
substrates. The availability of these tools and techniques will permit the initiation
of a number of direct structural and functional studies of normal and
mutant androgen receptor proteins, such as gel electrophoresis followed by
blotting with antibody, electrophoresis of affinity labeled receptors or
analyses of the DNA-binding capacity of partially proteolyzed receptors.
StatusFinished
Effective start/end date7/1/856/30/88

Funding

  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health

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Androgen Receptors
Androgens
Immunization
Electrophoresis
Antibodies
Assays
Binding Sites
Monoclonal Antibodies
Availability
Steroid Receptors
DNA
Chromatography
Labeling
Purification
Testosterone
Anti-Idiotypic Antibodies
Proteins
Peptide Hydrolases
Gels
Ligands