Cooper B, Gruber J A, McPhaul M J
Department of Internal Medicine, the University of Texas Southwestern Medical Center at Dallas, 75235-8857, USA.
J Steroid Biochem Mol Biol. 1996 Feb;57(3-4):251-7. doi: 10.1016/0960-0760(95)00273-1.
The ligand-binding properties of the human androgen receptor (AR) reside in the carboxy terminus of the receptor and are retained in fusion proteins encoding this domain. Despite a wealth of information pertaining to the importance of specific amino acid residues in the binding of ligands, the precise amino terminal boundary of this domain has not been determined for the androgen receptor. The current studies focused on the expression of a series of AR fusion proteins in bacteria. These experiments demonstrate that specific androgen binding is detected in a fusion protein encoding amino acid residues 675-917, although this binding is of lower affinity than that observed for the native AR. Inclusion of an additional segment (residues 622-675) restores the capacity of the hormone-binding domain to bind mibolerone with high affinity. Detailed studies of the ligand-binding properties and solubility of one AR fusion protein (625-917) indicate that although the levels of ligand binding and soluble receptor fusion protein usually vary in parallel, this is not true at all time points and the proportion of soluble AR fusion that is able to bind hormone is highest when the levels of soluble AR fusion protein are lowest. This observation, coupled with the changes in the quantity of soluble and insoluble fusion protein occurring at different times following induction at 12 degrees C, 22 degrees C, and 37 degrees C, suggests that the saturation of one or more steps is required for folding of the AR hormone-binding domain to a conformation that is both soluble and competent to bind hormone under conditions of high-level expression. Analysis of the solubility of six different fusion proteins containing different portions of the AR hormone-binding domain suggests that a discrete segment of this domain is not responsible for its synthesis as predominantly insoluble aggregates in bacteria when expressed at high levels.
