Abstract
Androgens, like progestins, are 3-ketosteroids with structural differences restricted to the 17beta substituent in the steroid D-ring. To better understand the specific recognition of ligands by the human androgen receptor (hAR), a homology model of the ligand-binding domain (LBD) was constructed based on the progesterone receptor LBD crystal structure. Several mutants of residues potentially involved in the specific recognition of ligands in the hAR were constructed and tested for their ability to bind agonists. Their transactivation capacity in response to agonist (R1881) and antagonists (cyproterone acetate, hydroxyflutamide, and ICI 176344) was also measured. Substitution of His(874) by alanine, only marginally impairs the ligand-binding and transactivation capacity of the hAR receptor. In contrast, mutations of Thr(877) and, to a greater extent, Asn(705) perturb ligand recognition, alter transactivation efficiency, and broaden receptor specificity. Interestingly, the N705A mutant acquires progesterone receptor (PR) properties for agonist ligands but, unlike wild type AR and PR, loses the capacity to repress transactivation with nonsteroidal antagonists. Models of the hAR.LBD complexes with several ligands are presented, which suggests new directions for drug design.
Highlights
From the :j:Small Molecule Drug Discovery Group, Genetics Institute, Cambridge, Massachusetts 02140 and the Wepartment of Human Genetics, Roswell Park Cancer Institute, Buffalo, New York 14263
The genomic clone differs from the cDNA clone isolated from HL-60 cells in that it encodes an extra copy of the decameric repeat located in the extracellular domain of P-selectin glycoprotein ligand (PSGL-l)
Further analysis indicated that the PSGL-l genes of HL-60 and U-937 cells contain repeats, whereas the PSGL-l genes ofpolymorphonuclear leukocytes, monocytes, and several other cell lines contain repeats
Summary
Vol 270, No 27, Issue of July 7, pp, 16470-16475, 1995 Printed in U.S.A. Genomic Organization and Chromosomal Localization of the Gene Encoding Human P-selectin Glycoprotein Ligand*. The organization of the PSGL-l gene closely resembles those of CD43 and human platelet glycoprotein GPIba, both of which have an intron in the 5'-noncoding region, a long second exon containing the complete coding region, and TATA-Iess promoters. Southern blot analysis indicated that the PSGL-l gene is present as a single copy in the human genome, suggesting that the two mRNAs are alternative transcripts derived from the same gene, resulting from either alternative splicing events or differential utilization of promoter and/or polyadenylation signal sequences [1]. The second class represents genes whose coding region consists of multiple exons, and the promoters of these genes generally (albeit with a few exceptions) resemble the typical eukaryotic promoter containing a TATA consensus sequence Examples of this class include glycophorin, CD4, and the L-selectin ligands GlyCAM-l and CD34 [9,10,11,12]. We report the isolation, genomic organization, and chromosomal mapping of the human PSGL-l gene
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