Immunoreactive AR and Genetic Alterations in Subjects with Androgen Resistance and Undetectable AR Levels in Genital Skin Fibroblast Ligand-Binding Assays

Abstract

Defects of the AR cause a wide range of abnormalities of male development, ranging from individuals with mild defects of virilization to those with complete female phenotypes. In parallel with this phenotypic spectrum, a large number of different mutations have been identified that alter the synthesis or functional activity of the receptor protein. This report aims to categorize the alterations of immunoreactive AR (IRAR) expression and the underlying genetic changes in a single category of patient: those in whom ligand binding is undetectable in genital skin fibroblasts. Our study found a wide range in the levels of IRAR that are detectable in fibroblast strains established from 27 such individuals. A large proportion (19 of 27) express significant amounts of AR protein, as detected using a sensitive Western blot technique. In a smaller number (8/27), AR expression was undetectable. Intact IRAR was identified in16 of the 19 fibroblast strains in which AR expression could be detected. The AR gene was analyzed in 14 strains from this group. In 13 instances, single amino acid substitutions were identified within the ligand-binding domain of the receptor protein. In three of the remaining patients (3 of 19), truncation of the receptor protein was suggested by the rapid migration of the IRAR in SDS-polyacrylamide gels. In those three patients, production of the shortened immunoreactive receptor was traced to mutations that interrupted the AR open reading frame. By contrast, only one of the eight patient samples with no detectable IRAR carried a mutation that resulted in a single amino acid substitution. An interruption of the AR open reading frame was identified in six of the eight strains in which immunoreactive receptor was absent. In the remaining strain, no mutation was present within or surrounding the eight coding exons. This study serves to define the effects that mutations of the AR have on the levels of expressed immunoreactive receptor protein. In addition, it demonstrates the type of information that can be obtained if an immunoblot assay were to be used as a component of a screening method to analyze samples from patients with defects of the AR. Finally, the study suggests that in some androgen-resistant patients, defects outside the AR open reading frame may result in major alterations of AR expression.

Changes in Androgen Receptor Nongenotropic Signaling Correlate with Transition of LNCaP Cells to Androgen Independence

“…In these cell lines, induction of ERK-1/2 phosphorylation by DHT was a function of AR expression. In strain 1017, in which we know that AR is not expressed due to a mutation at the splice acceptor site at the 5Ј boundary of exon 4 (38), no induction of ERK-1/2 phosphorylation was present (Fig. 10A).…”

“…Genital skin fibroblast strain 881 contains a mutation inactivating the DNA-binding domain of AR (C574R). Due to a mutation at a splice acceptor site at the 5Ј boundary of exon 4, immunoreactive AR was not detected in strain 1017 (38), however immunoreactive AR was present in both strains 1003 and 881 (40). For transcription assays, the following reporter plasmids were used: pSVOA⌬5Ј (a gift from Brent Cochram, Tufts University School of Medicine), consisting of a 379-bp murine c-fos promoter containing a cyclic AMP-responsive element driving a luciferase reporter gene (41); PRL-CMV-TK (Promega, Madison WI), which contains the Renilla luciferase cDNA under the control of the constitutively active CMV promoter; and PSA P/E-luc, in which the luciferase reporter is driven by the AR-dependent 2.4-kb PSA enhancer and 564-bp PSA promoter (31).…”

“…Genital skin fibroblasts strain 1003, 881, and 1017 were derived from patients with a normal male phenotype (1003) or from patients affected by a phenotype of complete androgen insensitivity (881 and 1017). The AR sequences of these cell lines and the functional characterization of the mutations detected have been published previously (38,39). Genital skin fibroblast strain 1003 has a wild-type AR sequence.…”

Novel Signaling Pathways That Control Ovarian Follicular Development, Ovulation, and Luteinization

“…Substitution of the same residue by histidine, a more conservative amino acid replacement, leads to normal levels of androgen binding in fibroblasts that displays a marked thermal lability in in vitro assays. Additional pedigrees have been described in which different amino acid substitution mutations at a single residue have differing effects on the binding of ligand by the receptor, depending of the nature of the amino acid substitution (Ris-Stalpers et al, 1991;Kazemi-Esfarjani et al, 1993;Beitel et al, 1994a).…”

Η Επίδραση Του Πολυμορφισμού CAG Του Ανδρογονικού Υποδοχέα Στα Επίπεδα Των Στεροειδών Ορμονών Και Των Λιπιδίων Του Αίματος Στους Άνδρες

“…Το μη φωσφορυλιωμένο, 110 kDa, ισομερές μετατρέπεται σε 112 kDa φωσφορυλιωμένο ισομερές. Η φωσφορυλίωση του ανδρογονικού υποδοχέα μπορεί να συσχετιστεί με την ενεργοποίηση της σύνδεσης της ορμόνης και με την ρύθμιση της σύνδεσης του με το DNA (Yong 1994, Avila 2002, ενώ υπάρχουν ενδείξεις ότι ενδεχομένως παίζει σημαντικό ρόλο στην ανεξάρτητη από την ορμόνη ενεργοποίησή του από πρωτεϊνικές κινάσες (Trifiro 1997). Φαίνεται δηλαδή ότι η φωσφορυλίωση τροποποιεί την μεταγραφική δραστηριότητα του ΑΥ (Lee 2001).…”