Subcellular distribution and glycosylation pattern of androgen receptor from sheep omental adipose tissue

Abstract

Sex steroids are known to have an influence on the distribution, metabolism and accretion of adipose tissue. These steroids carry out their function via specific receptors. We have previously reported the presence of oestrogen and progesterone receptors in sheep adipose tissues. In this study, we have tested the subcellular distribution of androgen receptor (AR) in sheep omental adipose tissue. Subcellular fractions -microsomes, plasma membrane and nuclei-cell debris -were isolated by differential and sucrose gradient centrifugation and confirmed by electron microscopy. The AR was determined in each fraction by Western blot analyses. As anticipated, the receptor was found in the cytosolic fraction, but a high concentration was also present in the microsomal fraction, a lesser amount in the plasma membrane fraction, and only a small amount was left in the nuclei-cell debris fraction. Two minor immunostaining bands with approximate molecular weights of 250 and 140 kDa and a major band at 110 kDa were detected in the cytosolic fraction, but only the 110 kDa band was detected in the membrane fractions. A 104 kDa band was observed on occasion and believed to be a degradation product.The cytosolic isoforms were tested for sensitivity to glycosidases. This treatment resulted in a decrease in the amount of the 250 and 140 kDa bands. To substantiate that the 250 and 140 kDa isoforms were glycoproteins, the cytosolic fraction was chromatographed on Concanavalin A-Sepharose. The 110 kDa band was eluted in the 0·4 M KCl salt wash while the 250 and 140 kDa bands were eluted with -methylmannoside. Treatment of the glycoprotein ( -methylmannoside) peak with glycosidases converted the 250 and 140 kDa bands to the 110 kDa band. These data confirm the presence of AR in subcellular fractions of adipose tissue and suggest that it exists in various glycosylated isoforms.

Androgen receptor: a new player associated with apoptosis and proliferation of pancreatic beta-cell in type 1 diabetes mellitus

“…Western blot analysis for AR from subcellular fractions of pancreatic beta-cells Isolation of subcellular fractions of pancreatic beta-cells was performed as previously described [25]. In brief, FACS purified beta-cells from controls and diabetic mice were homogenized at 4°C with Medium I (0.25 M sucrose, 1 mM EDTA, 10 mM Tris-HCl, pH 7.4) which contained protease inhibitors (aminoethyl-benzenesulfonyl fluoride, leupeptin, bacitracin, aprotinin and pepstatin).…”

Estrogen Activates Classical and Alternative Mechanisms to Orchestrate Neuroprotection

“…kDa proteins are related to nER. A possible explanation of the origin of these unconventional forms of ERs is that they may be the result of post-translational modifications, such as glycosylation, that might account for some differences on the molecular weight, as reported for androgen receptors (McCann et al, 2001). The presence of a subpopulation of O-linked N-acetylglucosamine modified forms of ERα (Jiang and Hart, 1997;Cheng and Hart, 2000) and ERβ (Cheng and Hart, 2001) has been reported as an additional way of receptor regulation of, both, its structural and functional preservation.…”

Expression of androgen receptor and its co-localization with estrogen receptor-alpha in the developing pituitary gland of sheep fetus

“…Androgen receptor immunohistochemical detection was performed using a polyclonal antibody (PG-21, Upstate, Lake Placid, NY, USA; diluted at 1:70 with 0.01 M PBS, pH 7.4), which has been used to detect sheep AR (McCann et al 2001). The procedure used to determine AR expression was performed as described previously (Goyal et al 1997).…”