Axel Alléra

Specific interaction of corticosteroids with components of the cell membrane which are involved in the translocation of the hormone into the intravesicular space of purified rat liver plasma membrane vesicles

Abstract The uptake of [ 3 H]-corticosterone was studied by a highly purified rat liver plasma membrane vesicle fraction. At 23°C, [ 3 H]-corticosterone is taken up very rapidly; equilibrium is reached as early as 5 s. At high concentrations of corticosterone (30–8100 nM) uptake occurs predominantly by a non-saturable process, but the presence of a saturable process is also detectable. When the intravesicular space is successively decreased by increasing the osmolarity of the external medium, uptake of [ 3 H]-corticosterone decreased. About 45% of the total steroid taken up is translocated into the lumen of the vesicle. In the presence of a 200-fold molar excess of non-labeled corticosterone about 40% of the total uptake of [ 3 H]-corticosterone is displaceable. At low concentrations of corticosterone (2—230 nM) and considering only the specific portion of uptake the presence of a high and a low affinity uptake system is observable. The K D of the two systems were 7.2 ± 2.0 nM and 234 ± 67 nM, respectively. The K D of the high affinity system corresponds to the concentration of free corticosterone (about 8 nM) in the plasma of the female rat. The maximal binding capacity was about 180 fmol/(mg membrane protein). Structural analogues of corticosterone reduce the displaceable portion of the uptake of [ 3 H]corticosterone; cortisol, progesterone. dexamethasone and 5α-dihydrocorticosterone are strong inhibitors. Among the sex hormones 5α-dihydrotestosterone and diethylstilbestrol are the most efficient inhibitors. These results suggest that corticosterone reacts in a specific manner with components of the plasma membrane which may function to translocate the steroid into the cell.

Significance of the CAG repeat length in the androgen receptor gene (AR) for the transactivation function of an M780I mutant AR.

Abstract Mutations in the androgen receptor gene (AR) cause a wide spectrum of androgen insensitivity syndromes (AIS). Mutation analysis of patients with AIS has revealed that the same missense mutation of the AR gene can give rise to strongly divergent phenotypes suggesting the influence of modifying factors. The polymorphic CAG repeat in the first exon of the AR gene may be such a modifying factor. The influence of the length of the CAG repeat on the transactivation function of the M780I-mutant AR (causing partial and complete AIS) has been determined by cotransfection of HeLa cells with various CAG-AR expression vectors and a highly androgen-responsive luciferase reporter gene construct. The transcriptional activity of the M780I mutant AR can be, in contrast to the wild-type AR, considerably enhanced by non-physiologically high androgen concentrations. Furthermore, an inverse relationship between the number of the CAG repeats in the mutant AR and its activity has been observed.

Glucocorticoid-recognizing and -effector sites in rat liver plasma membrane. Kinetics of corticosterone uptake by isolated membrane vesicles. I: Binding and transport

To gain insight into the mechanisms governing cellular uptake of glucocorticoids, we studied the binding and membrane transport of corticosterone (B) on a highly purified plasma membrane fraction from rat liver that was homogenized using a gentle, isotonic procedure. The fraction was mostly in the form of right-side out and osmotically active vesicles that were free of intracellular glucocorticoid receptors (GCR), transcortin (CBG) and ATP. Our uptake and binding studies carried out at 22 degrees C with [3H]B in physiological concentrations resulted in the following findings: (1) unlabeled B competed with [3H]B for uptake by the membrane vesicles; half-maximal competition of specific uptake was achieved with a 10- to 11-fold molar excess of unlabeled B. (2) [3H]B uptake was a saturable process of unusual kinetics (multiple sigmoidity); modified Scatchard plots revealed three significantly different apparent Kd-values of 1.3, 4.7 and 17.3 nM, corresponding to free B in the blood of non-stressed rats (4-16 nM). (3) Osmotic shrinkage of the vesicles led to a linear decrease in specific uptake, while non-specific uptake was independent of vesicle volume. Passive diffusion of [3H]B took place in leaky, but not in intact, vesicles. Reversible binding to, and mediated transport through, the membrane were interdependent parts of a strongly linked process. B was accumulated inside the vesicle up a concentration gradient by an active transport that followed first-order kinetics (Kt:3.9 nM); for its statistically reliable mathematical formulation and kinetic analysis, a replot was developed that revealed that relative accumulation increased with decreasing external hormone concentration. (4) Comparative binding studies disclosed that the apparent Kd-values (86.5 +/- 7.3 and 77.0 +/- 14.3 nM, respectively) of the [3H]B interactions with CBG and GCR did not differ (P greater than 0.3). These findings permit the conclusion that a plasma membrane-inserted carrier for B, effectively operating at physiological concentrations in the blood, is involved in a functional and regulatory manner in the biological action of glucocorticoids.