Chemical modification and structural analysis of the progesterone membrane binding protein from porcine liver membranes.
In addition to the classical genomic steroid actions on modulation of transcription and protein synthesis, rapid, nongenomic effects have been described for various steroids. These effects on cellular signaling and function are supposed to be transmitted by membrane binding sites unrelated to the classical intracellular receptors. Recently, a high affinity progesterone membrane binding protein (mPR) has been characterized in porcine liver membranes. In the present study, amino acid residues that are essential for progesterone binding to porcine liver microsomal mPR have been identified by the use of protein modifying reagents. Among all reagents tested, agents with specificity for carboxyl groups, methionine and tryptophan such as N,N'-dicyclohexylcarbodiimide, chloramine T and N-bromosuccinimide induced a reduction in [3H]progesterone binding. To evaluate the presence of essential disulfide bridges, porcine liver microsomes were incubated with the disulfide reducing agent dithiothreitol (DTT) and [3H]progesterone binding was measured. This treatment also resulted in a reduction of binding activity with an IC50 of 20 mM for DTT. Western-blotting analysis in the presence or absence of the reducing agent suggested that mPR--in its binding state--consists of at least two identical subunits with an apparent molecular mass of 28 kDa which are linked by a disulfide bridge. In conclusion, in the present study evidence for an involvement of carboxyl-, tryptophan- and methionine residues in [3H]progesterone binding to porcine liver microsomes is given. In addition, it is shown that mPR can form disulfide-linked homodimers.
Falkenstein
E
E
Institute of Clinical Pharmacology, Faculty of Clinical Medicine at Mannheim, University of Heidelberg, Germany.
Eisen
C
C
Schmieding
K
K
Krautkrämer
M
M
Stein
C
C
Lösel
R
R
Wehling
M
M
D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D001974:Bromosuccinimide / Q000494:pharmacology*; D002700:Chloramines / Q000494:pharmacology*; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology*; D019281:Dimerization; D004229:Dithiothreitol / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D008024:Ligands; D008565:Membrane Proteins / Q000378:metabolism*; D008862:Microsomes, Liver / Q000378:metabolism*; D011374:Progesterone / Q000378:metabolism*; D011485:Protein Binding; D011980:Receptors, Progesterone / Q000378:metabolism*; D012680:Sensitivity and Specificity; D013552:Swine; D014105:Tosyl Compounds / Q000494:pharmacology*
D002700:Chloramines; D008024:Ligands; D008565:Membrane Proteins; D011980:Receptors, Progesterone; D014105:Tosyl Compounds; C016300:chloramine-T; D011374:Progesterone; D004024:Dicyclohexylcarbodiimide; D001974:Bromosuccinimide; D004229:Dithiothreitol