Characterization of membrane estrogen binding proteins from rabbit uterus

Area of Science:

• Endocrinology
• Molecular Biology
• Cell Biology

Background:

• Estrogens mediate rapid, non-genomic effects in target tissues.
• These actions may involve cell membrane-associated receptors.
• The identity of these membrane-bound estrogen binding proteins remains largely uncharacterized.

Purpose of the Study:

• To identify and characterize membrane-associated 17beta-estradiol binding proteins in the rabbit uterus.
• To investigate the structural relationship of these proteins to the intracellular estrogen receptor (ER).

Main Methods:

• Radioligand binding assays using [3H]17beta-estradiol on uterine microsomes and cytosol.
• Competition assays with various steroids, 17alpha-estradiol, and tamoxifen.
• Enzyme assays to rule out cytosolic contamination.
• Immunoblot analysis using monoclonal antibodies against the nuclear estrogen receptor (ER).
• CHAPS detergent extraction of microsomes.

Main Results:

• Specific, saturable, high-affinity [3H]17beta-estradiol binding sites were detected in uterine microsomes.
• Binding affinity (Kd = 0.36 nM) and capacity were characterized.
• Microsomal binding sites were trypsin-sensitive and KCl-extractable.
• Immunoblot analysis revealed ER and five additional immunoreactive proteins in membranes, distinct from cytosol.
• Antibodies against the ER steroid-binding domain inhibited both cytosolic and membrane binding.

Conclusions:

• The rabbit uterus contains novel 17beta-estradiol membrane binding proteins.
• These proteins exhibit structural similarities to the intracellular estrogen receptor (ER).
• These findings suggest a role for these novel membrane proteins in mediating non-genomic estrogen actions.