The steroid 17 beta-estradiol (E2) acts to modulate transcription through c
lassical nuclear estrogen receptors (ER-alpha and ER-beta). However, E2 als
o induces a number of rapid responses (<10 min) within cells, including cel
ls devoid of classical ERs, consistent with the presence of a membrane rece
ptor for E2. Membrane impermeable steroids, typically bovine serum albumin
(BSA) conjugates, are commonly used to characterize these non-genomic actio
ns of E2 to exclude the involvement of nuclear ERs. Here we report that E2-
BSA conjugate preparations, but not unconjugated E2, activate extracellular
signal-regulated protein kinases (ERK1 and ERK2) in the SK-N-SH neuroblast
oma cell line, raising concerns regarding the use of these reagents as E2 m
imics. Freshly prepared solutions of E2-BSA were found to contain free immu
noassayable E2 (iE2), which could be removed by filtration. E2-BSA solution
s devoid of free iE2 failed to compete for binding of (125)[I]16 alpha-iodo
-E2 to ER-alpha or ER-beta. Furthermore, in contrast to E2, E2-BSA conjugat
es did not bind to ER-alpha or ER-beta as assessed by electrophoretic mobil
ity shift analyses. Protein analysis demonstrated that certain E2-BSA prepa
rations were of very high molecular weight, suggesting extreme protein cros
s-linking. These findings suggest that E2-BSA does not mimic E2 and is not
an appropriate ligand for investigating estrogen receptors. This underscore
s the need to design stable, cell impermeable analogs of estrogen for the c
haracterization of membrane estrogen receptors.