Free leucine dissociates homo- and heterodimers formed between proteins containing leucine heptad repeats.

A highly specific method for the dissociation of protein dimers has been developed. The method involves exposure of the dimers to free leucine at a concentration ranging between 3 and 10 mM. Using this method it has been possible to dissociate goat uterine oestrogen receptor homodimers, heterodimers formed between the non-activated oestrogen receptor (naER) and the oestrogen receptor activation factor (E-RAF) of the goat uterus, c-jun homodimers derived from bovine bone marrow and also glucocorticoid receptor homodimers isolated from rat liver cytosol. The pattern of dimer dissociation by leucine clearly differentiates two classes of proteins. The first is represented by steroid hormone receptors where dimerization is apparently contributed by both coiled-coil dimerization interfaces and the conserved heptad repeats of leucine. The second is represented by oncoproteins like c-fos and c-jun which dimerize through the exclusive involvement of leucine zippers. The patterns of dissociation of these two groups of proteins from the concerned affinity columns are distinctly different. This indicates a possibility that the elution pattern may be used as a yardstick to determine whether two proteins dimerize through the exclusive involvement of leucine zippers or whether coiled-coil interfaces are also involved in the dimerization process.

Protein-protein interactions that precede the nuclear entry of goat uterine estrogen receptor under cell-free conditions.

Mechanisms associated with a regulated nuclear entry of the goat uterine estrogen receptor (gER), under the influence of estradiol, have been examined using a cell-free system. The gER transport into the nucleus is mediated by a 55-kDa cytosolic protein, p55. Experimental evidence has been provided to demonstrate that p55 binds to the nuclear localization signals (NLS) on the gER. Under hormone-free conditions, a 28-kDa protein, p28, binds to the NLS and prevents the p55 interaction with the NLS. This inhibition is reversed by a 73-kDa protein, p73. It appears that p73 associates with the hormone binding domain (HBD) of the gER under hormone-free conditions. Estradiol binding to the HBD facilitates p73 interaction with p28. This leads to the dissociation of p28 from the NLS, which, in turn, facilitates the binding of p55 to the NLS on the gER. Both p28 and p55 cross-react with monoclonal antibodies against hsp-25 and hsp-70, indicating a possibility that p28 and p55 belong to a superfamily of molecular chaperones. J. Cell. Biochem. 78:650-665, 2000.