Stoichiometry and site-specific phosphorylation of human progesterone receptor in native target cells and in the...

Human progesterone receptor (PR) in T47D breast cancer cells is phosphorylated on nine different serine residues; three are hormone-inducible (Ser102, Ser294, and Ser345), while others are basal but hormone-stimulated. In the present study, we have compared the phosphorylation state of native and recombinant PR expressed in a baculovirus insect cell system. Stoichiometric measurements showed that unliganded native PR in T47D cells was approximately 50% phosphorylated ( approximately 4 phosphates/PR) and became essentially 100% phosphorylated ( approximately 9 phosphates/PR) when bound to hormone. Unliganded PR expressed in Sf9 insect cells was phosphorylated with a similar stoichiometry ( approximately 3 phosphates/PR), but the phosphate content did not change with hormone addition. Site-specific phosphorylation analyzed by tryptic phosphopeptide mapping and manual peptide sequencing revealed that expressed PR bound to hormone in the Sf9 insect cells was phosphorylated on all the same sites as hormone-treated PR in T47D cells. Only minor differences were detected in the relative proportion of three sites (two basal sites and Ser345) and phosphorylation did not occur on alternate sites. Interestingly, unliganded baculovirus-expressed PR was constitutively phosphorylated on hormone inducible sites and was phosphorylated on basal sites to the same extent as hormone treated PR. Thus, in the absence of hormone, the phosphorylation state of baculovirus-expressed PR resembled that of the hyperphosphorylated native PR. In contrast to native PR, the expressed receptor in cytosols of Sf9 cells did not form a large oligomeric complex suggesting that hyperphosphorylation may be due to dissociation of the complex in the absence of hormone. This study demonstrating phosphorylation on correct sites with a stoichiometry similar to that of native PR indicates that overexpressed PR in the baculovirus system is suitable for in vitro structure/function studies.