Abstract
Progesterone receptor (PR) isoforms, PRA and PRB, act in a progesterone-independent and dependent manner to differentially modulate the biology of breast cancer cells. Here we show that the differences in PRA and PRB structure facilitate the binding of common and distinct protein interacting partners affecting the downstream signaling events of each PR-isoform. Tet-inducible HA-tagged PRA or HA-tagged PRB constructs were expressed in T47DC42 (PR/ER negative) breast cancer cells. Affinity purification coupled with stable isotope labeling of amino acids in cell culture (SILAC) mass spectrometry technique was performed to comprehensively study PRA and PRB interacting partners in both unliganded and liganded conditions. To validate our findings, we applied both forward and reverse SILAC conditions to effectively minimize experimental errors. These datasets will be useful in investigating PRA- and PRB-specific molecular mechanisms and as a database for subsequent experiments to identify novel PRA and PRB interacting proteins that differentially mediated different biological functions in breast cancer.
Highlights
Background & SummaryEstrogen receptor (ER) and progesterone receptor (PR) are used to classify breast cancer histological subtypes and predict hormone therapy responsiveness[1]
PRB-AF3 domain contributes to strong transcriptional activity by suppressing the activity of an inhibitory domain (ID) within the common PRA/PRB N-terminal[12]
Since progestin-bound receptors get phosphorylated and degraded via a proteasome-dependent pathway and PRB rapidly degrades as compared to PRA (Fig. 1b)[26,27], we found fewer progestin-dependent PRB interacting partners compared to that of PRA
Summary
Estrogen receptor (ER) and progesterone receptor (PR) are used to classify breast cancer histological subtypes and predict hormone therapy responsiveness[1]. To PRB under progestin-independent condition[19] These two receptors exhibited distinct conformations and PRA contain an inhibitory domain (ID), prompting PRA to function as a strong ligand-dependent transdominant repressor of steroid hormone receptor transcriptional activity[25]. Ingenuity Pathway Analysis (IPA) showed that PRA and PRB interacting proteins enriched similar pathways but differences in significance value, except for the splicing of mRNA pathway that was only involved in unliganded-PRA, Fig. 5b. In the presence of progestin, proteins preferentially interacting with progesterone-bound PRA and PRB enriched similar pathways but showed differences in significance value, except for proteins in DNA damage and dysfunction of mitochondria pathways that were only interacting in progesterone-bound PRA, Fig. 6b. These new PRA and PRB interactome data will serve as molecular resources benefiting future interrogation into the PRA and PRB mediate breast cancer progression
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