Internalisation of membrane progesterone receptor-α after treatment with progesterone: Potential involvement of a clathrin-dependent pathway

Internalisation and recycling of seven transmembrane domain receptors is a critical regulatory event for their signalling. The mechanism(s) by which membrane progesterone receptor-alpha (mPR alpha) number is regulated on the cell surface is unclear. In this study, we investigated the cellular distribution of mPR alpha and mechanisms of mPR alpha trafficking using a cell line derived from a primary culture of human myometrial cells (M11) as an experimental model. RT-PCR and immunofluorescent analysis demonstrated expression of mPR alpha in M11 cells with mPR alpha primarily distributed on the cell surface under basal conditions. For the first time, plasma membrane localisation of mPR alpha was confirmed using immuno-gold transmission electron microscopy. Stimulation of M11 cells with progesterone (P4, 100 nM) resulted in internalisation of mPR alpha from the plasma membrane to the cytoplasm (10 min) and subsequent partial translocation back to the cell surface (20 min). We investigated potential endocytotic pathways involved in trafficking of mPR alpha after its internalisation. Partial co-localisation of clathrin with mPR alpha was obvious after 10 min of P4 treatment. Of note, chlorpromazine (inhibitor of clathrin-mediated pathway) inhibited the endocytosis of mPR alpha, whereas treatment with nystatin (inhibitor of caveolae-mediated pathway) did not affect internalisation. Collectively, these data suggest that mPR alpha is expressed on the cell surface of M11 cells and undergoes endocytosis after P4 stimulation primarily via a clathrin-mediated pathway.