Abstract. internalisation and recycling of seven transmembrane domain receptors is a critical regulatory event for their signalling. The mechanism(s) by which membrane progesterone receptor-α (mPrα) number is regulated on the cell surface is unclear. in this study, we investigated the cellular distribution of mPrα and mechanisms of mPrα 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 mPrα in M11 cells with mPrα primarily distributed on the cell surface under basal conditions. For the first time, plasma membrane localisation of mPrα was confirmed using immunogold transmission electron microscopy. Stimulation of M11 cells with progesterone (P4, 100 nM) resulted in internalisation of mPrα from the plasma membrane to the cytoplasm (10 min) and subsequent limited translocation back to the cell surface (20 min). We investigated potential endocytotic pathways involved in trafficking of mPRα after its internalisation. Partial co-localisation of clathrin with mPrα was obvious after 10 min of P4 treatment. of note, chlorpromazine (inhibitor of clathrin-mediated pathway) inhibited the endocytosis of mPrα, whereas treatment with nystatin (inhibitor of caveolae-mediated pathway) did not affect internalisation. collectively, these data suggest that mPrα is expressed on the cell surface of M11 cells and undergoes endocytosis after P4 stimulation primarily via a clathrin-mediated pathway.
IntroductionThe large group of seven transmembrane domain receptors (7TMRs) include the well-known superfamily of G protein coupled receptors (GPCRs) as well as the phylogenetically distinct family of membrane progesterone receptors (mPrs) (1,2). Trafficking of 7TMRs between the various cell compartments is crucial for their ligand-binding and signalling functions. internalisation of 7TMrs by endocytosis controls important receptor functions in the cell, fine tuning the binding characteristics of the receptor and regulating the activation of different signal transduction pathways. Some cell-surface receptors can also activate signal transduction pathways from intracellular compartments, suggesting that signalling can be regulated by compartmentalization (3).Internalisation of many GPCRs involves rapid clathrinmediated endocytosis (3-5). The agonist-occupied receptor is recruited into clathrin-coated pits (ccP), which then form vesicles for entry into the endocytic pathway. at this stage, vesicles and their cargo can be directed for either degradation or recycling back to the cell surface (6,7). For example, it has been shown that different internalisation routes seem to predetermine whether transforming growth factor β (TGF-β) receptors will trigger a signalling response or be degraded (3). The precise mechanisms influencing the fate of the CCPreceptor complex are not fully elucidated, but emerging data suggest that they are receptor specific and may vary between cell types (8). Another route by which GPCRs ca...