Endocrine Abstracts

Growth hormone receptor (GHR) internalisation plays a crucial role in GHR activation and signalling by controlling the availability of the receptor for binding growth hormone (GH), regulating the intensity and duration of the GH signal, initiating unique signalling pathways, and maintaining homeostasis in the GH signalling pathway. GHR internalisation occurs mainly through clathrin-mediated endocytosis. Upon GH binding, the GHR undergoes ubiquitination, which is a signal for internalisation. The GHR is then included in clathrin-coated pits and internalised into early endosomes. From here, GHR can be recycled back to the plasma membrane. Here we present a quantitative study of the internalisation of human GH, the long-acting GH analogue somapacitan and the GHR antagonist pegvisomant using human GHR-expressing baby hamster kidney fibroblasts (BHK21). Human GH, somapacitan and pegvisomant were conjugated with Alexa Fluor 647 dye to follow the internalisation. Receptor internalisation could be observed for all three compounds in the concentration range 0.005–10 nM. Cells were fixated at predetermined timepoints from 0–4 hours and fluorescence determined for each combination of time point and concentration level. Internalisation was reduced in the presence of a non-labelled competitor showing that uptake is specific to the receptor. The results showed that all three compounds were internalised by the GHR. The internalisation rate was highest for human GH followed by somapacitan and with pegvisomant having the slowest rate. Addition of non-labelled pegvisomant to cells together with labelled human GH or somapacitan showed a displacement effect of pegvisomant of both compounds on the GHR with a stronger effect on somapacitan. While the competition effect for GH is higher at 4 hours after addition of pegvisomant compared to 24 hours, the displacement effect for somapacitan lasted for 24 hours. In conclusion, somapacitan undergoes receptor-mediated internalisation by the GHR at a slower rate than human GH, but faster than pegvisomant. Somapacitan is more easily displaced from the GHR by pegvisomant compared to human GH.