Regulation of follitropin receptor cell surface residency by the ubiquitin-proteasome pathway

Little is known of the normal physiological processes that govern the cell surface residency of the human follitropin receptor (hFSHR), a G protein-coupled receptor expressed in the ovary and testis. In the hFSHR, the third intracellular (3i) loop is considered to be pivotal in attenuation of ligand activation, particularly internalization. To gain a better understanding of these processes, we used a yeast-based interaction trap to identify cytoplasmic proteins in a human ovarian cDNA library that interacted with the hFSHR 3i loop. Among the cDNA identified, four encoded isoforms of ubiquitin. Immunoprecipitated hFSHR probed with an antiubiquitin antibody revealed that the receptor is ubiquitinated, although not exclusively on the 3i loop. Cell-surface hFSHR levels increased when expressed at nonpermissive temperature in a temperature-sensitive, ubiquitination-defective cell line. Similarly, after treatment with proteasome inhibitors, HEK293 cells stably transfected with an hFSHR expression plasmid showed an increase in follitropin binding. Proteasome inhibitors did not affect the rate of FSH internalization when receptors were saturated before internalization was measured. In contrast, internalization decreased when binding experiments were performed under nonequilibrium conditions. A mutant hFSHR-K555R, which removes the only lysine in the 3i loop available for ubiquitination, was still ubiquitinated, illustrating that, although the third loop enables and interaction with ubiquitin, it is not the sole site of ubiquitination. These observations are consistent with a role for ubiquitination in the regulation of hFSHR cell surface residency. Additionally, it can be inferred that a sequence in the 3i loop is involved in regulating receptor ubiquitination and internalization.