GPR101 orphan receptor: a novel cause of growth hormone deregulation

抄録

Background:

　GPR101 is an orphan G-protein coupled receptor. Recently, a clinical study showed that GPR101 is strongly associated with X-linked acrogigantism syndrome (XLAG), a rare disorder characterized by excessive growth hormone (GH) secretion. Carriers of the GPR101 duplication on the X chromosome develop pituitary adenomas that do not respond to current therapies. The GPR101 function in growth regulation is elusive and this lack of precise information precludes its validation as a drug target. Therefore, we sought to characterize GPR101 signaling pathways.

Methods:

We cloned in expression vector the human GPR101 fused to an N terminus FLAG epitope and we generated cells stably expressing FLAG-GPR101. Subsequently, we determined the receptor precise cellular localization and trafficking by using flow cytometry and confocal microscopy. Then, we used several pharmacological transduction assays (cAMP PGlo sensor, IP1 accumulation) and we developed a bioluminescent beta arrestin complementation test to decipher GPR101 signaling pathways. We also screened for compounds (such as culture medium components) and we generated several mutants that can increase or alter GPR101 activity. Finally, we implemented transgenic mice to gain insight into the disease.

Results:

GPR101 is characterized by a very high constitutive activity. We detected important constitutive cAMP production that was linked to Gs activity, and we completed our study with an examination of receptor coupling to other G proteins and pathways. We also demonstrated that the basal recruitment of beta arrestins leads to constitutive internalization of the receptor and relocates the receptor into endosomes. Furthermore, we investigated the origin of constitutive activity and excluded a role of culture medium components. We applied targeted mutagenesis and identified putatively important residues (such as the E308) for constitutive signaling. Finally, several giant hallmarks were studied in our murine models (loss- and gain-of-function).

Conclusions:

The precise characterization of GPR101 signalling pathways constitute an absolute prerequisite to establish a strong mechanistic link between GPR101 activation in the pituitary and gigantism phenotype, and/or to treat people suffering from pituitary dysfunction.