Research
GPR101 is a driver of Growth Hormone secretion
Growth hormone (GH) is a key modulator of growth and GH over-secretion can lead to gigantism. One form is X-linked acrogigantism (X-LAG), in which infants develop GH-secreting pituitary tumors over-expressing the orphan G-protein coupled receptor, GPR101. The role of GPR101 in GH secretion remains obscure. We studied GPR101 signaling pathways and their effects in HEK293 and rat pituitary GH3 cell lines, human tumors and in transgenic mice with elevated somatotrope Gpr101 expression driven by the rat Ghrhr promoter (GhrhrGpr101). Here, we report that Gpr101 causes elevated GH/prolactin secretion in transgenic GhrhrGpr101 mice but without hyperplasia/tumorigenesis. We show that GPR101 constitutively activates not only Gs, but also Gq/11 and G12/13, which leads to GH secretion but not proliferation. These signatures of GPR101 signaling, notably PKC activation, are also present in human pituitary tumors with high GPR101 expression. These results underline a role for GPR101 in the regulation of somatotrope axis function.
Source: Abboud, D., Daly, A.F., Dupuis, N. et al. GPR101 drives growth hormone hypersecretion and gigantism in mice via constitutive activation of Gs and Gq/11. Nat Commun 11, 4752 (2020). https://doi.org/10.1038/s41467-020-18500-x
Discovery of ligands for the orphan GPCR GPR27
GPR27 is an orphan G protein coupled receptor characterized by a high level of conservation among vertebrates and a predominant expression in the central nervous system. It has recently been linked to insulin secretion. However, the absence of endogenous or surrogate ligands for GPR27 complicates the examination of its biologic function. Our aim was to validate GPR27 signaling pathways, and therefore we sought to screen a diversity-oriented synthesis library to identify GPR27-specific surrogate agonists. To select an optimal screening assay, we investigated GPR27 ligand-independent activity. Both in G protein-mediated pathways and in β-arrestin 2 recruitment, no ligand-independent activity could be measured. However, we observed a recruitment of β-arrestin 2 to a GPR27V2 chimera in the presence of membrane-anchored G protein-coupled receptor kinase-2. Therefore, we optimized a firefly luciferase complementation assay to screen against this chimeric receptor. We identified two compounds sharing a N-phenyl-2,4-dichlorobenzamide scaffold, which were selective for GPR27 over its closely related family members GPR85 and GPR173. The specificity of the activity was confirmed with a NanoLuc Binary Technology β-arrestin 2 assay, imaging of green fluorescent protein-tagged β-arrestin 2, and PathHunter β-arrestin 2 assay. Interestingly, no G protein activation was detected upon activation of GPR27 by these compounds. This study provides the first selective surrogate agonists for the orphan GPR27.
A dynamic and screening-compatible nanoluciferase-based complementation assay enables profiling of individual GPCR-G protein interactions
The effective identification of ligands for G protein-coupled receptors requires innovative assays that can detect and drive the design of compounds with novel or improved pharmacological properties. In particular, a functional and screening-compatible GPCR-G protein interaction assay is still unavailable. For this project, we report on a nanoluciferase-based complementation technique to detect ligands that promote a GPCR-G protein interaction. This system can be used to profile compounds with regard to the G proteins they activate through a given GPCR. Furthermore, we establish a proof of applicability of screening for distinct G proteins on dopamine receptor D2 whose differential coupling to Gαi/o family members has been extensively studied. In a D2-Gαi1 versus D2-Gαo screening, we retrieved five agonists that are currently being used in antiparkinsonian medications. We determined that in this assay, piribedil and pergolide are full agonists for the recruitment of Gαi1 but are partial agonists for Gαo, that the agonist activity of ropinirole is biased in favor of Gαi1 recruitment, and that the agonist activity of apomorphine is biased for Gαo. Thus, this newly developed assay could be used to develop molecules that selectively modulate a particular G protein pathway.
