GPR101 is a member of a large family of proteins found on the plasma membrane of cells known as G protein-coupled receptors (GPCRs) [1,2]. GPCRs are implicated in a myriad of physiological functions, such as mediating the biological effect of neurotransmitters and hormones. GPCRs are also involved in countless pathological conditions and are targeted by a large share of prescription drugs. From a structural perspective, GPCRs are constituted by a single polypeptide chain that spans the plasma membrane seven times, with seven alpha-helical structures, known as transmembrane domains (TMs). These TMs are connected by three extracellular loops (ECLs), and three intracellular loops (ICLs). The N-terminus is in the extracellular space, and the C-terminus is in the cytosol. For many GPCRs, the first segment of the C-terminus is an alpha-helical structure parallel to the plane of the plasma membrane known as helix 8 (H8) [[3], [4], [5], [6]]. See Fig. 3 in Trivellin et al. for a schematic 2D representation of the GPR101 structure [7].

GPR101 is implicated in a rare form of genetic gigantism known as X-linked acrogigantism, or X-LAG. Specifically, X-LAG patients harbor microduplications in the long arm of the X-chromosome that invariably include the gene GPR101. Duplications of the GPR101 gene lead to the formation of a new chromatin domain that causes over-expression of the receptor in the pituitary tumors of the patients [8,9]. GPR101 is a constitutively active receptor, which stimulates cells to produce the second messenger cyclic AMP (cAMP) in the absence of ligands [[8], [9], [10], [11]]. Moreover, GPR101 was recently reported to constitutively activate not only the cAMP pathway, but also other G protein subunits (Gq/11 and G12/13) [11]. Hence, chemicals that block the constitutive activity of GPR101, known as inverse agonists, have the potential to be useful for the development of pharmacological tools for the treatment of X-LAG.

To date, the natural, endogenous chemical that activates GPR101 is unknown. For this reason, GPR101 is classified as an orphan receptor. Literature reports indicate that the peptide GnRH-(1-5), a metabolite of the gonadotropin-releasing hormone (GnRH), might be a GPR101 ligand. In particular, the effect of GnRH-(1-5) on EGF release, which subsequently leads to EGFR phosphorylation, appears to be mediated by GPR101 via the metalloprotein MMP-9 [12,13]. Moreover, it has been reported that GNRH-(1-5) promotes β-arrestin recruitment by GPR101 [14]. The lipid n-3 docosapentaenoic acid-derived resolvin D5 (RvD5n-3 DPA) has also been reported to bind GPR101 and promote β-arrestin recruitment [15]. Beyond agonists, GPR101 blockers have been reported in two patents [16,17]. These include the drugs difeterol and vanoxerine [16] as well as a series of sulfonamides bearing aromatic rings [17]. In light of GPR101's high constitutive activity, it is possible that GPR101 does not have an endogenous ligand and fulfills in function in virtue of its ability to signal in its inactive state. It is also possible that GPR101 constitutive activity is modulated by endogenous inverse agonists, as it has been shown for several GPCRs [18].

In this study, we provide structural insights into the putative structure of GPR101 based on in-house built homology models, as well as third party models based on the machine learning methods AlphaFold and AlphaFold-Multistate. Moreover, we report a molecular dynamics study, meant to further probe the constitutive activity of GPR101. Finally, we provide a structural comparison with the closest GPCRs, which suggests that GPR101 does not share their natural ligands. As three-dimensional structures of proteins are valuable tools to generate mechanistic hypotheses on their functioning at the molecular level and to support ligand discovery campaigns, we made our GPR101 models publicly available to the scientific community with the intention of spurring research efforts that can shed light onto this elusive GPCR.