Abstract by Yufang Deng

G protein-coupled receptors (GPCRs) are the most studied membrane protein superfamilies that contain around 800 members in humans. GPCR receives a broad range of extracellular stimulation (e.g., small molecule, peptide, ion, and proton) and transduces the signal into the intracellular via interaction with a series of intracellular effectors such as G proteins and arrestins, leading to cellular responses and biological events. One-third of the marketed drugs target GPCR or its related signal pathways, making GPCR the most promising and productive drug target.

GPR15 is a prototypical class A GPCR identified in 1996 as a co-receptor for human immunodeficiency virus infection. Early studies indicated it is a potential therapeutic target for other immune disorders such as colitis, psoriasis, and multiple sclerosis. Moreover, GPR15 attracts increasing attention as a biomarker for tobacco smoking.

In the past decades, the molecular pharmacology characteristics of GPR15 remain largely unknown due to the lack of proper tool compounds. Until 2017, identifying the endogenous ligand GPR15L peptide by our group and others paved the way to study the GPR15 receptor further. The PhD project uses the GPR15L peptide as a pharmacological tool compound to characterize the GPR15 receptor functionally and pharmacologically.

As no small molecular ligand for GPR15 is available, Study I was a high throughput screening campaign aimed to identify small molecular ligands for GPR15 with a time-resolved fluorescence resonance energy transfer (TR-FRET)-based G_i cAMP assay on GPR15-HEK293A stable cells. However, all results were negative, with no ligand identified.

In Study II, we designed and obtained 44 GPR15L peptide analogues and dissected the structure-activity relationship of the GPR15L peptide with the TR-FRET-based G_i cAMP functional assay. The results showed that the C-terminal carboxy group, Leu⁷⁸, Pro⁷⁵, Val⁷⁴, and Trp⁷², were essential for the peptide activity. The GPR15 mutagenesis study was also performed with a bioluminescence resonance energy transfer (BRET)-based G protein activation assay in HEK293A cells transiently expressing GPR15. Lys192 and Glu272 were found to be residues that interact with GPR15L peptides and are important for receptor activity.

GPR15 was reported to couple to Gα_i/o protein in former studies. However, the subtype selectivity of GPR15 within Gα_i/o and its selectivity toward the three other Gα protein (Gα_s, Gα_q/11, and Gα_12/13) families are yet unknown. Study III determined the Gα protein coupling profiles of GPR15 with a BRET-based G protein activation and TR-FRET-based second messenger (cAMP/IP1) assays. Results showed that GPR15 exclusively couples to Gα_i/o and with similar subtype selectivity to Gα_i1, Gα_i2, Gα_i3, Gα_oA, and Gα_oB while with the least selectivity to Gα_z.

An earlier report suggests that GPR15 underwent constitutive internalisation. Nevertheless, the agonist-induced GPR15 internalisation and its molecular mechanism are unclear. Study IV delineated the endocytic proteins that regulate GPR15 internalisation by applying a real-time TR-FRET-based internalisation assay. Results showed that GPR15 internalisation is modulated by G protein-coupled receptor kinase, clathrin, dynamin, and caveolin. Notably, the arrestins seem not involved in GPR15 internalisation despite being recruited to the cell membrane upon agonist stimulation, as demonstrated by a real-time BRET-based bystander arrestin recruitment assay.

The PhD study has provided new insights into the future designing of the GPR15L peptide with improved pharmacological properties and increased the knowledge of the molecular mechanisms of the GPR15 receptor-mediated G protein signalling and the internalisation pathways.