title: Defining the patho-physiological roles of the receptor GPR35
Creators: name: Ganguly, Amlan
Publisher: name: University of Glasgow
Year: 2024

description: G-protein-coupled receptors (GPCRs) have tremendous potential as therapeutic targets for a wide range of disorders since they mediate most of our physiological responses to hormones, neurotransmitters, and environmental stimuli. GPR35 is a rhodopsin-like class A GPCR first identified over two decades ago. Even though GPR35 is currently a poorly characterised orphan receptor, it has significant therapeutic utilities for various ailments, including fatty liver disease, inflammatory bowel problem and different malignancies.

The pharmacology of the GPR35 orthologues in humans and rodents varies significantly and in human orthologue, there are 2 isoforms that are transcribed and translated from 3 variants of the human GPR35 gene. Although the pharmacology of the two human GPR35 isoforms is similar, agonist response efficacy is significantly lower in the longer isoform compared to the shorter one. From experimental studies, it was found that amino acid cysteine at position 27 of long isoform of GPR35 (hGPR35b) acted as a dampener of hGPR35b efficacy. Mutation of this cysteine to serine resulted in the efficacy of hGPR35b being equivalent to hGPR35a. In order to gain a deeper comprehension of the function of the N-terminal extension of hGPR35b, 10 further mutants containing cysteine in place of different residues were produced in the Cys27Ser hGPR35b backbone. Based on the findings, it was clear that almost all of the mutants showed G protein activation and arrestin-3 recruitment activity almost identical to hGPR35b after cysteine was added.

For investigating the post-translational states of GPR35, novel phospho-sitespecific antibodies were developed and employed. After conducting immunoblotting and immunocytochemical experiments with these phosphorylation specific antisera, it was clearly visible that these antisera acted as useful biosensors for assessing the activation status of the human and mouse orthologues of GPR35. Furthermore, these antibodies only recognised fully matured versions of GPR35 and might serve as helpful instruments for assessing target involvement in drug discovery and target validation operations. 

To pinpoint the specific contribution of individual GRK in GPR35 phosphorylation, a variety of complementary techniques, such as the production of genome-edited cell lines lacking the GRK isoforms GRK2, GRK3, GRK5, and GRK6, which are widely expressed, restoration of function using certain GRKs as well as the development and use of both selective small molecule GRK inhibitors were employed. The availability of cell lines generated from 293 lacking expression of several GRKs revealed that, whereas agonist-induced receptor-arrestin interactions were unaffected by the absence of either GRK2 or GRK3, they were nearly completely prevented by the absence of both GRK5 and GRK6. Furthermore, reconstitution investigations using individual GRK isoforms were made possible by the GRK2/3/5/6 HEK293 cells, which demonstrated that GRK5 and GRK6 functioned nearly equally well. My experimental results were supported by a molecular level and mechanistic investigation that used the ‘Alphafold’ deep learning algorithm and revealed that GRK5 and GRK6 interacted with hGPR35a more strongly and effectively than GRK2 and GRK3. Upon comparing the efficacies of GRK2/3 blockers and GRK5/6 inhibitors, it was clearly evident that agonist-induced interactions between human GPR35a and arrestin-2 or arrestin-3, as well as the identification of agonist-mediated phosphorylation of the human and mouse orthologue in immunoblotting and immunocytochemistry investigations, were effectively inhibited by GRK5/6 blockers. For further ensuring the predominant contribution of GRK5/6, hGPR35a phosphorylation was conducted in GRK2/3/5/6 293 cells with transient introduction of GRK2/3/5/6. Immunoblotting using antihGPR35a pSer300-pSer303 revealed that the presence of GRK5 and, in particular, GRK6 increased the phosphorylation of these sites, but GRK2 and GRK3 had no appreciable impact. 

Finally, the luciferase-based complementation assay also demonstrated that GRK5 and GRK6 could interact with GPR35 in a significant way, thereby supporting my recent findings.

In conclusion, the outcome of this thesis provides a clear insight into the isoform variation in human orthologues of GPR35. Moreover, this research project gives a clear idea about the phosphorylation and indeed, regulation of GPR35, a receptor that is currently gaining a lot of attention as a potential new therapeutic target for conditions like ulcerative colitis, inflammatory bowel diseases, fatty liver diseases, and different cancers. The outcomes of this research project will certainly aid in the discovery of novel therapeutics targeting the orphan receptor GPR35.