Comprehensive Assessment of GPR68 Expression in Normal and Neoplastic Human Tissues Using a Novel Rabbit Monoclonal Antibody

Abstract

GPR68 (OGR1) belongs to the proton-sensing G protein-coupled receptors that are involved in cellular adaptations to pH changes during tumour development. Although expression of GPR68 has been described in many tumour cell lines, little is known about its presence in human tumour entities. We characterised the novel rabbit monoclonal anti-human GPR68 antibody 16H23L16 using various cell lines and tissue specimens. The antibody was then applied to a large series of formalin-fixed, paraffin-embedded normal and neoplastic human tissue samples. Antibody specificity was demonstrated in a Western blot analysis of GPR68-expressing cells using specific siRNAs. Immunocytochemical experiments revealed pH-dependent changes in subcellular localisation of the receptor and internalisation after stimulation with lorazepam. In normal tissue, GPR68 was present in glucagon-producing islet cells, neuroendocrine cells of the intestinal tract, gastric glands, granulocytes, macrophages, muscle layers of arteries and arterioles, and capillaries. GPR68 was also expressed in neuroendocrine tumours, where it may be a positive prognostic factor, in pheochromocytomas, cervical adenocarcinomas, and endometrial cancer, as well as in paragangliomas, medullary thyroid carcinomas, gastrointestinal stromal tumours, and pancreatic adenocarcinomas. Often, tumour capillaries were also strongly GPR68-positive. The novel antibody 16H23L16 will be a valuable tool for basic research and for identifying GPR68-expressing tumours during histopathological examinations.

pH-Sensing G Protein-Coupled Receptor OGR1 (GPR68) Expression and Activation Increases in Intestinal Inflammation and Fibrosis

“…In the pancreas, OGR1-positive cells (Figure S3) revealed a similar staining pattern to those shown by Herzig [50]. This group elegantly demonstrated that the OGR1-positive cells in the pancreas are glucagon-producing islet cells [50]. In our current study, immunostaining of duodenum tunica muscularis revealed OGR1-positive staining in macrophages, granulocytes, endothelial cells, and fibroblasts (Figure 2D).…”

“…In the mucosal duodenum, OGR1-positive cells included macrophages, granulocytes, and endothelial cells (Figure 2C). In the pancreas, OGR1-positive cells (Figure S3) revealed a similar staining pattern to those shown by Herzig [50]. This group elegantly demonstrated that the OGR1-positive cells in the pancreas are glucagon-producing islet cells [50].…”

“…Recently, Herzig et al evaluated the OGR1 recombinant rabbit monoclonal antibody (16H23L16) in FFPE normal and neoplastic human tissue samples [50]. We tested this antibody in Caco-2 cells stably overexpressing OGR1.…”

Metabolite Sensing GPCRs: Promising Therapeutic Targets for Cancer Treatment?

“…Other studies showed GPR68 overexpression in human medulloblastoma tissue [172] and in head and neck squamous cancer [173]. Finally, immunohistochemical analysis detected significant GPR68 expression in endocrine or neuroendocrine tumors with origin in the pituitary, the thyroid gland, or the respiratory and digestive systems [174]. More detailed analysis on the expression of GPR68 in particular tumor cell types, showed a significant expression in cancer epithelial cells and fibroblasts of pancreatic ductal adenocarcinomas [171] and in hematopoietic and mesenchymal cells of colorectal tumors [175].…”

A review of antibody-based therapeutics targeting G protein-coupled receptors: an update

“…Such GPCRomics approaches [48] have highlighted potential therapeutic targets, such as GPR68 [47,49]. Research tools will be invaluable to further validate these potential targets for therapeutic relevance [50]. Therapeutic areas in which GPCRs have been identified as suitable targets for antibodybased molecules are shown.…”