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

Herzig Herzig,Evert Evert,Lupp Lupp,Kaemmerer Kaemmerer,Dasgupta Dasgupta,Schulz Schulz,Sänger Sänger

doi:10.3390/ijms20215261

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.

Highlights

GPR68, known as ovarian cancer G protein-coupled receptor 1 (OGR1), is a member of the proton-sensing G protein-coupled receptor (GPCR) family that contains three additional members: GPR4, GPR65, and GPR132
The specificity of anti-human GPR68 antibody 16H23L16 was first tested by Western blot analysis
GPR68 expression in BON-1 cells was silenced by a specific siRNA to further confirm the specificity of the antibody

Summary

Introduction

GPR68, known as ovarian cancer G protein-coupled receptor 1 (OGR1), is a member of the proton-sensing G protein-coupled receptor (GPCR) family that contains three additional members: GPR4, GPR65 (or T lymphocyte death-associated gene 8 protein, TDAG8), and GPR132 (or G2 accumulation protein, G2A). The pH window of GPR68 is quite narrow. The receptor seems to be inactive at pH 7.8 and fully active at pH 6.8 [1,2], but the opposite pattern has been shown [3]. The pH sensing ability of GPR68 is attributed to four histidines located in its extracellular loops. It is hypothesised that at a slightly alkaline pH, GPR68 is stabilised in an inactive state by hydrogen bonding of these residues. Protonation of the histidines at an acidic pH may cause loss of hydrogen bonding, allowing the receptor to adopt an active conformation [1]. In addition to direct activation by protons, ogerin and the benzodiazepine lorazepam have been shown to be positive allosteric modulators of GPR68 [4]. GPR68 is coupled to the Gq/11-phospholipase-C/inositol-triphosphate (IP3) pathway, and upon activation, calcium is released from the endoplasmic reticulum into the cytosol [1]

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