IBD-associated G protein-coupled receptor 65 variant compromises signalling and impairs key functions involved in inflammation

Cell Signal. 2022 May:93:110294. doi: 10.1016/j.cellsig.2022.110294. Epub 2022 Feb 24.

Abstract

Background and aims: Inflammatory bowel diseases (IBD) result in chronic inflammation of the gastrointestinal tract. Genetic studies have shown that the GPR65 gene, as well as its missense coding variant, GPR65*Ile231Leu, is associated with IBD. We aimed to define the signalling and biological pathways downstream of GPR65 activation and evaluate the impact of GPR65*231Leu on these.

Methods: We used HEK 293 cells stably expressing GPR65 and deficient for either Gαs, Gαq/11 or Gα12/13, to define GPR65 signalling pathways, IBD patient biopsies and a panel of human tissues, primary immune cells and cell lines to determine biologic context, and genetic modulation of human THP-1-derived macrophages to examine the impact of GPR65 in bacterial phagocytosis and NLRP3 inflammasome activation.

Results: We confirmed that GPR65 signals via the Gαs pathway, leading to cAMP accumulation. GPR65 can also signal via the Gα12/13 pathway leading to formation of stress fibers, actin remodeling and RhoA activation; all impaired by the IBD-associated GPR65*231Leu allele. Gene expression profiling revealed greater expression of GPR65 in biopsies from inflamed compared to non-inflamed tissues from IBD patients or control individuals, potentially explained by infiltration of inflammatory immune cells. Decreased GPR65 expression in THP-1-derived macrophages leads to impaired bacterial phagocytosis, increased NLRP3 inflammasome activation and IL-1β secretion in response to an inflammatory stimulus.

Conclusions: We demonstrate that GPR65 exerts its effects through Gαs- and Gα12/13-mediated pathways, that the IBD-associated GPR65*231Leu allele has compromised interactions with Gα12/13 and that KD of GPR65 leads to impaired bacterial phagocytosis and increased inflammatory signalling via the NLRP3 inflammasome. This work identifies a target for development of small molecule therapies.

Keywords: Actin remodeling; Bacterial phagocytosis; G proteins; GPR65; NLRP3 inflammasome.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • HEK293 Cells
  • Humans
  • Inflammasomes* / metabolism
  • Inflammation / metabolism
  • Inflammatory Bowel Diseases* / genetics
  • Inflammatory Bowel Diseases* / pathology
  • Interleukin-1beta
  • NLR Family, Pyrin Domain-Containing 3 Protein
  • Receptors, G-Protein-Coupled / genetics
  • Receptors, G-Protein-Coupled / metabolism*

Substances

  • GPR65 protein, human
  • Inflammasomes
  • Interleukin-1beta
  • NLR Family, Pyrin Domain-Containing 3 Protein
  • Receptors, G-Protein-Coupled